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Our data reveal that hRPA is able to bind and unfold a four-stranded intramolecular DNA quadruplex, forming at least two kinds of complexes in a multi-step mechanism. Thus, RPA may regulate the action of the telomerase during the cell cycle by opening G-quadruplex structures and maintaining them as ssDNA, thus facilitating the recruitment and binding of telomerase components onto telomeres.
|
16973897_p5
|
16973897
|
INTRODUCTION
| 4.284828
|
biomedical
|
Study
|
[
0.9995928406715393,
0.00024817336816340685,
0.0001589356252225116
] |
[
0.9987152814865112,
0.0007933599408715963,
0.0003705598646774888,
0.00012091578537365422
] |
en
| 0.999999
|
BSA was from Roche. [γ- 32 P]ATP (6 μCi/pmol) was from Amersham and T4 polynucleotide kinase from BioLabs. The oligonucleotides 5′-d(TTTTTTTTTTTTTTTTTTTTT)-3′(T21), 5′-d(GGGTTAGGGTTAGGGTTAGGG)-3′ (htelo),5′-d(CCCTAACCCTAACCCTAACCC)-3′ (21C), 5′-FLUO-(T21)-TAMRA-3′ (F-T21-T) and 5′-FLUO-(htelo)-TAMRA-3′(F-htelo-T) [where fluorescein (FLUO) and tetramethylrhodamine (TAMRA) are fluorescent dyes] were synthesized by Eurogentec (Seraing, Belgium). htelo and F-htelo-T oligonucleotides were based on the minimal human telomere repeat sequence capable of forming an intramolecular G-quadruplex structure. Recombinant hRPA was expressed in the Escherichia coli BL21 (DE3) (the three entire subunits p70, p32 and p14 were coexpressed with plasmid pET 11a hRPA generously provided by Dr Klaus Weisshart, IMB, Jena, Germany), and purified using Affi-Gel Blue, HAP and Q-Sepharose chromatographic columns according to Gomes et al. ( 25 ). hRPA was quantified using the Bradford assay.
|
16973897_p6
|
16973897
|
Materials
| 4.037882
|
biomedical
|
Study
|
[
0.9995793700218201,
0.0001458490442018956,
0.00027482782024890184
] |
[
0.9984424710273743,
0.0011324520455673337,
0.00033597732544876635,
0.00008909698226489127
] |
en
| 0.999996
|
Oligonucleotides were labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. 32 P-labeled oligonucleotides were purified using denaturing 15% PAGE. hRPA was diluted and pre-incubated (10 min at 4°C) in buffer containing 50 mM Tris–HCl (pH 7.5), 100 mM KCl, 1 mM DTT, 10% (v/v) glycerol, 0.2 mg/ml BSA and 0.1 mM EDTA. Radioactively labeled oligonucleotide (90 nM) was mixed with various amounts of protein in 10 μl of reaction buffer [25 mM Tris–HCl, pH 7.5, 1 mM EDTA, 2 mM MgCl 2 and 6% (v/v) glycerol] in the presence of 50 mM NaCl or KCl. hRPA–htelo binding reactions were conducted at 20°C for 10 min. Longer incubation times (up to 1 h) did not affect the band pattern or intensities, indicating that the systems had reached thermodynamic equilibrium in 10 min. Individual reaction mixtures were loaded onto a native 5% polyacrylamide gel in 0.5× TBE for 2.5 h at 7 V/cm and at 20°C. The gels were analyzed with a Phosphorimager STORM 860 instrument (Molecular Dynamics).
|
16973897_p7
|
16973897
|
hRPA–htelo binding
| 4.183855
|
biomedical
|
Study
|
[
0.999470055103302,
0.0003199691418558359,
0.00020988112373743206
] |
[
0.9991427659988403,
0.0004639637190848589,
0.0003155767044518143,
0.00007765652117086574
] |
en
| 0.999994
|
hRPA–htelo binding assays, prepared as described above, were loaded onto a native 5% polyacrylamide mini-gel in 0.5× TBE. The gels were stained by Coomassie blue (0.25%) for 45 min and then washed with a solution of 10% acetic acid and 30% ethanol. Colored bands were displayed and quantified with a G800 BIORAD™ densitometer and PDQuest 2-D Analysis Software™.
|
16973897_p8
|
16973897
|
Evaluation of hRPA in hRPA–htelo complexes
| 4.066625
|
biomedical
|
Study
|
[
0.9994656443595886,
0.00022244897263590246,
0.0003118652675766498
] |
[
0.9959318041801453,
0.003640176262706518,
0.0003213738964404911,
0.00010666765592759475
] |
en
| 0.999997
|
Protein–DNA complexes obtained in hRPA–htelo binding assays, prepared as described above, were cross-linked by the addition of 0.1% glutaraldehyde for 10 min. Longer treatments or 10 min incubation with 0.2% glutaraldehyde did not significantly improve cross-linking. Individual reaction mixtures were analyzed by non-denaturing 5% PAGE in the same conditions as described above.
|
16973897_p9
|
16973897
|
Cross-linking experiments
| 4.078095
|
biomedical
|
Study
|
[
0.9994887113571167,
0.00024717277847230434,
0.0002641975006554276
] |
[
0.9991840720176697,
0.0005147966439835727,
0.00024234030570369214,
0.00005868071093573235
] |
en
| 0.999996
|
The continuously decreasing mobility m C of C complex when r increased can be explained by a fast equilibrium [where the relaxation time τ of this equilibrium is much shorter than the migration time (2.5 h) between C I and C II complexes and free protein p]. C II ⇄ k − 1 p k 1 C I . If we define α and 1 − α as the fractions of C in the C I and C II forms, respectively, then m C = α m CI + ( 1 − α ) m CII and α = m C − m CII m CI − m CII . To take into account the smear observed at r = 0.5 and 1, we assumed that the 1:1 complex (in solution) partially dissociates in the gel. Thus, the smear is the result of the 1:1 complex dissociation. If f 0 , f 1 and f 2 are the relative weights in solution of free htelo, complex C I and complex C II , respectively, we can write f 0 + f 1 + f 2 = 1, with f 0 = w f , f 1 = w s + w CI + αw C and f 2 = (1 − α). w f , w s , w C and w CI , the weight of free htelo, smear, complex C and complex C I normalized with respect to total htelo, respectively, are calculated by Phosphorimager analysis from Figure 2. When applied to the data obtained in separate comparable experiments, f i values were calculated for each r -value.
|
16973897_p10
|
16973897
|
Quantification of f 0 , f 1 and f 2 fractions
| 4.279205
|
biomedical
|
Study
|
[
0.9990528225898743,
0.00026229445938952267,
0.0006849223864264786
] |
[
0.9994632601737976,
0.0002599540166556835,
0.00022918381728231907,
0.00004755912232212722
] |
en
| 0.999997
|
Fluorescence spectra at 20°C were recorded with a SpexFluoromax 3 instrument (Jobin-Yvon Horiba, Longjumeau, France), using 50 μl quartz cuvettes (Hellma, France) containing 90 nM F-htelo-T in 50 mM NaCl (or KCl), 2 mM MgCl 2 and 5 mM lithium cacodylate (pH 7.2). Concentrated protein aliquots (0.5 μl) were directly added to the F-htelo-T solution. The spectra were recorded between 490 and 660 nm while exciting at 470 nm, and corrected for background fluorescence, dilution factor and instrument response. The fluorescence intensity of individual fluorophores was estimated by averaging the fluorescence emission intensity in the 522–528 nm region for the donor (FLUO) and 584–590 nm for the acceptor (TAMRA; the low donor emission in that wavelength range was neglected). The ratio P was calculated as P = I D /( I D + I A ), where I D and I A are the average intensities of the donor and acceptor, respectively.
|
16973897_p11
|
16973897
|
Fluorescence spectroscopy
| 4.203834
|
biomedical
|
Study
|
[
0.9994468092918396,
0.00029585446463897824,
0.00025736558018252254
] |
[
0.9991922974586487,
0.0004060316423419863,
0.00033510950743220747,
0.0000665726765873842
] |
en
| 0.999998
|
Each solution containing 90 nM F-htelo-T in 2 mM MgCl 2 and 5 mM lithium cacodylate (pH 7.2) was mixed at time zero with a 5× molar excess of its complementary sequence 21C or hRPA. The kinetics were recorded at 20°C in 50 mM KCl. Fluorescence intensity at 516 nm was recorded at regular time intervals (1 s) using band slits of 5 nm. Data fitting was performed as described previously ( 27 ).
|
16973897_p12
|
16973897
|
Fluorescence kinetics
| 4.10372
|
biomedical
|
Study
|
[
0.9994487166404724,
0.0002463601704221219,
0.0003049208899028599
] |
[
0.9993933439254761,
0.0003448587085586041,
0.00020828229025937617,
0.000053492341976379976
] |
en
| 0.999997
|
The model sequence chosen for our study is a 21mer human telomeric repeat sequence (htelo): 5′-GGGTTAGGGTTAGGGTTAGGG-3′ which mimics the telomeric G-rich tail. It is well established that telomeric sequences may adopt several quadruplex conformations such as antiparallel ( 28 ) and parallel ( 29 ) G-quadruplex structures depending on the salt conditions . We selected a short fragment for our study, because four GGG blocks are sufficient for stable G-quadruplex formation in vivo ( 30 ).
|
16973897_p13
|
16973897
|
RESULTS
| 4.118021
|
biomedical
|
Study
|
[
0.9994670748710632,
0.0002112575020873919,
0.00032175093656405807
] |
[
0.9994039535522461,
0.0003298333322163671,
0.00021342850232031196,
0.00005288603642839007
] |
en
| 0.999996
|
To determine whether hRPA binds the htelo sequence, we first performed electrophoretic mobility shift assays (EMSA). In a standard experiment, 90 nM 32 P-labeled htelo was incubated for 10 min at 20°C with hRPA in the presence of 50 mM of either Na + or K + . Each mixture was defined by ‘ r ’, the ratio of hRPA added relative to htelo (expressed as a molar ratio).
|
16973897_p14
|
16973897
|
hRPA binds to G-quadruplexes and forms 1:1 and 2:1 complexes
| 4.101889
|
biomedical
|
Study
|
[
0.9994420409202576,
0.00022829962836112827,
0.00032972070039249957
] |
[
0.9993942975997925,
0.00037888868246227503,
0.00017941826081369072,
0.00004735734182759188
] |
en
| 0.999995
|
Figure 2a illustrates the results of EMSA experiments obtained when the binding reaction was performed in the presence of Na + . In the presence of hRPA, one or several retarded bands were obtained, demonstrating that this protein is able to form noncovalent complexes with htelo. Each complex (C) in a lane was defined by its relative mobility m C taking free htelo as reference, m f = 1 . At low protein/DNA ratios ( r = 0.5–1), a single well-defined complex, designated C I , with a relative mobility m CI = 0.27 ± 0.01, was detected. It was accompanied by free htelo and by a fast migrating smear. Increasing r from 2 to 5 progressively reduced the mobility of the main complex and quenched the smear, while free htelo vanished. For r -values >5, most of the htelo (80–90%) concentrated in a low-mobility band, designated C II , with a relative mobility m CII = 0.16 ± 0.01; its mobility remained unchanged upon increasing r up to 14 (data not shown).
|
16973897_p15
|
16973897
|
hRPA binds to G-quadruplexes and forms 1:1 and 2:1 complexes
| 4.220311
|
biomedical
|
Study
|
[
0.9992738366127014,
0.0003518768644426018,
0.00037425229675136507
] |
[
0.9994828701019287,
0.00019413007248658687,
0.000260957662248984,
0.00006208872218849137
] |
en
| 0.999996
|
To clarify the nature of these retarded complexes, we first determined their hRPA to htelo molar ratios (hRPA/htelo). In this case, after scanning of the radioactivity using a Phosphorimager , the gels were submitted to Coomassie blue staining; the intensity of the protein-containing bands was quantitatively analyzed with a densitometer . Results showed that free hRPA migrates as a low-mobility band, m P = 0.09, and that hRPA and htelo co-migrate in C complexes. A range of control experiments with Coomassie staining showed that the signal obtained was proportional to the amount of free hRPA loaded on the gel and that the presence of htelo did not alter protein detection and measurement. The data of the ratios obtained are summarized in Table 1 . When r varied from 1 to 7, the protein to DNA ratio increased continuously from ∼1 to >2, showing that C I is a 1:1 hRPA–htelo complex, while C II is a 2:1 complex accompanied possibly by higher-order complexes.
|
16973897_p16
|
16973897
|
hRPA binds to G-quadruplexes and forms 1:1 and 2:1 complexes
| 4.166251
|
biomedical
|
Study
|
[
0.999360978603363,
0.00030840604449622333,
0.00033064550370909274
] |
[
0.9995352029800415,
0.00016345891344826669,
0.0002499086840543896,
0.00005143860835232772
] |
en
| 0.999999
|
To further characterize the complexes at intermediate and high r values, protein–DNA cross-linking experiments with 0.1% glutaraldehyde were performed ( 31 ). Reaction mixtures were then loaded onto native gels. As shown in Figure 4a , glutaraldehyde pretreatment revealed two ( r = 1) or three well-defined bands ( r = 7) designated C I ′ , C II ′ , C III ′ . The covalent complexes C I ′ and C II ′ migrated as their non-cross-linked C I and C II homologues indicating that C I ′ and C II ′ are 1:1 and 2:1 covalent complexes, respectively. C III ′ was observed only at r ≥ 3; it may represent 3:1 hRPA–htelo complex pre-existing in solution, or result from an artifact induced by cross-linking. These cross-linking experiments show clearly that formation of 2:1 complex increases with r and becomes the major species for r = 7. Combining these data and the hRPA to htelo ratios found in C complexes ( Table 1 ), we concluded that C complexes observed at intermediate r -values that migrate with a relative mobility comprised between C I and C II are mixtures of 1:1 (C I ) and 2:1 (C II ) complexes; its continuously decreasing mobility observed when r increased can be explained by the increasing formation of 2:1 complex. Thus, we calculated for each r value the relative weights f 0 , f 1 and f 2 in solution of free htelo, complex C I and complex C II , respectively .
|
16973897_p17
|
16973897
|
hRPA binds to G-quadruplexes and forms 1:1 and 2:1 complexes
| 4.209646
|
biomedical
|
Study
|
[
0.9993686079978943,
0.0003297719522379339,
0.0003016585542354733
] |
[
0.9994367957115173,
0.00016347043856512755,
0.00033234874717891216,
0.00006724865670548752
] |
en
| 0.999996
|
Parallel experiments were performed in a buffer containing K + instead of Na + . hRPA binding was qualitatively similar in the presence of either ion . However, for the same r value the amount of C complexes was always lower in K + than in Na + . At r = 7, 60% of the htelo was involved in 2:1 complex in K + as compared to 90% in Na + , and a significant fraction of G-quadruplexes remained unbound (7%). These differences indicate a lower affinity of hRPA for htelo in the presence of K + , which is in line with the finding that in the presence of K + , G-quadruplex structures are more stable ( 32 ).
