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Quantitating Oligonucleotide Affinities for Duplex DNA: Footprinting vs Electrophoretic Mobility Shift Assays
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
244, 312–320 (1997)
AB969901
Quantitating Oligonucleotide Affinities for Duplex DNA: Footprinting vs Electrophoretic Mobility Shift Assays Matthew J. Ferber and L. James Maher, III1 Department of Biochemistry and Molecular Biology, Mayo Foundation, 200 First Street, SW, Rochester, Minnesota 55902
Received July 17, 1996
Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. Quantitative DNaseI footprinting assays (QDFA) offer a rigorous technique for this purpose. Electrophoretic mobility shift assays (EMSA) have proven to be simpler and more rapid. Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by QDFA. We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods. q 1997 Academic Press
Oligonucleotide-directed triple helix formation provides a strategy for designing potential artificial gene repressors (1–5). In vitro experiments suggest that triplexes can block DNA binding proteins (6, 7) and inhibit transcription initiation (2, 8–12). Recognition of homopurine/homopyrimidine sequences in duplex DNA involves hydrogen bonds between oligonucleotide 1 To whom correspondence should be addressed. Fax: 507-2842053. E-mail: [email protected].
bases and purine bases in the major groove. Triple helix formation occurs in two distinct patterns, termed the pyrimidine motif and purine motif (4, 5). In the pyrimidine motif, oligonucleotides bind parallel to the purine strand of the duplex by Hoogsteen hydrogen bonding to form TrArT and C/rGrC base triplets (1). In the purine motif, oligonucleotides bind antiparallel to the purine strand of the duplex by reverse Hoogsteen hydrogen bonding to form ArArT (or TrArT) and GrGrC base triplets (13). We are investigating triple helix formation as a possible approach to artificially regulate the expression of disease-related genes. Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. It is reasonable to assume that maximizing triplex stability will be important for practical applications. Equilibrium dissociation constants (Kd values) for triple-helical complexes have been estimated using several techniques. The most rigorous approaches involve quantitative affinity cleavage (14–18) or quantitative DNaseI footprinting assays (QDFA)2 (19). These procedures involve the preparation of an equilibrium mixture containing a trace amount of radiolabeled target DNA duplex and known concentrations of unlabeled oligonucleotides for triplex formation. In affinity cleavage assays, the oligonucleotide ligands are covalently modified to carry cleaving functions [e.g., EDTArFe(II)] such that a fraction of oligonucleotide binding events results in scission of the target duplex backbone. Cleavage is detected by the appearance of labeled DNA fragments after electrophoresis. In QDFA, triplex formation is detected by 2 Abbreviations used: DMS, dimethyl sulfate; DNaseI, deoxyribonuclease I; EMSA, electrophoretic mobility shift assay(s); Mops, 3[N-morpholino]propanesulfonic acid; QDFA, quantitative DNaseI footprinting assay(s); TBE, Tris–borate EDTA buffer; tRNA, transfer ribonucleic acid.
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0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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sequence-specific protection of the labeled DNA duplex from DNaseI cleavage. Though both of these analytical techniques can be made thermodynamically rigorous, each is relatively laborious. Both procedures require preparation of homogeneous DNA probes (typically hundreds of base pairs in length) with a single radiolabeled terminus. In addition, appropriate sequence standards must be created to allow interpretation of the results, and sequencing gels are typically required. Similar procedures are required for other footprinting agents such as DMS. Quantitative affinity cleavage assays further require the preparation of modified versions of the oligonucleotides of interest. Data analysis requires careful normalization of radioactive signals to internal standards. Simpler assays have been proposed for monitoring and quantitating triplex formation. Oligonucleotide binding can confer protection from DNA modification by restriction endonucleases and methylases that recognize sites that overlap the triplex (6). This observation provided an assay for thermodynamic and kinetic experiments with limited time resolution (7). Others have profitably exploited the differential affinity of single strands vs triplexes for nitrocellulose in filter binding assays (20), although the physical basis for this separation is not understood. Among the simplest and most convenient assays for triplex formation is the electrophoretic mobility shift assay (EMSA; 21), originally applied to triplex formation as a quantitative analytical tool by Hogan and coworkers (2) and subsequently applied to a variety of problems in triplex characterization (22–31). In this procedure, a trace amount of radiolabeled synthetic DNA duplex containing the target site of interest is incubated with known concentrations of oligonucleotides. Complexes are then separated by electrophoresis through a native polyacrylamide gel containing stabilizing cations such as Mg 2/. It is observed that triplehelical complexes migrate more slowly than the corresponding duplex. The mobility difference depends, in part, on the fraction of the length of the target duplex that is occupied by the third oligonucleotide strand at saturation. It should be noted that the physical basis for this electrophoretic retardation is unclear, as the formal charge/mass ratio of the triplex should be the same as for the duplex. Possible explanations involve the shape and flexibility of the resulting complexes and/ or the expectation that the high charge density of the triplex should enhance counterion condensation such that the fractional thermodynamic charge per phosphate is reduced (32). Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by footprinting methods such as QDFA.