|
16973897_p18
|
16973897
|
hRPA binds to G-quadruplexes and forms 1:1 and 2:1 complexes
| 4.135624
|
biomedical
|
Study
|
[
0.999359667301178,
0.00027379250968806446,
0.00036651818663813174
] |
[
0.9995562434196472,
0.00017318646132480353,
0.00022025886573828757,
0.00005040551695856266
] |
en
| 0.999997
|
As a control in these binding experiments, we tested a radiolabeled single-stranded 21mer oligodeoxythymidine T21 (90 nM). Sequential addition of hRPA in the presence of 50 mM Na + led to a different EMSA profile : (i) the weights of complexes observed on the gel without glutaraldehyde cross-linking at distinct r -values is slightly higher than for htelo, and (ii) the mobility of the C complexes was not reduced as much as for htelo. Glutaraldehyde cross-linking experiments showed that whatever the r -value, the C I ′ covalent complex predominates. All these results agree with previously reported data ( 23 ) showing that hRPA is an ssDBP which predominantly forms 1:1 complexes with short DNAs. Thus, it may be assumed that formation of major 2:1 complexes with htelo is governed by some peculiar properties of this short structured DNA, which assists in binding of two heterotrimeric molecules.
|
16973897_p19
|
16973897
|
The 1:1 complex is predominantly formed with a control oligonucleotide unable to form a G-quadruplex
| 4.217118
|
biomedical
|
Study
|
[
0.9993988275527954,
0.0002894880308303982,
0.00031165056861937046
] |
[
0.9994953870773315,
0.00020990028860978782,
0.00023195806716103107,
0.00006272410973906517
] |
en
| 0.999996
|
Fluorescence resonance energy transfer (FRET) experiments were used to study the conformation of htelo bound to hRPA ( 33 ). For this purpose, the htelo oligomer was labeled with two fluorophores, fluorescein (FLUO) and tetramethylrhodamine (TAMRA) attached to the 5′ and 3′ termini, respectively, leading to the dual-labeled oligonucleotide F-htelo-T . Fluorescence emission of FLUO (the donor, D) is efficiently quenched by TAMRA (the acceptor, A) if the distance D–A is short. If D and A are distant from one another, their respective emissions become independent of each other. Here the extent of energy transfer is represented by an empirical parameter P = I D /( I D + I A ), where I D and I A are the emission intensities of D and A, respectively (Materials and Methods). For free F-htelo-T, low P -values ( P Quad ) typical of G-quadruplex structures were obtained with P Quad = 0.18 ± 0.04 in the presence of Na + and P Quad = 0.25 ± 0.05 in the presence of K + ( 33 ). In others words, FRET efficiency in the absence of protein ( r = 0) is higher in Na + than in K + , in agreement with recent study ( 34 ). A 21mer duplex formed between F-htelo-T and its complementary sequence, designated 21C, served as a control for quadruplex opening . It was demonstrated previously that the stability of the F-htelo-T/21C duplex is higher than that of the quadruplex ( 35 ). Addition of a 5-fold molar excess of 21C over F-htelo-T (90 nM) led to a high P -value ( P Duplex ) of 0.83 ± 0.05 after equilibration, whatever the nature of the monovalent cation. This is consistent with the formation of a B-DNA duplex structure separating D and A by >70 Å .
|
16973897_p20
|
16973897
|
hRPA binding leads to G-quadruplex unfolding
| 4.408507
|
biomedical
|
Study
|
[
0.9992371797561646,
0.000465230958070606,
0.0002975573006551713
] |
[
0.9991938471794128,
0.000291075644781813,
0.0003962284536100924,
0.0001188939277199097
] |
en
| 0.999998
|
Fluorimetric titrations of F-htelo-T by hRPA were performed in the conditions used for hRPA–htelo binding in EMSA experiments. Figure 6a shows the data obtained in the presence of Na + . As the ratio r increases, FLUO emission is stimulated while TAMRA fluorescence decreases, indicating that FRET is suppressed and that hRPA is able to unfold quadruplexes. The absence of an isoemissive point and the biphasic variation of P as a function of r show that the binding process is complex and involves at least three species, in agreement with the formation of the C I and C II complexes with the free G-quadruplex structures. Thus, we can write (and calculate) that P = f 0 P 0 + f 1 P 1 + f 2 P 2 , where P 0 , P 1 and P 2 are P values for free htelo, C I complexes and C II complexes, respectively, and f 0 , f 1 and f 2 the previously calculated fractions of each species . To fit the experimental and calculated P curves, P 1 was considered as an adjustable parameter with P 0 = 0.18 and P 2 = 0.83. The agreement for both series of data is satisfactory when P 1 is taken as 0.55 ± 0.05.
|
16973897_p21
|
16973897
|
hRPA binding leads to G-quadruplex unfolding
| 4.244015
|
biomedical
|
Study
|
[
0.9993333220481873,
0.00034989402047358453,
0.00031680273241363466
] |
[
0.9994381070137024,
0.00017682844190858305,
0.00031562361982651055,
0.00006938999285921454
] |
en
| 0.999998
|
Since P 2 is nearly identical to P Duplex , the final conformation of DNA bound with two (or more) hRPA corresponds to an extended structure where both fluorophores are as far away as in the duplex B-DNA structure. The lower P -value for the 1:1 complexes indicates that the distance D–A is smaller in the 1:1 than in the 2:1 complexes. This could be the result of the coming together of the single-stranded tails triggered by the presence of only one bound hRPA.
|
16973897_p22
|
16973897
|
hRPA binding leads to G-quadruplex unfolding
| 4.239649
|
biomedical
|
Study
|
[
0.9993612170219421,
0.00023372031864710152,
0.0004050354764331132
] |
[
0.9989593029022217,
0.0006894294056110084,
0.00028508302057161927,
0.00006611336721107364
] |
en
| 0.999998
|
At first glance, titrations in the presence of K + are similar to those obtained with Na + . However, significant differences are observed: (i) for the same r -values, P in the presence of K + is always lower than P in the presence of Na + in agreement with the binding data discussed above, and (ii) the value of P in the presence of K + does not reach the P Duplex value at the highest r -value checked ( r = 7) as not all htelo is bound (7% remains free) and as a non-negligible amount of 1:1 complexes is still present as shown by the EMSA experiment .Taking P 0 = 0.25 and P 2 = 0.83, the best fit between the experimental and calculated data is obtained with P 1 = 0.6 ± 0.05.
|
16973897_p23
|
16973897
|
hRPA binding leads to G-quadruplex unfolding
| 4.181984
|
biomedical
|
Study
|
[
0.9992066025733948,
0.00026326763327233493,
0.000530150777194649
] |
[
0.9995311498641968,
0.00021217743051238358,
0.0002092933136736974,
0.00004735014226753265
] |
en
| 0.999995
|
One can therefore conclude that in the presence of either Na + or K + , G-quadruplex structures are unfolded in the hRPA–htelo complexes. In the 1:1 complexes, the oligonucleotide end-to-end distance is intermediate between the one observed in quadruplexes and the larger one ( d ≥ 70 Å) found in the 2:1 complexes.
|
16973897_p24
|
16973897
|
hRPA binding leads to G-quadruplex unfolding
| 4.21596
|
biomedical
|
Study
|
[
0.9993782043457031,
0.00022254345822148025,
0.0003992629935964942
] |
[
0.9989302754402161,
0.000657227705232799,
0.00035243973252363503,
0.00005999136556056328
] |
en
| 0.999996
|
To gain insight into the opening process of the hRPA-induced G-quadruplex, its kinetics were compared to those obtained during the formation of the duplex F-htelo-T/21C . Five equivalents of either hRPA or 21C were added to a solution of F-htelo-T and rapidly mixed. The kinetics were followed by measuring the emission spectra at 516 nm (FLUO emission) and are shown in Figure 7 . The experiments were performed in K + conditions in which the stability of the G-quadruplexes is highest ( 32 ). Duplex formation could not be properly fitted with a mono-exponential function, and was fitted with a bi-exponential model, as observed previously ( 27 , 36 ), with a fast phase ( k fast = 0.031 ± 0.003 min −1 ) accompanied by a much slower phase (data not shown). The fast phase was of the same order of magnitude as reported previously ( 36 , 37 ) for the unfolding step of G-quadruplex structures. A bi-exponential function was also required to fit the binding of hRPA to htelo, but the kinetics were obviously faster , with k fast = 0.81 ± 0.01 min −1 and k slow = 0.080 ± 0.001 min −1 . These relatively fast kinetics observed with hRPA as compared to complementary DNA highlight the active role of hRPA in quadruplex opening.
|
16973897_p25
|
16973897
|
hRPA efficiently unfolds G-quadruplexes
| 4.258861
|
biomedical
|
Study
|
[
0.9993866682052612,
0.0003639271017163992,
0.0002492938656359911
] |
[
0.9993891716003418,
0.00017910244059748948,
0.0003502945473883301,
0.00008145946776494384
] |
en
| 0.999996
|
Fluorimetric titrations of the dually labeled single-stranded oligonucleotide control F-T21-T by hRPA were performed in the presence of Na + (data not shown). hRPA has a greater affinity for the F-T21-T control sequence compared to F-htelo-T, since FRET titration is complete for r = 1 (with P increasing from 0.45 for r = 0 to 0.74 for r ≥ 1). Under the same conditions an important smear was observed in the EMSA experiment when radiolabeled T21 was mixed with one equivalent of hRPA . This observation indicates that the smear observed in the native gel at low r -values ( r = 0.5 and 1) represents 1:1 complex dissociation during migration . In addition, if we compare the P -values of the 1:1 complexes obtained with F-T21-T and F-htelo-T (0.74 ± 0.01 and 0.55 ± 0.05, respectively), we can conclude that in the 1:1 complexes with hRPA the DNA conformations of T21 and htelo differ. This is in agreement with a different mode of binding of hRPA to the quadruplex-forming substrate, as discussed above. Moreover, kinetic experiments of the dually labeled single-stranded oligonucleotide control F-T21-T by hRPA showed that <30 s were necessary to obtain maximal fluorescence emission (data not shown). This time scale agrees with the one observed previously for hRPA binding to ssDNA ( 38 , 39 ).
|
16973897_p26
|
16973897
|
hRPA efficiently unfolds G-quadruplexes
| 4.2593
|
biomedical
|
Study
|
[
0.9994007349014282,
0.0003510731621645391,
0.00024814694188535213
] |
[
0.9993775486946106,
0.00021009318879805505,
0.00033323923707939684,
0.00007915767491795123
] |
en
| 0.999996
|
From the data presented, it can be concluded that hRPA is able to bind 21mer G-quadruplex structures by forming 1:1 complexes and 2:1 complexes, and possibly higher order complexes. The 2:1 complexes are presumably stabilized by cooperative interactions between the two hRPA molecules ( 26 , 31 ) leading to the very stable noncovalent complexes observable by electrophoresis. In comparison with hRPA binding to the single-stranded T21 oligomer, which mainly displays 1:1 complexes, it is clear that the hRPA-binding mode differs depending on the nature of the DNA. It is well known that RPA exhibits relatively low specificity for nucleic acid sequences with a 50-fold preference for polypyrimidine tracks ( 22 – 24 , 40 ). This sequence preference is similar to those of other nonspecific ssDBP, e.g. the E.coli ssDBP ( E.coli ssDBP) ( 41 ). However it was clearly demonstrated recently that there is a general influence of the nucleic acid sequence itself on the binding interactions with ssDBPs: ssDNA binding is influenced by base stacking and the nearest-neighbor (nucleotide sequence in DNA) dependence of this stacking ( 42 ). Nevertheless, formation of major stable 2:1 complexes with a 21mer DNA strongly suggests that unlike T21, hRPA binding to G-quadruplex structures is directed by the structure itself with a sequential binding mode. This implies that the first hRPA binds to one extremity of the DNA, allowing binding of the second hRPA. In this case, as htelo is a 21mer, both hRPA should bind htelo by its 8–10 nt binding mode ( 22 – 26 ).
|
16973897_p27
|
16973897
|
DISCUSSION
| 4.479909
|
biomedical
|
Study
|
[
0.999251663684845,
0.0004211040504742414,
0.00032716678106226027
] |
[
0.9987868666648865,
0.0003647815028671175,
0.0007278024568222463,
0.0001205654043587856
] |
en
| 0.999997
|
Duplex formation with a G-quadruplex prone sequence is a slow process, especially in the presence of K + ( 35 ). Opening of the quadruplex is a prerequisite for duplex formation, and the kinetics are dictated by quadruplex unfolding rather than by bimolecular association. In contrast, FRET and kinetics experiments demonstrate that hRPA acts rapidly and efficiently promotes G-quadruplex opening, as a few minutes only are necessary to open the htelo G-quadruplex structure(s). The fast action of hRPA might be required in cases where quadruplex lifetime is long compared to key cellular processes such as replication. Preliminary experiments with an ssDBP such as E.coli ssDBP suggest that under similar conditions the binding mode and kinetics of E.coli ssDBP are very different compared to hRPA (T. R. Salas, unpublished data), arguing for a specific effect of the hRPA on the G-quadruplex. Other ssDBP or nucleic acids chaperones will be tested to determine if hRPA possesses a unique mode of action on the G-quadruplex.
|
16973897_p28
|
16973897
|
DISCUSSION
| 4.30358
|
biomedical
|
Study
|
[
0.9994416832923889,
0.00029489235021173954,
0.00026346297818236053
] |
[
0.9991508722305298,
0.000330014358041808,
0.000439613766502589,
0.00007948849815875292
] |
en
| 0.999999
|
Taken together, these data lead to the sequential model of hRPA binding summarized in Figure 8 . F-htelo-T only displays three short ‘single-stranded’ regions corresponding to the TTA loops ; it is not clear if these short single-stranded regions are sufficient for the initial binding of hRPA. Alternatively, it has been shown that G-quadruplex structures are in equilibrium with partially unfolded G-quadruplex species (F-htelo-T′) where some Hoogsteen hydrogen bonds might be transiently opened ( 43 ). These single-stranded regions generated at the extremities could be accessible and rapidly trapped by hRPA. This first binding step should destabilize the hydrogen bonds between the remaining Gs of the proximal quartet, permitting the initial conformational change observed by FRET (1:1 complexes). This unfolded DNA bound with one hRPA molecule could facilitate binding of a second hRPA molecule. Thus hRPA would form two distinct complexes in which DNA is maintained in different unfolded conformations.
|
16973897_p29
|
16973897
|
DISCUSSION
| 4.413849
|
biomedical
|
Study
|
[
0.9993226528167725,
0.00039526703767478466,
0.00028212586767040193
] |
[
0.9987286925315857,
0.0006117919692769647,
0.000537514453753829,
0.00012196227180538699
] |
en
| 0.999998
|
The idea that hRPA can remove secondary and tertiary DNA structures by a simple destabilization process was reported previously ( 44 , 45 ). The recent model of a multi-step helix destabilization process by hRPA described by Binz et al. ( 46 ) supports our results. Unlike hRPA, human helicases such as BLM and WRN, that unwind G-quadruplex structures with a 3′–5′ polarity, require ATP, Mg 2+ and at least one single-stranded 3′ tail ( 47 , 48 ). In our system, neither ATP nor 3′ tail is present. In addition, even if there is no evidence of polarity of hRPA binding to G-quadruplex structures, it is well known that hRPA binds ssDNA with the opposite 5′–3′ polarity ( 49 , 50 ). Clearly, more detailed studies are needed to unravel the mechanism by which hRPA binds and opens G-quadruplex structures. This binding proceeds with high efficiency and significant specificity for G-quadruplexes, suggesting that it has an important biological role in telomere maintenance.
|
16973897_p30
|
16973897
|
DISCUSSION
| 4.498098
|
biomedical
|
Study
|
[
0.9994045495986938,
0.00033254153095185757,
0.00026297097792848945
] |
[
0.9979977011680603,
0.00033664083457551897,
0.0015213430160656571,
0.00014429670409299433
] |
en
| 0.999997
|
G-quadruplex structures may be important for a number of biological processes and disease-related mechanisms. Particularly, it has been shown that they inhibit telomerase activity by impeding the recruitment and binding of telomerase components to telomeres. We have revealed by this investigation that G-quadruplex structures are specific targets for hRPA and that this protein is able to bind and open G-quadruplex structures much faster than the complementary DNA strand. There is good reason to believe that opening G-quadruplex structures by RPA takes place in order to maintain the telomeric G-overhang in a single-stranded conformation, compatible with telomerase activity. Cohen et al. ( 21 ) showed that depending on its concentration, hRPA may exert either stimulatory or inhibitory effects on telomerase; it would be important to correlate this observation with the formation of 1:1 or 2:1 complexes. Interestingly, Zaug et al. ( 51 ) indicated that human POT1 is able to disrupt telomeric G-quadruplexes and that extension by the telomerase depends on the relative position of POT1 on htelo. Thus, the details of the architecture protein–DNA complexes may play an essential role in telomerase activity. We are now investigating whether similar conclusions may be reached with hRPA.
|
16973897_p31
|
16973897
|
DISCUSSION
| 4.363136
|
biomedical
|
Study
|
[
0.9993767142295837,
0.0003767606394831091,
0.00024656191817484796
] |
[
0.9990278482437134,
0.00031427634530700743,
0.0005511246854439378,
0.00010673458746168762
] |
en
| 0.999997
|
The use of structurally separate domains to make up the function of a protein may be best illustrated by the general class of nucleic acid binding proteins. Domains providing target specificity are clearly separable from other domains. Specificity and affinity for a target may be provided by a properly arranged stretch of basic residues ( 1 , 2 ). For example, the basic leucine zipper family (bZIP), one of the best-characterized families of DNA binding motifs, consists of a N-terminal basic region and a C-terminal leucine zipper dimerization region ( 3 , 4 ). Mix-and-match experiments have demonstrated that the sequence specificity of bZIP depends entirely on the N-terminal basic region ( 5 – 7 ). This region retains some sequence specificity even when the leucine zipper is removed ( 8 ). Thus, the bZIP family is ideal for protein engineering designed to obtain new sequence-specific DNA-binding proteins.
|
16982643_p0
|
16982643
|
INTRODUCTION
| 4.354049
|
biomedical
|
Study
|
[
0.9993257522583008,
0.00027420889819040895,
0.00040003436151891947
] |
[
0.9415751695632935,
0.0018652210710570216,
0.05631479248404503,
0.0002448650193400681
] |
en
| 0.999995
|
In another example, arginine-rich RNA binding regions of RNA binding proteins are found in many viruses, as in the Rev protein of human immunodeficiency virus (HIV). This region is not only responsible for target specificity, but also retains binding affinity as tight as that of the isolated intact protein ( 9 – 11 ). Short α-helical peptides corresponding to the arginine-rich RNA binding domain from the Rev protein of HIV are able to bind the rev responsive element (RRE) specifically, and are sufficient for a high binding affinity, comparable to that of Rev ( 10 ).
|
16982643_p1
|
16982643
|
INTRODUCTION
| 4.32158
|
biomedical
|
Study
|
[
0.9995954632759094,
0.00018801525584422052,
0.00021646534150931984
] |
[
0.9984018206596375,
0.0005153724923729897,
0.0010054538724943995,
0.00007738364365650341
] |
en
| 0.999995
|
Many small peptides, either artificially designed or derived from natural nucleic binding proteins, have been tested for sequence or conformational specificity ( 8 , 12 – 17 ). One such family, short peptides of alternating lysine, has been shown to bind to and stabilize poly(dG–d5meC) in the Z conformation under nearly physiological conditions ( 12 , 13 ). These peptides can also stabilize triplex-helical nucleic acids at millimolar concentrations and low pH ( 18 ).
|
16982643_p2
|
16982643
|
INTRODUCTION
| 4.134803
|
biomedical
|
Study
|
[
0.9995971322059631,
0.0001555800554342568,
0.00024727691197767854
] |
[
0.9982802867889404,
0.00029131220071576536,
0.0013724120799452066,
0.00005608106584986672
] |
en
| 0.999995
|
Not all amino acids alternating with lysine result in Z-DNA binding peptides. Only peptides containing small and relatively flexible amino acids such as alanine and glycine have an ability to induce the B–Z transition. Bulky amino acids such as tyrosine, phenylalanine or valine interfere with binding ( 13 ). Proper spacing between the positively charged lysines is therefore important to the activity of the peptide. A model for the binding of peptide can be derived from the conversion of poly(dG–d5meC) to the Z form in the presence of high salt or small positively charged molecules such as spermidine ( 19 ). Both multivalent cations and spermidine stabilize the Z-DNA conformation of poly(dG–d5meC) because they shield the more closely spaced phosphates in the Z-DNA backbone ( 20 ). Takeuchi et al . ( 12 , 13 ) have proposed that the cooperative shielding of phosphate backbones of Z-DNA by properly spaced lysines can stabilize the Z-DNA conformer, and thereby alter the B–Z equilibrium of poly(dG–d5meC).
|
16982643_p3
|
16982643
|
INTRODUCTION
| 4.36401
|
biomedical
|
Study
|
[
0.9995558857917786,
0.0002166595950257033,
0.00022750858624931425
] |
[
0.9987099170684814,
0.0003788313188124448,
0.000830860051792115,
0.00008041821274673566
] |
en
| 0.999996
|
In this report, we replaced the basic region of the consensus bZIP with the heptamer KGKGKGK to produce an artificial Z-DNA binding domain, KGZIP. The Z-DNA binding activity of the peptide was characterized by circular dichroism, gel mobility shift assay and surface plasmon resonance (Biacore AB). KGZIP binds to Z-DNA specifically and with high affinity, even in the presence of a large excess of B-DNA competitor. Binding of poly(dG–d5meC) by KGZIP is sufficient to convert the DNA to the Z conformation. The heptapeptide (KGKGKGK) may be sufficient to provide Z-DNA specificity to many proteins that contain it.
|
16982643_p4
|
16982643
|
INTRODUCTION
| 4.262393
|
biomedical
|
Study
|
[
0.999476969242096,
0.00031098045292310417,
0.0002121074649039656
] |
[
0.9992467164993286,
0.000350256625097245,
0.0003015636757481843,
0.00010140400263480842
] |
en
| 0.999998
|
An alternating lysine heptamer, Lys-Gly-Lys-Gly-Lys-Gly-Lys (KGKGKGK), was synthesized using F-Moc chemistry. KGZIP, consisting of KGKGKGK and the consensus leucine zipper sequence deduced from the work of O'Neil et al . ( 21 ) connected by a four-glycine linker , was synthesized by the same method. Peptides were further purified by HPLC. The quality of the peptides was confirmed by MALDI mass spectroscopy. The concentrations of peptides were determined by amino acid analysis.
|
16982643_p5
|
16982643
|
Preparation of peptides
| 4.12806
|
biomedical
|
Study
|
[
0.9995661377906799,
0.00018596492009237409,
0.00024783299886621535
] |
[
0.9992383718490601,
0.0004920627688989043,
0.00021321493841242045,
0.00005632407555822283
] |
en
| 0.999996
|
Peptides were dissolved in TE buffer [10 mM Tris–HCl (pH7.4) and 1 mM EDTA]. Poly(dG–d5meC) (Pharmacia) was dissolved in TE buffer and its concentration determined by absorbance at 255 nm . DNA was diluted at least 20-fold in buffer A [10 mM Tris–HCl (pH 7.4) and 10 mm KF] for analysis. CD spectra were taken on an AVIV model 202. The background CD spectrum of buffer A was taken for base line calibration, before adding nucleotides and peptides. The measurements were carried out using 100 μM (nucleotide) of DNA at 25°C in a 5 mm quartz cell. Spectra were recorded at 1 nm interval averaged over 3 s. The peptide was then added to the sample from the concentrated stock solution. The maximum volume of peptide added to the sample did not exceed 5% of the total volume. For equilibrium measurements, samples were heated at 50°C for 5 min then cooled to 25°C for 10 min before CD spectra were taken. As described elsewhere ( 12 , 13 ), heating is essential to achieve quick conformational equilibration. Kinetic measurements of time-dependent conformational change of the polynucleotides by KGZIP were carried out using 100 μM DNA and 16 μM KGZIP. After mixing the DNA and peptide, CD spectra were taken at 25°C immediately and after several time intervals.
|
16982643_p6
|
16982643
|
Circular dichroism spectra measurement
| 4.21027
|
biomedical
|
Study
|
[
0.9994704127311707,
0.00031022189068607986,
0.00021939007274340838
] |
[
0.9991956353187561,
0.0003830505011137575,
0.00034374892129562795,
0.00007758977153571323
] |
en
| 0.999999
|
The assay was carried out using d( 5Br C-G) 20 as a Z-DNA substrate, which is stable in the left-handed Z-DNA conformation under all conditions uses in these studies, as determined by circular dichroism spectroscopy ( 22 , 23 ). Briefly, a short DNA primer, d(G- 5Br C) 6 , was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase (New England Biolab) for 1 h at 37°C. The synthetic DNA oligomer, d(G- 5Br C) 20 served as a template. Typically, primer and template were mixed at 25°C for 20 min prior to adding Klenow DNA polymerase. DNA polymerization was then carried out in the presence of d 5Br CTP (Roche) and dGTP at 25°C for 1 h. The labeled Z-DNA was separated from unincorporated nucleotides and short DNA species by nondenaturing PAGE in a 6% gel. The labeled Z-DNA typically migrates as a single band and comigrates with 40–60 bp markers. The labeled Z-DNA was purified from the gel and used for gel mobility shift assays in this study.
|
16982643_p7
|
16982643
|
Gel mobility shift assay
| 4.187225
|
biomedical
|
Study
|
[
0.9994910955429077,
0.00027967625646851957,
0.0002292584249516949
] |
[
0.9992615580558777,
0.0003709765151143074,
0.00029416067991405725,
0.00007329748768825084
] |
en
| 0.999996
|
Various concentrations of KGZIP were mixed with <10 pM of Z-DNA substrate in binding buffer B [10 mM Tris–HCl (pH 8.0), 50 mM KCl, 5 mM DTT, 5% glycerol and 50 μg/ml BSA]. The reaction was incubated at 22°C for 30 min. Then the mixture was analyzed on a 5% native polyacrylamide gel run in 0.5× TBE buffer (22.5 mM Tris–borate and 1 mM EDTA). After electrophoresis, the gel was dried and autoradiographed at −70°C on Kodak X-OMAT film.
|
16982643_p8
|
16982643
|
Gel mobility shift assay
| 4.15489
|
biomedical
|
Study
|
[
0.9994391798973083,
0.00035075368941761553,
0.00021001201821491122
] |
[
0.9954954385757446,
0.003797233337536454,
0.0005277562304399908,
0.00017957252566702664
] |
en
| 0.999997
|
The binding affinity of artificial Z-DNA binding peptides (KGZIP) was measured using a BIAcore 2000 (Biosensor Inc.). Response units (RU) (450) of biotinylated poly(dG–dC), stabilized in the Z-DNA conformation by chemical bromination ( 24 ), were immobilized on a streptavidin coated SA chip (Biosensor Inc.). All measurements were performed as described in Herbert et al . ( 24 ). Specifically, KGZIP was injected for 180 s at 20 μl/min, followed by a dissociation step without KGZIP at the same flow rate. Four different concentrations of KGZIP were applied to measure kinetic constants. The equilibrium binding constant ( K D ) was calculated from the association rate constant ( k on ) and the dissociation rate constant ( k off ) using BiaEVAL software (Biosensor Inc.).
|
16982643_p9
|
16982643
|
BIAcore measurement
| 4.138502
|
biomedical
|
Study
|
[
0.9995505213737488,
0.00022833855473436415,
0.00022115801402833313
] |
[
0.9993640780448914,
0.00024400235270150006,
0.000331861519953236,
0.00006006258990964852
] |
en
| 0.999995
|
KGZIP (50 nM) and <10 pM Z-DNA substrate were incubated in binding buffer B in the presence of sheared salmon sperm DNA (B-DNA competitor) at various concentrations (0.56 μM to 3.67 mM of base pairs, 3-fold serial dilution). The reactions were analyzed under the same conditions used for the gel mobility shift assay. After electrophoresis, gels were dried, exposed to a phospho-imager screen and the signals were quantified (Molecular Dynamics). The binding affinity of KGZIP in competition with B-DNA was calculated indirectly as described by Greisman and Pabo ( 25 ). The experiments were replicated three times and averaged to determine the binding affinity.
|
16982643_p10
|
16982643
|
B-DNA competition assay
| 4.129966
|
biomedical
|
Study
|
[
0.9994860887527466,
0.0002672822738531977,
0.00024663456133566797
] |
[
0.9993537068367004,
0.0003209226415492594,
0.00026486211572773755,
0.000060594033129746094
] |
en
| 0.999998
|
We were interested in whether KGKGKGK could function as an autonomous domain and direct Z-DNA binding in the context of a larger sequence. This heptapeptide has been shown by Takeuchi et al . ( 12 , 13 ) to induce the formation of Z-DNA by poly(dG–d5meC). The bZIP family of DNA-binding motifs includes a basic subdomain that provides binding specificity; therefore it was reasonable to determine the specificity and affinity of a synthesized bZIP motif including the KG peptide. KGZIP is made up of the alternating lysine sequence connected via a Gly 4 linker to an idealized leucine zipper ( 21 ) . KGZIP is highly soluble and forms a α-helix as demonstrated by negative peaks at 208 and 222 nm in a CD spectrum . The basic domains of members of the bZIP family often form α-helices when the protein is bound to DNA ( 1 , 26 , 27 ). Examination of the signature wavelengths for α-helix, 222 and 255 nm, reveal no increase in the amount of ellipticity for KGZIP in the presence of Z-DNA (data not shown).