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Whereas a footprinting reaction is terminated minutes after addition of the cleaving agent (providing a ‘‘snapshot’’ of the equilibrium mixture), EMSA continues the binding experiment from the initiation of the binding reaction to the completion of gel electrophoresis. During this time (which may be many hours), the samples experience a series of environments that differ from the conditions in the initial binding reaction. To apply EMSA as an alternative to more complex assays such as QDFA, one would like to assume that the electrophoresis portion of EMSA simply reports the extent of triplex formation in the sample that was initially loaded into the gel. To what extent is this true? In EMSA experiments with proteins and other ligands, complexes appear to persist during electrophoresis much longer than predicted by their anticipated lifetimes in free solution. This effect has been attributed to both caging and sequestration in the gel matrix. Caging maintains high local concentrations of dissociation products so that collisions between the dissociated partners are favored. Sequestration isolates each complex from interaction with other molecules, reducing exchange reactions catalyzed by collisions with other complexes or reagents (33, 34). Thus, by isolating and insulating complexes from one another, EMSA has the potential to preserve the distribution of labeled molecules between bound and free forms throughout the electrophoresis experiment. Because the EMSA technique has proven to be rapid and simple, we wished to compare Kd estimates from EMSA with estimates obtained in parallel using a more rigorous QDFA procedure. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods. REAGENTS
Buffers. Reactions included either buffer A (20 mM Mops, pH 7.1, 100 mM KCl, 5 mM MgCl2 , 1 mM spermine) or buffer B (buffer A lacking KCl). Plasmid DNA. Plasmid pJ010 contains Ç500 bp of the human FasL gene, extending upstream from the translation initiation codon into the promoter region. The fragment is cloned into pCR-Script SK(/) (Stratagene). Supercoiled plasmid DNA was isolated from bacteria using the alkaline lysis method and purified using Qiagen columns as directed by the manufacturer (Qiagen). Oligonucleotides and analogs. Oligonucleotide sequences are shown in Fig. 1A. Synthetic oligonucleotides and oligonucleotide analogs were synthesized on ABI synthesizers using phosphoramidite reagents. Protected 5-methylcytosine phosphoramidite derivatives (2*-deoxy or 2*-O-methyl), protected thymidine phos-
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phoramidite derivatives (2*-deoxy or 2*-O-methyl), other 2*-O-methyl ribonucleoside phosphoramidites, and acridine phosphoramidite (hexamethylene tether) were obtained from Glen Research. Oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis, eluted from gel slices, and desalted by SepPak C18 cartridge chromatography (Waters). Oligonucleotides were quantitated by absorbance at 260 nm using molar extinction coefficients (M01 cm01) of 15,400 (dA), 11,700 (dG), 7300 (dC), 5700 (dMe5C), and 8800 (dT), assuming no hypochromicity. Enzymes and reagents. Restriction endonucleases and the Klenow fragment of DNA polymerase I were obtained from New England Biolabs. RQ1 DNaseI was purchased from Promega. Spermine tetrahydrochloride was obtained from Sigma. Formic acid and piperidine were purchased from Aldrich. METHODS