|
16982643_p11
|
16982643
|
Design of a Z-DNA specific peptide, bZIP
| 4.257302
|
biomedical
|
Study
|
[
0.9993625283241272,
0.0002674766583368182,
0.00037000025622546673
] |
[
0.99953293800354,
0.00020819697238039225,
0.00020157005928922445,
0.00005724985749111511
] |
en
| 0.999995
|
When poly(dG–d5meC) is incubated with KGZIP, a protein dependent change in the CD spectrum is observed . This change is indicative of a shift of the DNA from the B to the Z conformation. At a protein:DNA ratios of 0.16, the spectrum is identical to that of Z-DNA induced in poly(dG–d5meC) by 3M NaCl (data not shown). KGZIP behaves very similarly to the KGKGKGK peptide in its ability to induce the B–Z transition. The midpoint of the transition is effected at a protein:DNA ratio of 0.06 (KGZIP versus:nucleotide DNA) . This is the same as that seen for KGKGKGK (data not shown) under the same conditions, and as previously reported result by Takeuchi et al . ( 12 , 13 ).
|
16982643_p12
|
16982643
|
Z-DNA formation by poly(dG–d5meC) in the presence of KGZIP, as measured by circular dichroism
| 4.214772
|
biomedical
|
Study
|
[
0.9994799494743347,
0.00024697548360563815,
0.0002730543783400208
] |
[
0.9993392825126648,
0.0003201226645614952,
0.00027876600506715477,
0.00006182337529025972
] |
en
| 0.999997
|
The B–Z transition of a sample of poly(dG–d5meC) in the presence of KGZIP proceeds slowly at room temperature, and is accelerated by heat. The kinetics of the B–Z transition induced by KGZIP at 25°C is shown in Figure 3 . The transition has a half-life of ∼75 min under the conditions studied in here. This is in striking contrast to another well-characterized naturally occurring Z-DNA binding protein domain, Zab ( 22 ), which induces the B–Z transition in this substrate almost instantaneously (data not shown). The difference in the conversion kinetics of KGZIP and Zab may be related to the necessity for structural rearrangement of the peptide. Unlike Zab, which has a well-defined and fixed Z-DNA binding site ( 22 ), KGZIP must reposition the lysine residues as it binds to Z-DNA, in order to maximize contacts and enhance binding energy. That process is likely to contribute to the longer time required for KGZIP to convert B-DNA to Z-DNA It is likely that KGZIP induces the B–Z transition by binding to Z-DNA formed transiently by Brownian motion, thereby shifting the B–Z equilibrium; a similar model has been suggested for an anti-Z-DNA antibody ( 28 ).
|
16982643_p13
|
16982643
|
Z-DNA formation by poly(dG–d5meC) in the presence of KGZIP, as measured by circular dichroism
| 4.420061
|
biomedical
|
Study
|
[
0.9992759823799133,
0.0004083129169885069,
0.00031564917298965156
] |
[
0.9991459846496582,
0.0003042026946786791,
0.00045155902625992894,
0.00009826260793488473
] |
en
| 0.999996
|
CD studies confirm that KGKGKGK retains its ability to bind Z-DNA when fused to a leucine zipper. The affinity of that binding was measured by the gel mobility shift assay. A preformed Z-DNA substrate, d( 5Br C-G) 20 , was incubated with KGZIP under nearly physiological conditions, 50 mM KCl, pH 8.0, at 22°C. Under these conditions, KGZIP binds Z-DNA with a K D of ∼30 nM .
|
16982643_p14
|
16982643
|
Z-DNA binding by KGZIP, as measured by gel mobility shift assays and BIAcore
| 4.209135
|
biomedical
|
Study
|
[
0.9994947910308838,
0.0002552922233007848,
0.00024987448705360293
] |
[
0.9993079900741577,
0.00039233363349922,
0.00022759189596399665,
0.0000720356110832654
] |
en
| 0.999997
|
Real-time kinetics of binding and an accurate measure of binding affinity can be determined using the BIAcore system. Poly(dG–dC), stabilized in the Z conformation by chemical bromination ( 29 ) was attached to a SA-chip using a biotin-streptavidin linkage. KGZIP binding was monitored , showing that both association and dissociation are fast. In these experiments a K D of 27 nM was measured. The affinity and kinetic constants of KGZIP are similar to those of Zα, a Z-DNA binding domain whose specificity is the result of a well-tailored binding ( 24 , 30 ). The affinity of KGZIP is striking in light of the relative simplicity and small size of the binding domain and the few possible protein–nucleic acid contacts.
|
16982643_p15
|
16982643
|
Z-DNA binding by KGZIP, as measured by gel mobility shift assays and BIAcore
| 4.251495
|
biomedical
|
Study
|
[
0.9994986057281494,
0.0002806613629218191,
0.0002206718927482143
] |
[
0.9993016719818115,
0.0003189374110661447,
0.0003015808470081538,
0.00007777485734550282
] |
en
| 0.999997
|
The heptapeptide KAKAKAK is also capable of binding to Z-DNA ( 12 ). KAZIP has a K D of binding to brominated poly(dG–dC) comparable to that of KGZIP, as measured by BIAcore (data not shown); however, the association and dissociation rates are faster, suggesting that a slight difference in the position of the lysines may affect the interaction with DNA.
|
16982643_p16
|
16982643
|
Z-DNA binding by KGZIP, as measured by gel mobility shift assays and BIAcore
| 4.146906
|
biomedical
|
Study
|
[
0.9994920492172241,
0.000185390017577447,
0.00032248307252302766
] |
[
0.998994767665863,
0.0007401139009743929,
0.00019672168127726763,
0.00006839273555669934
] |
en
| 0.999997
|
Most DNA is right-handed. For a Z-DNA binding peptide to be biologically relevant, it must not only bind Z-DNA with high affinity, but also exhibit high specificity for Z-DNA over B-DNA. The binding of KGZIP to Z-DNA was assayed in the presence of increasing amounts of sheared salmon sperm DNA, a non-specific B-DNA competitor . When 50 nM KGZIP is incubated with radio-labeled Z-DNA probe in the absence of competitor, >50% of the probe migrates as a complex . In the presence of increasing B-DNA competitor, the binding is gradually reduced. However, a significant amount of KGZIP is still bound to Z-DNA probe in the presence of B-DNA up to 1.23 μM. Using the method of Greisman and Pabo ( 25 ), a K D of ∼50 μM can be calculated for the binding of KGZIP to B-DNA. This is a difference of more than three orders of magnitude from the binding to Z-DNA. Therefore it is reasonable that KGKGKGK can recognize Z-DNA even in the context of a vast excess of B-DNA, as is found in cells.
|
16982643_p17
|
16982643
|
B-DNA competition assay
| 4.339022
|
biomedical
|
Study
|
[
0.9995611310005188,
0.00023049541050568223,
0.00020844125538133085
] |
[
0.9989629983901978,
0.0004880033666267991,
0.00045538219274021685,
0.00009359211253467947
] |
en
| 0.999996
|
Molecular modeling studies of peptide binding to Z-DNA were also carried out, revealing both hydrophobic and hydrogen bond interactions. These results, which will be published elsewhere, suggest that KGZIP changes conformation to accommodate binding to Z-DNA.
|
16982643_p18
|
16982643
|
B-DNA competition assay
| 3.750476
|
biomedical
|
Study
|
[
0.999015212059021,
0.00027234942535869777,
0.0007123883697204292
] |
[
0.9978827834129333,
0.001593050197698176,
0.00040810173959471285,
0.00011601856385823339
] |
en
| 0.999997
|
Basic peptides containing alternating lysines have been shown to stabilize the Z-DNA conformation of poly(dG–d5meC) under physiological conditions and at neutral pH by binding preferentially to Z-DNA. This binding has been proposed to be the result of the alignment of the peptide to the distinctive zigzag phosphate backbone in the Z conformation ( 12 , 13 , 20 ). Modeling suggests that favorable hydrogen bonding between the peptide and the backbone of the nucleic acid substrate can occur (H.-J. Park and Y.-G. Kim, unpublished data). KGZIP binds Z-DNA with an affinity and specificity comparable to that of human Zα ADAR1 , a Z-DNA binding domain of the editing enzyme double-stranded RNA adenosine deaminase. This is striking because Zα is a highly organized 77 amino acid domain, which makes multiple contacts with Z-DNA in the context of a precisely fitted binding surface ( 30 ). In contrast, the binding site of KGZIP is only seven amino acids, and both the number and the nature of protein–DNA contacts are limited by this size. There are differences between the binding of KGZIP and Zα, most notably the kinetics of the conversion of poly(dG–d5meC) from the B-form to the Z-form in the presence of protein. This may reflect a difference in the mechanism of these reactions such as the difference between the key and lock model and the induced fit model, respectively.
|
16982643_p19
|
16982643
|
DISCUSSION
| 4.480469
|
biomedical
|
Study
|
[
0.9994000196456909,
0.000363915809430182,
0.00023601138673257083
] |
[
0.9986461997032166,
0.0004231061029713601,
0.0008006066200323403,
0.0001301108131883666
] |
en
| 0.999996
|
In naturally occurring members of the bZIP family, the basic region is responsible for specific binding to a target, while the leucine zipper dimerizes, thereby locating two binding domains near each other. Dimerization increases binding affinity and specificity ( 31 ). The binding affinity of KGKGKGK for d( 5Br C-G) 20 observed by affinity co-electrophoresis is above 10 μM (data not shown). In KGZIP, dimerization does not appear to increase the rate at which the Z-conformation of poly(dG–d5meC) is formed but does add to the affinity of KGKGKGK for Z-DNA. By linking KGKGKGK to the N-terminus of a leucine zipper, we have created a novel Z-DNA specific binding protein.
|
16982643_p20
|
16982643
|
DISCUSSION
| 4.220654
|
biomedical
|
Study
|
[
0.9994699358940125,
0.00018205457308795303,
0.00034803146263584495
] |
[
0.9989756345748901,
0.0007352395914494991,
0.00021706082043237984,
0.00007210228795884177
] |
en
| 0.999997
|
In order to determine whether alternating lysine sequences occur in nature, the protein database was searched with BLAST. KGKGKGK is found in a wide variety of proteins from Arabidopsis , Drosophila and vertebrates. Fibrillarin from Euglena contains an unusual nine repeats of Lys-Gly. KAKAKAK is less common than KGKGKGK, and is found most often in ribosomal proteins. The most striking occurrence of KG repeats is in the family of eukaryotic DNA (cytosine-5-)-methyltransferases as indicated previous reports ( 13 , 32 ). A conserved region of (KG) 5–7 K found in the linker region between the two domains is found in enzymes from human, mouse, rat, chicken, sea urchin, zebrafish and frog . In addition, the region just N-terminal to the KG repeats is also highly conserved. This region maps between the two defined functional domains of DNA cytosine methyltransferase, the N-terminal regulatory domain and the C-terminal catalytic domain. This occurrence of the KG repeat differs from the peptide tested here because it is embedded within the protein sequence. However, protein structure predictions using PROF ( 33 ) suggest that the conserved region N-terminal to the KG repeat, as well as the repeat itself are part of a large loop. Interestingly, a program that identifies conformational switch regions ( 34 ) predicts that the KG repeat is in the middle of a long, extensively solvent exposed, confomationally variable region. Therefore it is reasonable that the KG repeat is flexible and able to bind Z-DNA. Although the conservation of the KG repeat domain suggests a functional role, no such role has been identified to date. This enzyme is involved in maintaining the methylated state of cytosines after DNA replication. It is possible that interaction with Z-DNA formed by supercoiling of CpG islands is involved in targeting the enzyme. In the experiments reported here, we have used completely methylated poly(dG–d5meC) in order to stabilize the Z conformation. In vivo , negative supercoiling would have the same stabilizing effect. KGZIP would be likely to bind the stabilized Z-form.
|
16982643_p21
|
16982643
|
DISCUSSION
| 4.4912
|
biomedical
|
Study
|
[
0.9992285966873169,
0.0004499885253608227,
0.0003213439485989511
] |
[
0.9990299940109253,
0.0003692311584018171,
0.0004709643835667521,
0.00012975491699762642
] |
en
| 0.999997
|
The possibility that the sequence KGKGKGK is sufficient to form a Z-DNA binding domain raises a number of interesting possibilities. This is an unusually compact domain, and is found in a wide variety of proteins. In addition, KGZIP is a starting point for the creation of Z-DNA specific reagents, for the study of or manipulation of this unusual conformation in vitro and in vivo .
|
16982643_p22
|
16982643
|
DISCUSSION
| 3.938001
|
biomedical
|
Study
|
[
0.9989449381828308,
0.0001644934673095122,
0.0008906054426915944
] |
[
0.8961074352264404,
0.09976214915513992,
0.003732298268005252,
0.00039802558603696525
] |
en
| 0.999996
|
Central to research into cell survival, growth and differentiation in normal and diseased states is the ability to quantify altered patterns of gene expression. Oligonucleotide ( 1 , 2 ) and cDNA ( 3 ) hybridization microarrays have emerged as the leading quantitative tool for analyzing transcription of many thousands of genes in a sample simultaneously ( 4 ) yet have known limitations in analytical performance and sample throughput ( 5 – 7 ). Real time or quantitative polymerase chain reaction (qPCR) ( 8 ) is the superior alternative because of its high accuracy, precision and dynamic range and, as a consequence, is the reference assay for calibration and validation of microarray data ( 9 ). However, scaling qPCR to analyze larger numbers of genes and samples simultaneously is intrinsically prohibited by the logistics and cost of the assay in its current microliter format in 96- or 384-well microplates.
|
17000636_p0
|
17000636
|
INTRODUCTION
| 4.151849
|
biomedical
|
Study
|
[
0.9992029070854187,
0.00026543752755969763,
0.0005316720344126225
] |
[
0.8438387513160706,
0.003478121943771839,
0.15242800116539001,
0.0002551503712311387
] |
en
| 0.999997
|
High throughput PCR strategies have focused on smaller reaction volumes and follow one of two fluidics methods. Fast sequential analysis is exemplified by monolithic, functionally integrated lab-on-a-chip devices that flow a sample bolus through fixed temperature zones of a micromachined channel for target sequence PCR amplification, followed by sequence specific capture by hybridization and electrochemical detection ( 10 ), fluorescence detection ( 11 ) or electrophoretic separation with fluorescent detection ( 12 ). With quantitative performance similar to a microarray, detection sensitivity is further constrained by sample throughput and the increased potential for cross-contamination from processing samples in a common microchannel.