QDFA. A 350-bp StuI–EcoRI fragment was isolated from plasmid pJ010 (Fig. 1A) by native polyacrylamide gel electrophoresis, extracted from the gel slice by incubation overnight at 377C in extraction buffer (0.375 M ammonium acetate, 1 mM EDTA, 0.1% SDS), and purified by phenol/chloroform extraction and sequential precipitations from ethanol and spermine (35). The top strand of this fragment was uniquely labeled at the EcoRI site by end filling with the Klenow fragment of DNA polymerase I and [a-32P]dATP in the presence of 0.1 mM dTTP. The resulting labeled restriction fragment was purified by precipitation with spermine. To provide sequence markers, an A / G ladder was created by treatment of a sample of labeled restriction fragment with formic acid, followed by precipitation and cleavage with piperidine (36). Triplex binding reactions (25 mL) contained Ç4 1 104 cpm (Ç0.2 pmol) labeled FasL restriction fragment together with the indicated concentration of oligonucleotide. Reactions included either buffer A or buffer B. After incubation at 227C for at least 3 h, reactions were supplemented with 2.5 mL CaCl2 solution (100 mM) and treated with 0.3 units RQ1 DNase at 227C for 2 min. Reactions were terminated by the addition of 50 mL of a solution containing 0.2 M NaCl, 30 mM EDTA, 1% sodium sarcosinate, and 100 mg/mL yeast tRNA. Samples were precipitated from ethanol, resuspended in a denaturing buffer containing formamide and tracking dyes, heated to 907C for 4 min, cooled on ice, and loaded onto 5% denaturing polyacrylamide sequencing gels (19:1 acrylamide:bisacrylamide) prepared and electrophoresed in 0.51 TBE buffer (36). The resulting gels were dried and imaged. EMSA. Oligonucleotides comprising the target duplex were annealed as follows: 500 pmol each of oligo-
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nucleotides 5 and 6 was mixed with 2 mL of 5 M NaCl and brought to a total volume of 42 mL with H2O. This annealing reaction mixture was incubated at 757C for 12 min and then gradually cooled to 257C. Thirty picomoles of the resulting oligonucleotide duplexes was radiolabeled using the Klenow fragment of DNA polymerase I and [a-32P]dATP in the presence of 0.1 mM each dGTP, dTTP, and dCTP. The resulting labeled duplex oligonucleotides were purified by Sephadex G-50 spin column chromatography. Triplex binding reactions (10 mL) contained 1 mg yeast tRNA and Ç6 1 104 cpm (Ç0.03 pmol) labeled FasL duplex 5 / 6 (Fig. 1B) together with the indicated concentration of oligonucleotide. Reactions included either buffer A or buffer B. After incubation at 227C for at least 3 h, 1 mL 80% (v/v) glycerol was added to each 10-mL reaction, and samples were loaded onto a 20% native polyacrylamide gel (19:1 acrylamide:bisacrylamide) prepared and electrophoresed in a buffer containing 50 mM Mops, pH 7, 5 mM MgCl2 . Electrophoresis was performed with recirculation at 47C for 15 h at 12 V/cm. Image analysis. Electrophoretic gels were imaged by storage phosphor technology using a Molecular Dynamics Storm 840 phosphorimager under the control of an Apple Macintosh 7100/80 microcomputer. Images were analyzed using ImageQuant software (Molecular Dynamics). Data analysis and estimation of Kd . To quantitate oligonucleotide binding to the target site in the FasL promoter using QDFA, radioactive signal was measured for two regions of each gel lane. One region (corresponding to box x in lane 16 of Fig. 2A) represents the extent of DNaseI cleavage within the promoter site targeted for triplex formation. A second region (corresponding to box y in lane 16 of Fig. 2A) represents the extent of DNaseI cleavage within an irrelevant sequence not targeted for triplex formation. The amount of DNaseI cleavage within the target sequence was first normalized using the signal in the nontargeted region of each gel lane: Snorm Å Sx /Sy ,
[1]
where Snorm is the normalized radioactive signal at the triplex target sequence of each lane and Sx and Sy are the radioactive signals in boxes x and y of each lane (see example in lane 16 of Fig. 2A). The footprinting data were then scaled relative to the maximum value of Snorm (obtained in the absence of added oligonucleotide) and the minimum value of Snorm (obtained for saturating amounts of added oligonucleotide) using the relation
u Å 1 0 [(Snorm 0 Snorm, sat)/(Snorm, 0 0 Snorm, sat), [2]
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where u is the fractional saturation of the DNA target site, Snorm, sat is the normalized radioactive signal within the triplex target site at saturating levels of oligonucleotide, and Snorm, 0 is the normalized radioactive signal within the triplex target site in the absence of added oligonucleotide. Values of the apparent triplex dissociation constant, Kd, QDFA , were then obtained by least-squares fitting of the experimental data to the binding isotherm
u Å ([O]n/K dn)/(1 / [O]n/K dn),
[3]
where [O] is the oligonucleotide concentration, and n is the Hill coefficient (37). Curve fitting was performed using Kaleidagraph (Abelbeck Software). To quantitate oligonucleotide binding to the target site in the FasL promoter using EMSA, radioactive signal was measured for two regions of each gel lane. One region (corresponding to box T in lane 8 of Fig. 3A) represents the radioactive signal due to triple-helical complex formation. A second region (corresponding to box D in lane 8 of Fig. 3A) represents the radioactive signal associated with free duplex probe 5 / 6. The fractional saturation, u, of the DNA duplex in each gel lane is then given by
u Å Striplex/(Striplex / Sduplex),
[4]
where Striplex and Sduplex are the radioactive signals associated with the triplex and duplex complexes, respectively (see example in lane 8 of Fig. 3A). Values of the apparent triplex dissociation constant, Kd, EMSA , were then obtained by least-squares fitting of the experimental data using Eq. [3]. RESULTS AND DISCUSSION
Model system. To compare QDFA and EMSA methods for estimating Kd values for triple-helical complexes, we selected a previously unexplored model system involving DNA sequences from the promoter of the human Fas ligand gene, FasL. The FasL promoter contains a consensus TATA box (Fig. 1A). Located 31 bp upstream from the TATA box is a 29-bp homopurine/ homopyrimidine sequence containing a single TrA interruption (Fig. 1A). This sequence is not highly conserved between human and mouse (38) and is of unknown regulatory significance. We are studying whether oligonucleotide binding to this sequence will alter expression of the FasL gene. In anticipation of these studies, we synthesized oligonucleotides 1–4 (Fig. 1A) to compare their binding affinities for the FasL target. These oligonucleotides contain several modifications intended to enhance their affinities for duplex DNA. All oligonucleotides contain 5-methylcy-
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FIG. 1. Experimental system. (A) Duplex DNA target used in QDFA. A 350-bp StuI–EcoRI restriction fragment containing a segment of the human FasL gene contains a nearly perfect homopurine/ homopyrimidine sequence (indicated by R and Y and boxed duplex sequence below), upstream of the TATA box and translation start codon (ATG). Synthetic oligonucleotides 1–4 were designed to bind to the homopurine target sequence in the pyrimidine triple helix motif. 5-Methylcytosine residues are indicated by italics. 2-O-Methyl backbones are indicated by underlining. An intercalating acridine residue is indicated by acr. (B) Synthetic duplex DNA target used for EMSA. Homopurine/homopyrimidine target sequence is boxed. The duplex was made blunt-ended during the labeling procedure.