|
17000636_p1
|
17000636
|
INTRODUCTION
| 4.113943
|
biomedical
|
Study
|
[
0.9995394945144653,
0.00013428508827928454,
0.00032624322921037674
] |
[
0.9755124449729919,
0.003447846509516239,
0.020895354449748993,
0.00014431003364734352
] |
en
| 0.999997
|
Many of these problems are mitigated in a parallel fluidics approach. Miniaturized versions of microplates based on high-density arrays of wells etched in a planar substrate is the basis for nanoliter- ( 13 – 16 ) or picoliter-scale PCR ( 17 ) in an array format. Other embodiments include PCR in microdroplets on a patterned hydrophobic–hydrophilic surface ( 18 ) or in a 2D array of communicating microchannels ( 19 – 21 ). Reports of quantitative nucleic acid measurement in these devices have focused on limiting dilution schemes ( 22 – 24 ), which is clearly not high-throughput. Achieving high areal densities of physically independent reaction containers (>4/mm 2 ) requires stringent fluidic isolation between adjacent containers and a high degree of environmental control to prevent cross-contamination and evaporative loss during temperature cycling. Despite these challenges, parallel micro- or nanofluidics offers throughput advantages by thermal cycling and imaging many reactions at once to quantify target copy number in multiple genes and samples, simultaneously. Imaging reactions in parallel allows for longer integration times, improves detected signal-to-noise ratios and benefits PCR specificity and sensitivity by requiring fewer temperature cycles to detect a given target copy number. Shorter cycle times are facilitated by rapid heat transfer across proportionally larger surface areas as the reaction volume is reduced.
|
17000636_p2
|
17000636
|
INTRODUCTION
| 4.388043
|
biomedical
|
Study
|
[
0.9992992877960205,
0.00033889879705384374,
0.00036179431481286883
] |
[
0.9343212246894836,
0.0011755049927160144,
0.06423472613096237,
0.0002685344952624291
] |
en
| 0.999996
|
Intuitively, an approach to high-throughput qPCR grounded in a high-density array of nanoliter reactions is attractive because it combines the high precision, accuracy and dynamic range of qPCR with the parallelism of a microarray for simultaneous quantification of gene expression across multiple genes and samples. For this to occur, two challenges need to be overcome. The first is creation of a simple interface for precise and accurate transfer of liquids between the wells of a microplate to those of a nanoplate. The second is achieving the accuracy, precision and sensitivity demanded by qPCR in a 96- or 384-well microplate but in a substantially reduced reaction volume. Discovery of a facile interface for speedy transfer of liquids between micro- and nanoplates and identifying a robust approach to ensure qPCR assay performance at the nanoliter-scale has been at the leading edge of our development efforts.
|
17000636_p3
|
17000636
|
INTRODUCTION
| 4.142585
|
biomedical
|
Study
|
[
0.9995555281639099,
0.0001765108754625544,
0.00026797238388098776
] |
[
0.9697311520576477,
0.008685545064508915,
0.021373338997364044,
0.00020990644406992942
] |
en
| 0.999997
|
We have solved these problems with an approach based on through-hole arrays ( 25 – 27 ). Effectively thought of as a high-density version of a microplate, our nanoplates combine the high-throughput and reagent savings of a nanofluidic system with the macroscale performance of qPCR in microplates. A stainless steel (317 stainless steel) platen the size of a microscope slide (25 mm × 75 mm × 0.3 mm) is photolithographically patterned and etched to form a rectilinear array of 3072, 320 μm diameter through-holes. The through-holes are grouped in 48 subarrays of 64 holes each and spaced on a 4.5 mm pitch equal to that of wells in a 384-well microplate . A series of vapor and liquid deposition steps covalently attaches a PCR compatible polyethylene glycol (PEG) hydrophilic layer amine-coupled to the interior surface of each through-hole, and a hydrophobic fluoroalkyl layer to the exterior surface of the platen. The differential hydrophilic–hydrophobic coating facilitates precise loading and isolated retention of fluid in each channel. Primer pairs stored in 384-well microplates are transferred into individual through-holes by an array of 48 slotted pins manipulated by a 4-axis robot ( XYZθ ) in an environmentally controlled chamber to prevent evaporative loss during loading. Once a platen is fully populated with primer pairs, the solvent is evaporated in a controlled manner leaving the primers immobilized in a PEG matrix on the inside surface of each through-hole. The array loaded with primer is stored in an evacuated Mylar™ bag at −20°C, ready for sample addition.
|
17000636_p4
|
17000636
|
INTRODUCTION
| 4.273071
|
biomedical
|
Study
|
[
0.9994144439697266,
0.00032352193375118077,
0.0002620717859826982
] |
[
0.997514009475708,
0.0018438546685501933,
0.000526796851772815,
0.00011535026715137064
] |
en
| 0.999997
|
Up to 48 different, previously prepared cDNA samples at a concentration of 32 ng/μl are mixed with off-the-shelf qPCR reagents for SYBR Green PCR (see Materials and Methods; PCR Mix) and dispensed into each sub-array (one sample per sub-array) with an automated 48 pipette tip dispensing device. A slotted cassette for holding the platen is assembled by sandwiching a U-shaped glass-reinforced epoxy polymer spacer between two microscope slides patterned with an opaque ink to optically mask background autofluorescence from the spacer. A degassed, immiscible perfluorinated liquid (Fluorinert™) is dispensed into the cassette, the platen inserted and the assembly hermetically sealed with a plug of ultraviolet (UV) curable epoxy.
|
17000636_p5
|
17000636
|
INTRODUCTION
| 4.140471
|
biomedical
|
Study
|
[
0.9992340803146362,
0.00044147134758532047,
0.00032443797681480646
] |
[
0.9535714387893677,
0.045110125094652176,
0.0009126908262260258,
0.0004057125188410282
] |
en
| 0.999996
|
Real time PCR (RT-PCR) occurs in a computer-controlled imaging thermal cycler whose essential components are two pairs of off-axis, high energy light emitting diode (LED) excitation sources, a thermoelectric flat block holding up to three encased arrays, two emission filters in a computer-controlled filter wheel and a thermoelectrically-cooled CCD camera. Under software control, the real time method for 9216 PCR amplifications and dissociation curves is implemented in <4 h. Post-acquisition data processing generates fluorescence amplification and melt curves for each through-hole in the array, from which cycle threshold (C T ) and melt temperature ( T m ) are computed. All data are stored in a flat file ( * .csv) format for ready export to a database or third party software for further analysis.
|
17000636_p6
|
17000636
|
INTRODUCTION
| 4.107648
|
biomedical
|
Other
|
[
0.9978341460227966,
0.0009983237832784653,
0.0011674737324938178
] |
[
0.29712697863578796,
0.6980081796646118,
0.003943843767046928,
0.0009209882118739188
] |
en
| 0.999997
|
Sheets comprised of 12 arrays attached by thin tabs to a support frame were purchased from Tech-Etch Inc. (Plymouth, MA). The arrays are fabricated by double-sided wet-etching of a photolithographically-patterned 300 μm sheet of 317 stainless steel resulting in the hole pattern shown in Figure 1 . The sheets are cleaned for 2 h in 10% RBS 35 (Pierce) at 50°C, rinsed in reverse osmosis de-ionized (RODI) salt water and dried with a stream of dry nitrogen gas. A vinyl-terminated silane monolayer (7-octenyltrimethoxysilane, Gelest) is vapor deposited [5 h, 100°C in a vacuum oven (VWR)] followed by a 30 min NH 3 (g) cure.
|
17000636_p7
|
17000636
|
Through-hole array fabrication
| 3.962983
|
biomedical
|
Study
|
[
0.9938878417015076,
0.00025890825781971216,
0.005853347480297089
] |
[
0.9762284159660339,
0.02325504645705223,
0.00038469902938231826,
0.00013174452760722488
] |
en
| 0.999998
|
Next, the vinyl groups inside the through-holes are selectively oxidized by first immersing the sheet in a 1l bath of ethanol to overcome the surface tension of the hydrophobic coating followed by immersion in 1L of RODI water. The sheet is next slowly passed through a layer of 30 ml of an oxidation solution (5 mM KMnO 4 and 19.5 mM NaIO 4 ) floating on 1l of Fluorinert™ , incubated for 2 h, rinsed in RODI water and dried in a stream of dry nitrogen gas. After oxidation, a hydrophilic PEG layer is deposited inside the through-holes by repeating the previous steps except replacing the oxidation solution with 30 ml of 15 mg/ml EDC (Pierce) and 5 mg/ml PEG 5000 (Nektar-Synasia) in HEPES buffer (pH 7.5). After incubation for 2 h, the sheet is removed and dried overnight at 100°C under vacuum. A second hydrophobic layer is added by vapor deposition of heptadecafluorotriethoxysilane (Gelest) for 2 h, 150°C in a vacuum oven followed by a 30 min cure with NH 3 (g). Finally, the array sheets are rinsed in RODI water to remove the physisorbed PEG layer, thus exposing the underlying covalently linked hydrophilic PEG.
|
17000636_p8
|
17000636
|
Through-hole array fabrication
| 4.289731
|
biomedical
|
Study
|
[
0.9986935257911682,
0.0005903025157749653,
0.000716193753760308
] |
[
0.9844187498092651,
0.014071028679609299,
0.0012354230275377631,
0.0002747301768977195
] |
en
| 0.999997
|
RNA samples were converted to randomly primed first strand cDNA using High Capacity cDNA Archive Kit (Applied BioSystems, Foster City, CA). To reduce non-specific product formation during qPCR, the cDNA sample was heated to 75°C for 10 min to inactivate the reverse transcriptase; snap chilled on ice for 5 min, then treated 1 h with 1.3 U/μl Exonuclease I (Amersham Biosciences, Piscataway, NJ). The Exonuclease I is heat inactivated at 85°C for 10 min and the resulting cDNA solution is stored at −20C.
|
17000636_p9
|
17000636
|
cDNA preparation
| 4.07248
|
biomedical
|
Study
|
[
0.9990284442901611,
0.0006021051667630672,
0.00036944402381777763
] |
[
0.864955484867096,
0.1328192800283432,
0.0014584338059648871,
0.0007669013575650752
] |
en
| 0.999995
|
The PCR master mix consists of 1× LightCycler™ FastStart DNA Master SYBR Green I (Roche Applied Science, Indianapolis, IN), 0.2% (w/v) Pluronic F-68 (Gibco, Carlsbad, CA), 1 mg/ml BSA (Sigma–Aldrich, St. Louis, MO), 1:4000 SYBR Green I (Sigma–Aldrich), 0.5% (v/v) Glycerol (Sigma–Aldrich), 8% (v/v) Formamide (Sigma–Aldrich) and sample. For each kinase test of 507 assays, 66 μl of reaction mix was required.
|
17000636_p10
|
17000636
|
PCR mix
| 3.847634
|
biomedical
|
Study
|
[
0.998778760433197,
0.0005025090067647398,
0.0007186256698332727
] |
[
0.6496134996414185,
0.34864920377731323,
0.0008834455511532724,
0.0008538637775927782
] |
en
| 0.999996
|
Kinase genes were selected based on their classification in Gene Ontology ( ) and their presence in the RefSeq database ( ). Primer pairs biased towards the 3′ end of these genes were obtained from either PrimerBank ( 28 ) and or designed using Primer3 [ ]. Primers were ordered from a commercial supplier (Sigma-Genosys, ) and their performance was validated with the following process. An ABI 7900 (Applied BioSystems Inc., Foster City, CA) was used to test if each primer set run against either five tissue cDNA library (qPCR Human Reference cDNA, BD BioSciences, Franklin Lakes, NJ) or 37 tissue cDNA library (BD Quick-Clone II Human Universal cDNA, BD BioSciences, Franklin Lakes, NJ) could generate a product rising above 0.2 ΔR n . Amplicon mobility in 4% agarose E-gels (Invitrogen, Carlsbad, CA) was measured from images captured with a gel imager (AlphaImager, AlphaInnotech, San Leandro, CA) and processed by software (Quantity One 1-D Analysis Software; BioRad, Hercules, CA) to confirm the primer set made a product within 10% of the predicted length. Amplicons generated from primer pairs passing the above criteria were pooled and gel purified to remove fragments <80 and >400 bp in size. This pool was used as a source of template in subsequent PCR array validation experiments. All amplicons were sequenced; ∼70% matched all or part of the expected target sequence while although the remainder could not be confirmed by sequencing, these amplicons nonetheless matched the expected size of the predicted amplified product.
|
17000636_p11
|
17000636
|
Primer design
| 4.133322
|
biomedical
|
Study
|
[
0.9995043277740479,
0.00028247712180018425,
0.00021319848019629717
] |
[
0.9993839263916016,
0.0002607457572594285,
0.0002870469179470092,
0.00006836355169070885
] |
en
| 0.999998
|
The primer concentration where each primer set fails to produce a product in the PCR array was determined. The working primer concentration was adjusted to 8-fold above this concentration to ensure that small quantities of primer carryover between holes during sample loading will reduce interference with PCR in adjacent holes. Assays were demonstrated to have at least a 95% confidence of detecting 4-fold change at >100 copies by first measuring a <0.5 C T STD across greater than five replicates and then demonstrating a ΔC T shift of 2 +/−1 cycle for a sample diluted 4-fold. The template for these experiments was the pooled amplicon template in 1 ng total RNA equivalent randomly primed liver cDNA, with an average of 200 +/−100 starting copy number. A pass rate of 60% was observed with this validation process.
|
17000636_p12
|
17000636
|
Primer validation
| 4.126201
|
biomedical
|
Study
|
[
0.999580442905426,
0.00021995244605932385,
0.00019954795425292104
] |
[
0.9991348385810852,
0.0005489586619660258,
0.0002460411924403161,
0.00007010182162048295
] |
en
| 0.999996
|
The PCR array thermal cycling protocol consisted of 10 min, 92°C polymerase activation step followed by 35 cycles of 15 s @ 92C, 1 min @ 55°C and 1 min @ 72°C (imaging step). Following amplification, amplicon dissociation was measured by cooling the PCR array to 65°C then slowly heated to 92°C @ 1°/min, with images collected every 0.25°C.
|
17000636_p13
|
17000636
|
Real time thermal cycler protocol
| 4.07892
|
biomedical
|
Study
|
[
0.998927652835846,
0.0007230916526168585,
0.00034930731635540724
] |
[
0.866510272026062,
0.13149620592594147,
0.001212442060932517,
0.0007809942471794784
] |
en
| 0.999995
|
Pooled HUVEC and Endothelial Cell Medium-2 (EGM(tm)-2) were purchased from CAMBREX BIO SCIENCE (Walksersville, MD). Recombinant Human TNF-α (10 ng/ml in EGM™-2), purchased from R & D Systems Inc. (Minneapolis, MN) was added to HUVEC cultures that were ∼50% confluent in T150 tissue culture flasks. Cultures were incubated for 4 h at 37C, 5% CO 2 . Medium was removed and 2.5 ml of TRI Reagent® (Molecular Research Center) was added to lyse cells. RNA was purified using an RNeasy Mini Kit (Qiagen Inc.) and reverse transcription was done using a High Capacity cDNA Archive Kit (Applied Biosystems). Samples were treated with Exonuclease I (10 U/μl) (Amersham Biosciences).