tosine substituted for cytosine (39). Oligonucleotides 1, 3, and 4 were synthesized with 2*-O-methylated RNA backbones with the goal of greater triplex stability (40). The single TrA interruption in the FasL homopurine target sequence is accommodated by a G residue in oligonucleotide 1, with the goal of forming a GrTA base triplet at this position (41). Oligonucleotide 4 contains a 5*-acridine modification, intended to stabilize triplex formation by intercalation (3). For QDFA, a 350-bp FasL promoter fragment was isolated and labeled on the purine-rich strand, 3* to the homopurine element (Fig. 1A). This labeled DNA duplex was incubated with various concentrations of oligonucleotides 1–4 in buffer A or buffer B (buffer A lacking KCl), treated with DNaseI, and electrophoresed on sequencing gels as described under Methods. For EMSA, synthetic DNA duplex 5 / 6 (Fig 1B) was radiolabeled, incubated with various concentrations of oligonucleotides 1–4 in buffer A or buffer B, and loaded onto native polyacrylamide gels in the presence of Mg 2/ as described under Methods. QDFA data. An example of a footprinting result in buffer B is shown in Fig. 2A. Lane 1 displays an A
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FIG. 2. Example of results of QDFA using a 350-bp fragment of the human FasL gene. (A) Gel result. This example depicts oligonucleotide binding in buffer B. Lane 1 contains an A / G sequence ladder. The sample in lane 2 was not exposed to DNaseI. Samples in lanes 3–23 were exposed to DNaseI after incubation with 0.012, 0.06, 0.3, 1.5, or 7.5 mM concentrations of oligonucleotides 1–4. Bracket at left indicates position of homopurine/homopyrimidine target sequence. Boxes x and y in lane 16 exemplify regions quantitated in each gel lane to calculate normalized radioactive signal within the triplex target site, as described under Methods. (B) Estimation of Kd, QDFA . Analysis of QDFA data was performed as described under Methods.
/ G sequence ladder that identifies the triplex target sequence in the FasL promoter fragment. DNaseI was added to samples in lanes 3–23. No oligonucleotide was added to the samples in lanes 1–3. Increasing concentrations of the indicated oligonucleotides were added to samples in the remaining gel lanes. Footprints for oligonucleotide 1 (Fig. 2A, lanes 4–8) involve the entire homopurine target sequence, while complexes with the shorter oligonucleotides 2–4 protect only the 3* half of the site, as expected (Fig. 2A, lanes 9–23). Careful examination of the footprints also reveals that the triplex formation is accompanied by enhanced DNaseI cleavage at the 3* end of the homopurine target sequence. In all cases, the highest oligonucleotide concentration tested (7.5 mM) is sufficient to saturate the homopurine target site. The electrophoretic data from multiple QDFA experiments of this type were quantitated as described under Methods. As an example, the data from the gel in Fig. 2A are shown in Fig. 2B. Leastsquares curve fitting suggests similar Kd, QDFA values in the 1007 M range, with some variation in the optimum value of the Hill coefficient. The entire Kd, QDFA data set is included in the fifth column of Table 1. The QDFA data may be summarized as follows. Binding affinities for oligonucleotides 1–4 are between 60 and 300 nM for experiments performed in buffer B.
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Oligonucleotide 4 displays the highest affinity under these conditions. In the presence of physiological concentrations of KCl (buffer A), triplex formation is inhibited by 7- to 43-fold, consistent with previous observations (7). Binding affinities in buffer A range between 0.39 and 5.2 mM, with oligonucleotide 4 again forming the tightest complex. For both buffers A and B, the range of estimated Kd, QDFA values for oligonucleotides 1–4 spans less than one order of magnitude. EMSA data. An example of an electrophoretic gel mobility shift result in buffer A is shown in Fig. 3A. No oligonucleotide was added to the sample in lane 1. Increasing concentrations of the indicated oligonucleotides were added to samples in the remaining gel lanes. Triple-helical complexes for oligonucleotide 1 (Fig. 3A, lanes 2–5) have migrated more slowly than complexes with the shorter oligonucleotides 2–4 (Fig. 3A, lanes 6–17). In buffer A, the highest oligonucleotide concentration tested (7.5 mM) is sufficient to saturate the homopurine target site for oligonucleotides 2–4 (Fig. 3A, lanes 9, 13, and 17) and nearly saturates in the case of oligonucleotide 1 (Fig. 3A, lane 5). The electrophoretic data from multiple EMSA experiments of this type were quantitated as described under Methods. As an example, the data from the gel in Fig. 3A are shown in Fig. 3B. As for the QDFA example in Fig. 2, least-
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Kd Values for Triple-Helical Complexes Oligomer
Buffer
1 2 3 4 1 2 3 4
A A A A B B B B
Kd,EMSA (mM) 1.2 0.18 0.29 0.10 0.22 0.025 0.097 0.061
{ { { { { { { {
0.16 0.018 0.050 0.009 0.090 0.010 0.050 0.024
n
Kd,QDFA (mM)
4 4 2 2 4 3 2 2
5.2 0.82 3.3 0.390 0.12 0.096 0.30 0.060
{ { { { { { { {
0.90 0.049 0.10 0.16 0.080 0.017 0.26 0.022
n 4 4 2 2 2 2 2 2
Kd,QDFA/Kd,EMSA 4.3 4.3 11.4 3.9 0.6 3.8 3.1 1.0
{ { { { { { { {
0.95 0.48 2.0 0.39 0.43 1.7 3.1 0.53
Note. Buffer compositions are given under Materials and Methods. Data are presented as Kd { standard deviation from the indicated number of experimental replications (n). Standard deviations for the final data column were calculated by conventional error propogation methods (42).