|
17000636_p14
|
17000636
|
Human umbilical vein endothelial cells (HUVEC sample preparation
| 4.118532
|
biomedical
|
Study
|
[
0.9995531439781189,
0.0002464655553922057,
0.0002004471461987123
] |
[
0.9962683916091919,
0.0032525467686355114,
0.00035404125810600817,
0.00012501285527832806
] |
en
| 0.999996
|
To examine amplification uniformity, three arrays were uniformly loaded with PCR mastermix, CycA primer pairs and amplicon, resulting in an average of 500 starting copies of amplicon per through-hole. OD 260 of purified amplicon was used to independently confirm the number of starting copies per hole. An image generated by subtracting pixel values of the first and 19th cycling image provides a visualization of amplicon replication in each through-hole of the array. Dark holes resulting from a sample loading failure are detected by the absence of SYBR Green fluorescence. Fluidics errors are typically <2% and are stochastically distributed amongst the through-holes as failed PCRs . The dimmer holes along the first and last column of the array image are not from reduced yield of PCR product but rather from reduced SYBR fluorescence intensity from non-uniformities in LED excitation, imaging field of view and slight variations in optical path at the refractive index boundaries across the array. These fluorescent signal differences (≈6% CV, data not shown) are corrected for in the instrument calibration. Residual optical differences are taken into account by fluorescent baseline normalization of the amplification curve for each through-hole prior to crossing threshold (C T ) calculation. The mean C T for 500 starting copies is 15.3 cycles, on average about 11 cycles earlier than microplate-based qPCR systems. The C T shift results from a higher concentration of amplified products expected for PCR at reduced volumes ( 22 ) and an improved C T calling algorithm we developed by combining baseline intercept with numerical modeling of the exponential amplification phase. The instrument precision at 500 starting copies was +/−0.16 C T (or <12% CV on copy number), estimated from the C T STD for >9100 amplification reactions . The C T distribution follows a Gaussian distribution slightly skewed towards delayed (higher) C T and with 0.6% of the through-holes having outlier C T s > 3 STD from the mean. While the C T map indicates a small C T gradient spanning 0.32 cycles diagonally from the middle of the array, outliers map stochastically (data not shown). We speculate that the reduced PCR efficiency of the outlier population may arise from random micro-scale defects of the interior polymer surface coating and/or contamination by random interfering particulates, but their impact on data quality is substantially reduced with replicate assays.
|
17000636_p15
|
17000636
|
Baseline qPCR performance: uniformity, precision, accuracy, sensitivity and dynamic range
| 4.37332
|
biomedical
|
Study
|
[
0.999285876750946,
0.0004809346282854676,
0.00023326314112637192
] |
[
0.9991124272346497,
0.00033423921559005976,
0.00042574593680910766,
0.00012755999341607094
] |
en
| 0.999998
|
PCR array thermal uniformity was examined by melt curve analysis of the amplicon products shown in Figure 3 . Following PCR, the array temperature was equilibrated to 65C, slowly ramped to 92°C at 1°C per minute, and SYBR Green fluorescence images were collected every 0.25°C to record SYBR Green dye quenching on amplicon dissociation. The product melt temperature, T m , for each through-hole is derived as the maximum of −d F /d T , where T is the array temperature and F is the fluorescence emission from a through-hole at each temperature ( 29 ). The T m distribution across the PCR array indicates the array center was 1° cooler than at its edge. The T m computed across three arrays produced a similar T m distribution and mean T m , with an array-to-array T m STD equal to ±0.28°C .
|
17000636_p16
|
17000636
|
Baseline qPCR performance: uniformity, precision, accuracy, sensitivity and dynamic range
| 4.153411
|
biomedical
|
Study
|
[
0.9995346069335938,
0.00026105379220098257,
0.00020435627084225416
] |
[
0.9993056058883667,
0.00030955314286984503,
0.0003165644302498549,
0.00006832606595708057
] |
en
| 0.999998
|
The dynamic range and accuracy of the instrument was measured by performing RT-PCR on a CycA amplicon titration. Figure 4A depicts 12 replicate amplification profiles for each log dilution, ranging from 10 7 to 1 starting copies per hole and a no template control. The high quality curves showed >150-fold signal-to-noise, and >4 cycles exponential-phase amplification. The C T computed from these curves were linear over 6 log amplicon concentrations , and near perfect amplification efficiency based on slope of C T versus log starting copy plot. The precision for measurements ≥1000 starting copies was equivalent to the earlier PCR uniformity experiment, averaging around 0.17 STD C T . The no template control had no detectable product, indicating carryover between subarrays is below the limit of detection.
|
17000636_p17
|
17000636
|
Baseline qPCR performance: uniformity, precision, accuracy, sensitivity and dynamic range
| 4.166362
|
biomedical
|
Study
|
[
0.999494194984436,
0.0003068209334742278,
0.00019899864855688065
] |
[
0.9993096590042114,
0.0002389763976680115,
0.0003742641129065305,
0.00007705793541390449
] |
en
| 0.999998
|
Sensitivity can be estimated by measuring the frequency of PCR positive reactions near the limit of assay detection. The decreasing precision below 1000 copies appears to follow the noise contribution based on the Poisson effect . Holes predicted to have an average of a single starting copy produced 49 of 64 positive assays (77%); reasonably close to the 63% predicted by Poisson statistics. Taken together, the through-hole array demonstrated single copy sensitivity. Additional thermal cycling of these arrays show no increase in assay positive holes, indicating that there is no intra subarray carryover of primers at the level of single copy sensitivity.
|
17000636_p18
|
17000636
|
Baseline qPCR performance: uniformity, precision, accuracy, sensitivity and dynamic range
| 4.161124
|
biomedical
|
Study
|
[
0.9995002746582031,
0.0002454603381920606,
0.0002542127331253141
] |
[
0.9991264939308167,
0.0005044916179031134,
0.0002986173494718969,
0.00007043280493235216
] |
en
| 0.999996
|
To assess the performance of the system with a biologically relevant example, an array was constructed with primer pairs targeting expression of 508 human kinases, 13 endogenous (housekeeping) controls in quadruplicate and 208 negative controls for analyzing four samples per array (see Materials and Methods, Primer Validation). Normal human heart and liver RNA from a commercial source was reverse transcribed into cDNA equivalent and divided into two aliquots of 0.25 and 1 ng cDNA per through-hole. The pipette sample loader transferred sample mixed with PCR mastermix into arrays pre-loaded with the assay primer sets and the arrays were processed according to the real time procedure described in the Methods section.
|
17000636_p19
|
17000636
|
qPCR measurement of differential expression of human kinase genes between human heart and human liver RNA samples
| 4.095192
|
biomedical
|
Study
|
[
0.9996210336685181,
0.00018917027045972645,
0.000189856524229981
] |
[
0.9991543292999268,
0.0005212182877585292,
0.0002588318893685937,
0.000065684798755683
] |
en
| 0.999997
|
Correlation of C T technical replicates for the heart and liver samples at 1 and 0.25 ng of cDNA per through-hole shows high assay precision and accuracy across a large dynamic range of responses. With 1 ng cDNA per through-hole, 78% of the assays for the heart sample have STD <0.5 while for liver, 72% of the assays had this precision or better . Assay accuracy was estimated from computing the mean C T difference between two different cDNA concentrations for the heart and liver samples. A histogram of ΔC T for all assays in both tissues shows a median C T of 1.8 ± 0.48 for heart and 1.7 ± 0.61 for the liver sample, indicative of a 4-fold change in cDNA concentration. Negative controls in each subarray showed no detectable carry-over between through-holes .
|
17000636_p20
|
17000636
|
qPCR measurement of differential expression of human kinase genes between human heart and human liver RNA samples
| 4.13984
|
biomedical
|
Study
|
[
0.9995618462562561,
0.00025733481743372977,
0.0001808060915209353
] |
[
0.9992771744728088,
0.0003016879200004041,
0.0003517944714985788,
0.00006933999247848988
] |
en
| 0.999997
|
Differential gene expression between the heart and liver samples at 1 ng cDNA per through-hole was determined by comparing the difference in mean C T s for each gene in the liver and heart assay populations . Positive assays for which an accurate C T could be measured passed the following criteria: (i) the amplicon product was detected and confirmed by melt curve analysis; (ii) target expression greater than single copy in both tissues (CT < 25) and (iii) each assay had a technical replicate ≥2. Eighty-three assays were rejected based on these criteria and the remaining 442 positive assays were normalized relative to the geometric mean of the same 13 housekeeping genes for each tissue. Two subpopulations are observed when the difference in expression for the same gene is compared between the two tissues: (i) tissue-specific expression where the gene expressed at less than 10 copies in one tissue but not the other and (ii) the gene expressed in both tissues with an abundance >10 copies but there is a significant difference in expression between the tissues based on Student's t -test (two-tailed distribution and two-sample unequal variance) with a P -value <0.005. Segmentation of the assay population based on these criteria is not surprising given the physiological differences between heart and liver tissue, even for common functional pathways.
|
17000636_p21
|
17000636
|
qPCR measurement of differential expression of human kinase genes between human heart and human liver RNA samples
| 4.206144
|
biomedical
|
Study
|
[
0.9994727969169617,
0.00031590680009685457,
0.00021137217117939144
] |
[
0.999239444732666,
0.0002717074239626527,
0.0004170940665062517,
0.00007167092553572729
] |
en
| 0.999997
|
Consider the well-studied glycolysis pathway as a point of comparison where multiple kinases play fundamental roles in the multi-step oxidation of glucose to pyruvate and ATP ( 30 ). The first step in this process is ATP-dependent phosphorylation of glucose to the intermediate glucose-6-phosphate by the liver-specific hexokinase isozyme glucokinase . We detected insignificant expression levels in the heart sample (<1 copy) compared with substantially higher levels in the liver sample (>200 000-fold higher). A similar pattern of tissue-specific expression was recorded for the liver and muscle isoforms of phosphofructose kinase; and the muscle and liver isoforms of pyruvate kinase. In contrast, PGK1 , one of two isoforms of phosphoglycerate kinase, shows a small yet significant difference in expression between the heart and liver sample (4.7-fold; P = 6 × 10 −7 ) yet is expressed at high levels in both tissues, indicative of its known ubiquitous expression. Expression of the second isoform, PGK2 , was not detected in either sample; this is not surprising given that PGK2 is only expressed in meiotic spermatocytes and postmeiotic spermatids during spermatogenesis ( 31 ).
|
17000636_p22
|
17000636
|
qPCR measurement of differential expression of human kinase genes between human heart and human liver RNA samples
| 4.330553
|
biomedical
|
Study
|
[
0.9994663596153259,
0.0002772913430817425,
0.0002564141759648919
] |
[
0.9992700219154358,
0.00023892226454336196,
0.00041720151784829795,
0.00007390822429442778
] |
en
| 0.999998
|
To externally validate assay predictions, 21 assays showing higher, equal and lower expression in liver than heart were run in a microplate on a real time thermal cycler . The resulting measurements showed a high degree of concordance with the PCR array results ( R 2 = 0.98) with equivalent precision .
|
17000636_p23
|
17000636
|
qPCR measurement of differential expression of human kinase genes between human heart and human liver RNA samples
| 4.076395
|
biomedical
|
Study
|
[
0.9995392560958862,
0.00023304609931074083,
0.00022771315707359463
] |
[
0.9992446899414062,
0.0003753297496587038,
0.0003114183200523257,
0.00006863346789032221
] |
en
| 0.999996
|
HUVEC TNF-α response was examined using both a kinase array and a custom array. The custom array contained 26 TNF response pathway assays selected from a library of 2800 validated primer pairs (see Materials and Methods; Primer Validation). The layout of the custom array allows 48 samples per PCR array and the data analysis was similar to the heart-liver comparison. Figure 7A depicts the ΔC T between the TNF-α treated and vehicle treated HUVEC (see Materials and Methods). Of the 533 targets screened, 406 were positive in both samples. Negative controls in each subarray showed no detectable carry-over between through-holes . As predicted, the TNF-α treatment increased selectin E and vascular cell adhesion molecule 1 expression level >100-fold ( 32 , 33 ). The dynamic range of SELE was compared between microplate and PCR array by diluting the sample and determining if the resulting housekeeping-normalized ΔC T gave the same measurement across the dilution series. The PCR array produced a similar ΔC T until the SELE starting copy number reached single copy in the PCR array hole, ∼0.03 ng/hole.
|
17000636_p24
|
17000636
|
qPCR measurement of differentially expressed genes in TNF-α stimulated NK-β pathway
| 4.147134
|
biomedical
|
Study
|
[
0.9995114803314209,
0.0002691951231099665,
0.0002193545369664207
] |
[
0.9994651675224304,
0.00018824067956302315,
0.0002822861715685576,
0.0000643496387056075
] |
en
| 0.999996
|
A nanofluidic system for performing solution-phase RT-PCR in an array of isolated through-holes overcomes limitations of existing micro- or nanofluidic devices by implementing a parallel approach to fluidic handling that lends itself well to interfacing with microtiter plates for simple and efficacious transfer of liquid samples and reagents into the nanofluidic structure using arrays of slotted pins or pipette tips. The reaction containers are kept separate and distinct by selective and controlled modification of the platen surface to make the inside surfaces of the through-holes chemically distinct from the outside surfaces. In the system described here, the interior surface of each hole is modified to be hydrophilic and the exterior surface hydrophobic.
|
17000636_p25
|
17000636
|
DISCUSSION
| 4.128643
|
biomedical
|
Study
|
[
0.9993979930877686,
0.0002719102194532752,
0.00033013784559443593
] |
[
0.9778698086738586,
0.02029499039053917,
0.0016118948115035892,
0.0002232999395346269
] |
en
| 0.999998
|
We validated the system performance by carrying out thousands of real time SYBR Green PCR assays for a number of sample sets. With amplicon as the target, the baseline performance of the system had the same dynamic range, sensitivity and precision as PCR in a microplate but in 64-fold smaller reaction volumes. The increase in C T variability with decreasing number of target amplicon was shown to follow a Poisson distribution at low copy number, demonstrating the system is capable of single copy detection. Further, the thermal uniformity was demonstrated by the small spread in C T and T m in a uniformly loaded array. With volume miniaturization, we identified the need to increase sample concentration to maintain a constant number of target molecules in the reaction volume to obtain the high quality PCR observed.
|
17000636_p26
|
17000636
|
DISCUSSION
| 4.133408
|
biomedical
|
Study
|
[
0.9995105266571045,
0.00028422020841389894,
0.00020528699678834528
] |
[
0.9992910623550415,
0.00024571927497163415,
0.0003913421824108809,
0.00007190713949967176
] |
en
| 0.999996
|
When challenged with cDNA prepared from normal human heart and liver RNA, the high replicate precision and accuracy enabled detection of tissue-specific expression of kinase genes and discrimination in differential kinase expression between the two tissue types. A large number of genes showed small differences in expression (<2-fold) with a high degree of significance ( P < 0.001), suggesting the system is potentially capable of revealing new patterns of transcription across large numbers of genes. The data from TNF-α stimulation of the NF-β pathway in HUVEC cells demonstrates this point. The high sample throughput of over 27 000 RT-PCR analyses per person per day, based on a workflow of 3 h to prepare three arrays and three processing runs per day per person, is a 24-fold increase in analytical throughput based on current 384-well microplates and opens new possibilities in genetic system analysis not currently possible. Clearly, the nanofluidic PCR array has the potential to span the space of genomic applications requiring the parallelism of hybridization microarrays with the specificity, accuracy, dynamic range and precision of solution phase PCR.
|
17000636_p27
|
17000636
|
DISCUSSION
| 4.288488
|
biomedical
|
Study
|
[
0.9996175765991211,
0.00023718498414382339,
0.00014527699386235327
] |
[
0.9961995482444763,
0.000992654007859528,
0.002660483354702592,
0.000147238839417696
] |
en
| 0.999998
|
Histones are among the most conserved proteins in the eukaryotic kingdom. Their amino acid sequences are highly conserved as well as the coding sequence suggesting a strong selection pressure on the secondary structure of the corresponding mRNAs ( 1 , 2 ). In contrast, sequences of the 5′- and 3′-untranslated regions (5′- and 3′-UTRs) are more divergent. They are believed to be modern acquisitions that restrict the histone biosynthesis to the S phase of the cell cycle. Different strategies have been developed by eukaryotes for that purpose. In yeast and plants, histones are produced from classical polyadenylated mRNAs. In metazoans, a sophisticated machinery involving many factors and ribonucleoprotein particles (RNPs) is used for the histone synthesis ( 3 , 4 ). In the latter case, histone mRNAs are not polyadenylated but instead end in a highly conserved hairpin structure. The histone mRNAs are synthesized as precursor mRNAs with a 3′ extension. The mature histone mRNAs are generated by a single endonucleolytic cleavage occurring between two cis -acting elements: a highly conserved hairpin structure upstream of the cleavage site and a so-called histone downstream element . These two sequences are the scaffold for the assembly of the whole processing machinery. The highly conserved hairpin structure is bound by the stem–loop binding protein (SLBP, also called HBP for hairpin binding protein) ( 5 , 6 ). Downstream of the cleavage site, the HDE anneals to the 5′ end of the minor U7 snRNP. Then, the Zinc-Finger rich Protein (ZFP100) bridges the two cis -elements by interacting on one hand with the U7-specific protein Lsm11 and on the other hand with SLBP bound to its target hairpin ( 7 – 9 ). Results from recent UV-crosslinking studies suggest that CPSF-73, a known component of the cleavage/polyadenylation machinery, is the cleavage factor, which is recruited in a U7-dependent manner ( 10 ). CPSF-73 would act in a larger processing complex comprising eight proteins of the cleavage/polyadenylation machinery including the heat labile factor (HLF) also called Symplekin ( 11 ). The mature histone mRNAs are exported to the cytoplasm for translation. Therefore, the 3′ end processing of histone pre-mRNAs is the cornerstone of histone biosynthesis. Early in vitro processing experiments have suggested that the hairpin for SLBP binding is required for efficient 3′ end processing. However, the effect of loss of binding mutants of the hairpin can be compensated by optimizing the base pairing between the HDE and the U7 snRNP ( 12 ). Furthermore, the distance between the hairpin and the HDE is critical for efficient processing ( 13 – 15 ). In vitro experiments also showed that efficient processing of mouse H2a-614 mRNA can be observed in absence of the SLBP whereas processing of mouse H1t mRNA is fully dependent on the presence of the SLBP on its target hairpin because of weak base-pairing with U7 ( 16 ). In vivo experiments on mouse histone mRNA H2a-614 showed that an intact hairpin target for the SLBP is required for efficient 3′ end processing ( 17 ). Recently, this has been validated using the nowadays-available knockout techniques and indeed the SLBP is an essential protein in Caenorhabditis elegans ( 18 , 19 ) and in HeLa cells ( 20 , 21 ). On the other hand, Drosophila histone genes are special in that they contain polyadenylation sites downstream to the normal 3′ end site. Therefore, the SLBP depletion by RNAi in Drosophila is not lethal but only causes infertility because the histone mRNAs become polyadenylated. This allows in vivo examination of 3′ processing reaction, which shows that the reaction is strongly affected in the absence of the SLBP ( 22 ). Altogether, these data suggest that SLBP is required for efficient histone 3′ end processing, in facilitating U7 snRNP binding by protein–protein contacts mediated through ZFP100 ( 8 ).
|
16982637_p0
|
16982637
|
INTRODUCTION
| 5.004141
|
biomedical
|
Study
|
[
0.9967467784881592,
0.0020163054578006268,
0.0012369786854833364
] |
[
0.970651388168335,
0.002326502464711666,
0.025633016601204872,
0.0013890617992728949
] |
en
| 0.999995
|
The rationale of the present study was to investigate whether the effect of the SLBP on the 3′ end processing could also be mediated by other mechanisms involving RNA structure. For this, we investigated the secondary structure of the 3′-UTR of histone H4-12 pre-mRNAs, which is processed in vitro in a SLBP-dependent manner ( 6 , 12 ). Our results show that SLBP induces conformational rearrangements of the HDE that enables U7 snRNP anchoring. This is also observed for the histone gene H1t, which displays SLBP-dependent processing but not for H2a-614 ( 16 ). The implications for histone biosynthesis in metazoan are discussed.
|
16982637_p1
|
16982637
|
INTRODUCTION
| 4.201234
|
biomedical
|
Study
|
[
0.9994568228721619,
0.00028663125704042614,
0.00025653550983406603
] |
[
0.9994288086891174,
0.0002258838212583214,
0.00028134571039117873,
0.00006399851554306224
] |
en
| 0.999996
|
Templates for T7 in vitro transcription of the 3′-UTRs and U7 snRNA have been cloned into the SmaI site of pUC19. Sequences of the 3′-UTRs of mouse H4–12 , H2a-614 , H1t gene and mouse U7 snRNA have been reconstructed by shotgun oligonucleotides ligation. All constructs were verified by DNA sequencing.
|
16982637_p2
|
16982637
|
Construction of templates
| 4.108634
|
biomedical
|
Study
|
[
0.9995313882827759,
0.00017506854783277959,
0.00029360296321101487
] |
[
0.9911283254623413,
0.008227669633924961,
0.0004803144547622651,
0.00016368173237424344
] |
en
| 0.999997
|
In vitro transcription of the 3′-UTRs was performed as previously described ( 23 ). Transcripts were separated by denaturing 10% PAGE and electro-eluted from gel slices using a Biotrap apparatus (Schleicher and Schuell). The pure uncapped transcripts were dephosphorylated using bovine alkaline phosphatase (Fermentas) and 5′-radiolabelled with T4 polynucleotide kinase (New England Biolabs). Radioactive transcripts were further purified by denaturing 10% PAGE and eluted overnight at 4°C in buffer A (0.3 M NaCl, 0.5 mM EDTA, 10 mM Tris–HCl pH 7.5). Before use, transcripts were folded in water by incubation at 80°C for 2 min, slow cooling to 35°C and kept on ice.
|
16982637_p3
|
16982637
|
In vitro transcription and labelling
| 4.157318
|
biomedical
|
Study
|
[
0.9995150566101074,
0.00027342670364305377,
0.0002115137904183939
] |
[
0.9981670379638672,
0.001242947531864047,
0.0004806500510312617,
0.00010936029138974845
] |
en
| 0.999997
|
Enzymatic and chemical structural probing of RNA were conducted by established procedures ( 24 ). Probing mixtures (15 μl) containing radioactive transcripts (50 000 c.p.m) were incubated for 20 min on ice in buffer B (10 mM Tris–HCl pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 2 mM vanadyl ribonucleoside RNase inhibitor (New England Biolabs), 30 U RNasin (Promega) and 10 pmoles of yeast bulk tRNA. Statistical RNA cleavage was achieved after 10 min incubation at 20°C with 5.9.10 −5 U of RNase V1, 1.27 U RNase T1 or 5.6 U of RNase T2. Nucleases V1, T2 and T1 were from Kemotex, Invitrogen and BRL, respectively, and lead acetate from Merck. Reactions were stopped by rapid cooling on ice and addition of 15 μl of stop mix A (0.6 M sodium acetate pH 6, 3 mM EDTA and 0.1 mg/ml total tRNA). Lead probing required a 5 min incubation at 20°C with 10.7 mM final concentration of a freshly prepared lead-acetate solution. Rapid cooling on ice and addition of EDTA (17 mM final concentration) was used for quenching the reactions. After phenol–chloroform extraction, RNA was ethanol precipitated in the presence of glycogen (1 μg) and washed twice with 80% ethanol. Pellets were dried and dissolved in formamide dye. Samples were migrated on 10% denaturing-PAGE in parallel with RNase T1 and alkaline ladders of the corresponding end-labelled RNA for band assignments. RNase T1 ladders were made as previously described ( 25 ). Alkaline ladders were obtained by incubation of the labelled transcript with 1 μg of yeast bulk tRNA for 10 min at 80°C in a buffer containing 80 mM Na 2 CO 3 /NaHCO 3 pH 9. As control, we compared probed RNAs with RNAs treated similarly but without probe.
|
16982637_p4
|
16982637
|
Enzymatic and chemical RNA probing
| 4.26431
|
biomedical
|
Study
|
[
0.9993427395820618,
0.0003945080388803035,
0.0002626574714668095
] |
[
0.9989332556724548,
0.00044820812763646245,
0.0005280472105368972,
0.00009054186375578865
] |
en
| 0.999995
|
Footprinting reactions were done as described above in the presence of the SLBP. Recombinant human His-tagged SLBP was obtained as previously described ( 26 ). The amount of SLBP used for each footprint was determined by EMSA (see below) in order to obtain at least 80% of the transcript (50 000 c.p.m) shifted. Before use, appropriate amounts of SLBP were dialysed against buffer B and then mixed with the radioactive transcripts (50 000 c.p.m).
|
16982637_p5
|
16982637
|
Enzymatic and chemical footprinting
| 4.078712
|
biomedical
|
Study
|
[
0.9994878768920898,
0.00020320324983913451,
0.0003089185629505664
] |
[
0.9991146922111511,
0.0006229536957107484,
0.00020881961972918361,
0.000053514057071879506
] |
en
| 0.999995
|
For electrophoretic mobility shift assay (EMSA), 50 000 c.p.m of 5′-labelled U7 snRNA transcript were incubated in buffer B on ice for 30 min, with H4-12 transcript (0.01 μM) in the presence or in absence of the SLBP (10 μM). The competitions experiments were conducted by pre-annealing the competitor HDE oligonucleotide (5′-AAGAGCTGTAACACTT-3′) with U7 snRNA transcript or by pre-incubating the SLBP with competitor stem–loop RNA (wild-type stem–loop competitor: 5′-GGAGCUCAACAAAAGGCCCUUUUCAGGGCCACCC-3′; * mutated stem–loop competitor: 5′-GGAGCUCAACAAAA CCGGAAAGCCUUCCGG A CCC-3′, with an underlined stem–loop sequence completely mutated) on ice for 30 min. The complexes were separated by native 5% PAGE and visualized by phosphorimaging.
|
16982637_p6
|
16982637
|
Electrophoretic mobility shift assay with U7 snRNA transcripts
| 4.170393
|
biomedical
|
Study
|
[
0.9995088577270508,
0.00026745814830064774,
0.00022362252639140934
] |
[
0.998822033405304,
0.0007527372799813747,
0.0003486739587970078,
0.00007656779780518264
] |
en
| 0.999998
|
Three transcripts corresponding to the 3′-UTR of histone pre-mRNAs H4-12, H1t and H2a-614 (94, 85 and 113 nt, respectively) have been synthesized by T7 run-off transcription. These sequences start downstream of the stop codons and end after the HDE sequences. They were chosen according to previous studies, which showed that they were active during processing ( 12 , 16 ) . For better T7 transcription yield, two G residues have been inserted at the 5′ end of the sequence. For run-off transcription, C residues were added at the 3′ ends in order to create a SmaI restriction site . The structure of the three RNA has been probed by RNases T1, T2, V1 and by lead. RNase T2 and lead cut preferentially after unpaired residues and RNase T1 cuts after unpaired G residues. RNase V1 cuts in double-stranded sequences or higher order structures. The background hydrolysis by water or traces of contaminating metal cations were discriminated from probe induced-cuts by control experiments performed without probes. As an additional control, we checked by chemical and enzymatic probing that the folding of the 3′-UTR was identical in full-length pre-mRNAs and that the structure of the 3′-UTR was not influenced by sequences located into the open reading frame. For this, the probed uncapped pre-mRNAs were extended by reverse transcription using a primer annealed to the 15 last residues at the 3′ end of the transcript (data not shown).
|
16982637_p7
|
16982637
|
Methodological considerations
| 4.211456
|
biomedical
|
Study
|
[
0.999380350112915,
0.00032246182672679424,
0.0002970787463709712
] |
[
0.999427318572998,
0.0002167366910725832,
0.0002903882705140859,
0.00006566365482285619
] |
en
| 0.999997
|
The 94 nt-long T7 transcript from the H4-12 3′-UTR was synthesized and 5′-end labelled by T4 polynucleotide kinase. The 3′-UTR was probed by lead and RNase T2, T1, V1 (see above). As the probes exhibit different specificities, a map of the single stranded and double stranded regions of the RNA molecule could be drawn. These results were used as constraints for the M-fold software ( 27 ). The resulting 2D-model of the 3′-UTR of H4-12 precursor mRNA is shown in Figure 2 . It contains a cloverleaf structure comprising four domains named I, II, III and IV. The presence of the highly conserved hairpin structure (domain I), which is bound by the SLBP, is clearly visible. This hairpin is highly stable since no T1 cuts are detected after the five G residues of stem I (G37, G38, G48, G49 and G50) even under denaturing conditions . More strikingly, the HDE is embedded in two hairpin structures named domains III and IV. The 5′ part of the HDE is involved in domain III, a 6 bp-stem and 4 nt-loop. The stem from domain III is hardly cut by RNase V1 and T1. V1 cuts are only detected on one side of the helix. On the other side of the helix, three G residues from the HDE (G71, G72 and G74) are not cleaved at all by T1, even under denaturing conditions, indicating inaccessibility of domain III-stem to RNase hydrolysis and high stability in denaturing conditions. Such behaviour suggests that the stem of domain III may be buried in the tertiary structure of the H4 UTR. In contrast G77 which lies in the 3′ part of HDE, in the stem of domain IV, is readily cut by T1 under denaturing conditions suggesting that stem IV can unfold more easily than stem III. The processing site clearly lies in a single-stranded sequence as indicated by the presence of lead, T1 and T2 cuts and the absence of V1 cuts.
|
16982637_p8
|
16982637
|
The H4-12 3′-UTR contains four structured domains
| 4.503337
|
biomedical
|
Study
|
[
0.9990707635879517,
0.0005823885439895093,
0.0003468061040621251
] |
[
0.998946487903595,
0.00044052847079001367,
0.0004498741473071277,
0.00016315137327183038
] |
en
| 0.999997
|
Using recombinant SLBP produced in baculovirus, we performed footprinting of the SLBP–3′-UTR complex . As expected, the SLBP binding prevents the V1 cleavages that occur in the 5′ part of the stem I and the single-strand specific cleavages in loop I (lead and T2). More surprisingly, the SLBP also protects residues 20–36 in domain II upstream of its target domain I indicating that SLBP also interacts with domain II .
|
16982637_p9
|
16982637
|
The SLBP footprint experiments reveal structural rearrangements in H4-12 HDE
| 4.212195
|
biomedical
|
Study
|
[
0.9994816184043884,
0.0002484997676219791,
0.00026987053570337594
] |
[
0.9993718266487122,
0.0003578911127988249,
0.00020524831779766828,
0.0000649831781629473
] |
en
| 0.999998
|
The binding of SLBP to the 3′-UTR not only leads to protection but also induces the appearance of new cleavages or enhancement of existing cuts. This shows that binding of the SLBP induces structural rearrangements of the 3′-UTR precursor . All these SLBP-enhanced cuts are exclusively located in domains III and IV containing the HDE. In the well-defined stem–loop III, V1 and lead reactivity increase is clearly visible. This variation in the accessibility to the probes suggests that the global conformation of the UTR has changed. On the contrary, the structure of domain IV is significantly altered by the binding of the SLBP. The strong lead cleavages that appear on both sides of stem IV indicate that the stem is progressively melted on SLBP binding. Altogether, these data suggest that the reactivity changes observed after SLBP binding might result from significant conformational changes, which lead to a more accessible 3′ end of the HDE.
|
16982637_p10
|
16982637
|
The SLBP footprint experiments reveal structural rearrangements in H4-12 HDE
| 4.416956
|
biomedical
|
Study
|
[
0.9993625283241272,
0.0003750996838789433,
0.0002622909378260374
] |
[
0.9990181922912598,
0.0003477487771306187,
0.0005389272700995207,
0.00009517509170109406
] |
en
| 0.999997
|
One obvious consequence of the conformation changes observed on SLBP binding would be to facilitate U7 snRNP anchoring. To validate this hypothesis, we performed EMSAs with the 3′-UTR of H4-12 pre-mRNAs transcript and [ 32 P]-labelled U7 snRNA transcript. The H4-12 3′-UTR and U7 snRNA were mixed in the presence or absence of the SLBP and complexes separated by native PAGE. Figure 3 shows that incubation of the H4-12 3′-UTR and U7 snRNA with the SLBP improves the anchoring of the U7 snRNA (lane 5) since more U7 is shifted in the SLBP/H4-12/U7 ternary complex in the presence of the SLBP. The ternary complex formation was prevented using a RNA stem–loop corresponding to the SLBP binding site (lane 9) and by a DNA-oligonucleotide corresponding to the HDE sequence (lane 7). This provided unambiguous ternary complex U7/3′-UTR/SLBP identification. In addition, a mutated RNA stem–loop was unable to displace the SLBP from the complex (lane 11).
|
16982637_p11
|
16982637
|
The SLBP enables U7 snRNA anchoring on H4-12 3′-UTR
| 4.246832
|
biomedical
|
Study
|
[
0.9994080066680908,
0.0003468207723926753,
0.00024516397388651967
] |
[
0.9994022846221924,
0.00024205551017075777,
0.00027949915966019034,
0.00007617479423061013
] |
en
| 0.999997
|
Next, we performed similar experiments on two other histone precursor 3′-UTR, namely H1t and H2a-614. These histone genes have been selected for this study because they show different behaviour with respect to the influence of the SLBP as shown by previous in vitro processing experiments ( 16 ). The in vitro 3′ end processing of histone H1t pre-mRNAs fully requires the SLBP whereas the processing of H2a-614 does not need the presence of the SLBP.
|
16982637_p12
|
16982637
|
The HDE of H1t and H2a-614 3′-UTR are also embedded in hairpins
| 4.105683
|
biomedical
|
Study
|
[
0.9992923736572266,
0.00025147732230834663,
0.00045611854875460267
] |
[
0.9994611144065857,
0.00024501854204572737,
0.00024403000134043396,
0.00004986129715689458
] |
en
| 0.999997
|
The probing of the H1t 3′-UTR pre-mRNAs has been performed using the same probes and the 2D model also contains four domains that have been named I to IV in analogy to H4-12. Domain I, the stem–loop structure bound by the SLBP, is highly stable as shown by the absence of reactivity to G residues of the stem under denaturing conditions and by strong stacking at positions 44–48 in the alkaline ladder . Stem I is accessible to V1 on both sides in contrast to the half-accessibility observed in the case of H4-12. This suggests that the 3′ part of stem I in H4-12 might be masked by another RNA domain or might adopt a specific conformation inaccessible to RNases. Domains II of H4-12 and H1t are very similar whereas domain III is completely unpaired in H1t. The HDE is almost completely embedded in a structured domain IV.
|
16982637_p13
|
16982637
|
The HDE of H1t and H2a-614 3′-UTR are also embedded in hairpins
| 4.392399
|
biomedical
|
Study
|
[
0.9993683695793152,
0.00032740161987021565,
0.00030430141487158835
] |
[
0.9991945624351501,
0.00034328975016251206,
0.00037271351902745664,
0.00008942928252508864
] |
en
| 0.999999
|
Like for H4-12, the SLBP footprint on the H1t 3′-UTR shows protection in domain I and II but only the 5′ half of stem I is protected . This is in good agreement with previous experiments which showed that the SLBP interacts only with the 5′ part of domain I to allow binding of another protein, the 3′ hExo, on the second part ( 28 ). Binding of the SLBP led to appearance of lead cleavages in stem II and adjacent residues from the upper part of stem IV, suggesting opening of these double stranded regions. Similarly, an enhancement of accessibility to the probes in the 3′ part of domain IV was observed. Like for H4-12, these results suggest that SLBP induces conformational rearrangements resulting in increases of the probe-reactivity of residues from the HDE. However, in the case of the H1t, it is the 5′ part of the HDE that opens on SLBP binding whereas the 3′ part of the HDE is opened in the case of H4-12. To conclude, one can say that the overall 2D-structure of the H1t 3′ UTR is more accessible than H4-12 but the SLBP also induces structural rearrangements in both domains IV.
|
16982637_p14
|
16982637
|
The HDE of H1t and H2a-614 3′-UTR are also embedded in hairpins
| 4.557912
|
biomedical
|
Study
|
[
0.9990690350532532,
0.00046441814629361033,
0.0004666562017519027
] |
[
0.9986833930015564,
0.000520596222486347,
0.000668909284286201,
0.00012699734361376613
] |
en
| 0.999996
|
Probing of the H2a-614 reveals a rather different organization . Only domain I displays clear V1 cuts on both sides of stem I and single strand-specific cuts in loop I. Domains II and III are completely unpaired while domain IV, which contains the HDE, is composed of 9 bp interrupted by two bulges. The 5′ and 3′ ends of the HDE are unpaired. Similar to H4-12 and H1t, binding of the SLBP induces protections exclusively on domains I and II; however no significant changes are observed in domain III and IV, except five lead cleavages at positions 53, 78, 86, 89 and 105 flanking stem IV . In contrast to the previous histone 3′-UTRs no changes in the HDE are detected on SLBP binding to the H2a-614 3′-UTR. Altogether, these data suggests that the 3′-UTR of H2a-614 is less structured than H4-12 and H1t and that binding of the SLBP has no effect on the structure of the HDE.
|
16982637_p15
|
16982637
|
The HDE of H1t and H2a-614 3′-UTR are also embedded in hairpins
| 4.422898
|
biomedical
|
Study
|
[
0.999268114566803,
0.00035136702354066074,
0.00038042658707126975
] |
[
0.9991249442100525,
0.0003962884075008333,
0.0003770276380237192,
0.00010163697152165696
] |
en
| 0.999999
|
We have previously shown that the conformation changes observed on SLBP binding on the H4-12 3′-UTR facilitates U7 snRNP anchoring. We repeated the same experiment with H1t and H2a-614 transcripts. Despite the fact that we observed conformation changes in the H1t 3′-UTR on SLBP binding, we did not observe any improvement of the U7 snRNA hybridization (data not shown). The same absence of stimulation was observed with H2a-614 3′-UTR, which did not display any conformation change on SLBP binding (data not shown).
|
16982637_p16
|
16982637
|
SLBP-binding does not improve the U7 snRNA anchoring on H1t and H2a-614 3′-UTR
| 4.072437
|
biomedical
|
Study
|
[
0.9993078708648682,
0.0002170812658732757,
0.0004750479420181364
] |
[
0.9995198249816895,
0.0002769559796433896,
0.00015447843179572374,
0.000048796347982715815
] |
en
| 0.999998
|
These data, together with footprint results suggest the following hierarchy of SLBP-dependency for U7 snRNA anchoring. The SLBP induces significant conformation changes in H4-12 3′-UTR and consequently favours the anchoring of the U7 snRNA to H4-12. With H1t, significant conformational changes are observed on SLBP binding but no improvement of the U7 snRNA could be detected. With H2a-614, changes could be detected neither on the 2D structure nor on U7 snRNA binding. These results are in good agreement with previous experiments showing that H4-12 and H1t are SLBP-dependent for efficient processing whereas H2a-614 does not require SLBP for in vitro processing ( 6 , 16 ).
|
16982637_p17
|
16982637
|
SLBP-binding does not improve the U7 snRNA anchoring on H1t and H2a-614 3′-UTR
| 4.268417
|
biomedical
|
Study
|
[
0.9994345307350159,
0.00026012014131993055,
0.00030538419377990067
] |
[
0.9993282556533813,
0.00030537473503500223,
0.0003021415905095637,
0.00006424014281947166
] |
en
| 0.999999
|
The 3′ end processing of histone pre-mRNAs is an intricate process because it requires at least three proteins (SLBP, ZFP100, CPSF-73) and the minor U7 snRNP. Numerous RNA–RNA and RNA–protein interactions enable assembly of the processing machinery. In addition, the unique U7 snRNP triggers 3′ end processing of many different histone pre-mRNAs by annealing to distinct HDEs. In order to decipher the function of each component, we investigated the influence of the SLBP on the anchoring of the U7 snRNA by chemical and enzymatic probing.
|
16982637_p18
|
16982637
|
DISCUSSION
| 4.277254
|
biomedical
|
Study
|
[
0.9994385838508606,
0.0002945555606856942,
0.0002669264213182032
] |
[
0.9991612434387207,
0.00039056004607118666,
0.000369473040336743,
0.00007876444578869268
] |
en
| 0.999996
|
The structural probing led to three 2D models in which resemblances are found. In the three cases, the stem–loop dedicated to the SLBP binding (domain I) is clearly present, followed by a single stranded region carrying the cleavage site. Remarkably, nucleotides from the three HDE are embedded in duplex structures, which is unexpected for a sequence dedicated to U7 snRNA hybridization. Duplexes harbouring the HDE nucleotides are formed with proximal sequences, as in stem III of H4-12 pre-mRNAs and by long-distance interactions with sequences located upstream of the SLBP hairpin-binding site (as in stem IV of the three pre-mRNAs).
|
16982637_p19
|
16982637
|
DISCUSSION
| 4.379219
|
biomedical
|
Study
|
[
0.9993997812271118,
0.0003380390116944909,
0.0002621744351927191
] |
[
0.9989670515060425,
0.00048028628225438297,
0.0004529511497821659,
0.00009968494123313576
] |
en
| 0.999998
|
Footprint experiments revealed that significant probe-reactivity changes occur in H4-12 and H1t 5′-UTR on SLBP binding. First, RNA protections were unambiguously detected in domain I and in adjacent domain II of the three 3′-UTR. In fact, the SLBP interacts mainly with the 5′ half of the hairpin I and with the upstream region of domain II. This is in good agreement with previous experiments showing that the 3′ hExo can bind to the already formed SLBP–hairpin complex and form a ternary complex ( 28 ). This implies that the 3′ hExo can start trimming before or even without the release of the SLBP. More surprising was the protection induced by the SLBP on the upstream domain II. It was already known that the four residues preceding stem I are essential for SLBP binding ( 29 , 30 ). We show here that SLBP can protect more residues, from 15 residues for H4-12 to only 6 for H2a-614. This variation also suggests some plasticity in the RNA-protein binding mode, according to the sequence diversity observed in the pre-mRNAs upstream of the canonical stem–loop I.
|
16982637_p20
|
16982637
|
DISCUSSION
| 4.386299
|
biomedical
|
Study
|
[
0.9992961883544922,
0.0003992300189565867,
0.0003046217607334256
] |
[
0.9992132186889648,
0.0002578810090199113,
0.00043563058716244996,
0.00009323386620962992
] |
en
| 0.999997
|
In addition to RNA protection, the binding of the SLBP- induces increased reactivity that can be interpreted as a progressive opening of the duplexes containing H4-12 and H1t HDE. The reactivity changes in H4-12 affect stem–loop III, which becomes more accessible to double-strand specific probes and to lead. The adjacent stem IV is characterized by new lead cuts which suggests melting of the stem. In the case of H1t, reactivity changes are less pronounced but a clear increase of the accessibility of stem IV containing the HDE is observed. Stem IV becomes reactive to single-strand specific probes, suggesting melting of the upper part of the stem, whereas the lower part becomes more accessible to V1 cuts. Thus, in both cases, the reactivity changes observed on SLBP binding reveal significant structural changes and dynamic rearrangements. One obvious consequence of the HDE melting would be to favour U7 snRNA hybridization and we showed that these rearrangements actually facilitate the anchoring of the U7 snRNA in vitro . However, improvement of the U7 snRNA binding is only observed with H4-12 and not with H1t although it displays also the HDE melting. On the other hand, the H2a-614 3′-UTR is much more static and shows no significant conformation change on SLBP binding. These results are in good agreement with previous investigations that showed that the 3′ end processing of H1t is more dependent than H2a on SLBP ( 16 ). Altogether, the results observed with the three different UTRs suggest that distinct mechanisms govern the first step of the processing reaction. However, we cannot exclude that the presence of other trans -acting factors like ZFP100 or Sm/Lsm proteins in U7 snRNP might improve the efficiency of U7 hybridization with H1t and H2a-614 and reach a level comparable to that of H4-12.
|
16982637_p21
|
16982637
|
DISCUSSION
| 4.618997
|
biomedical
|
Study
|
[
0.9989564418792725,
0.0006059089209884405,
0.00043764914153143764
] |
[
0.9985997080802917,
0.0004549407458398491,
0.0007686041644774377,
0.0001766839559422806
] |
en
| 0.999998
|