squares curve fitting of the EMSA data in Fig. 3A suggests similar Kd, EMSA values. The entire Kd, EMSA data set is also included in the third column of Table 1. The EMSA data may be summarized as follows. Binding affinities for oligonucleotides 1–4 are between 0.1 and 1.2 mM for experiments performed in buffer A (containing 100 mM KCl). As observed in QDFA, oligonucleotide 4 displays the highest affinity under these conditions. In the absence of KCl (buffer B), triplex formation is enhanced by 1.6- to 7.6-fold. Binding affinities in buffer B range between 25 and 220 nM, with oligonucleotide 2 forming the tightest complex. As observed for the QDFA data, the range of estimated Kd, EMSA values for oligonucleotides 1–4 spans approximately one order of magnitude in both buffers A and
B. As previously observed by QDFA, it is noteworthy that the 2*-O-methylated RNA backbone of oligonucleotide 3 did not stabilize triplexes. Complexes involving oligonucleotide 3 were two- to threefold less stable than those involving DNA oligonucleotide 2 (Table 1). This unexpected result suggests that triplex stabilization by RNA-like backbones may be sequence-dependent. Data comparison. The main goal of this work was to compare Kd estimates obtained by QDFA and EMSA. This comparison is presented in the last column of Table 1 and in Fig. 4. The data demonstrate that QDFA and EMSA techniques consistently provide Kd estimates that differ by less than one order of magnitude (final column of Table 1). In addition, the degree of agreement between techniques depends on buffer con-
FIG. 3. Example of results of EMSA using a radiolabeled 42-bp duplex carrying the homopurine/homopyrimidine target sequence. (A) Gel result. This example depicts oligonucleotide binding in buffer A. Lane 1 contained no added binding oligomer. Samples in lanes 2–17 were electrophoresed after incubation with 0.012, 0.06, 0.3, or 1.5 mM concentrations of oligonucleotides 1–4. Mobilities of duplex (D) and triplex (T) species are indicated at left. Rectangles T and D in lane 8 exemplify regions quantitated in each gel lane to calculate the fractional saturation of the radiolabeled duplex DNA probe by added oligonucleotide, as described under Methods. (B) Estimation of Kd, EMSA . Analysis of EMSA data was performed as described under Methods.
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FIG. 4. Comparison of Kd, QDFA and Kd, EMSA data. Binding data from QDFA (stippled bars) and EMSA (open bars) as a function of oligonucleotide (indicated below chart) and binding buffer condition (indicated above chart). Error bars indicate standard deviations.
ditions. Kd estimates differ by 4- to 11-fold in Buffer A (containing 100 mM KCl), but are identical (within experimental error) for oligonucleotides 1, 3, and 4 in buffer B (Fig. 4). The overall conclusion is that Kd, EMSA estimates are in reasonable agreement with Kd, QDFA . Despite the reasonable agreement between Kd values obtained from QDFA and EMSA, two questions were raised by these data. First, why is the agreement between Kd estimates closer for experiments in buffer B? Second, why might EMSA underestimate Kd values (i.e., overestimate oligonucleotide affinities) relative to QDFA in buffer A? We hypothesize that answers to these questions are related to the fact that QDFA involves constant reaction buffer conditions throughout the experiment, while EMSA samples experience a buffer change between the binding and electrophoresis phases of the experiment. In particular, the EMSA gel buffer (50 mM Mops, pH 7, 5 mM MgCl2) is very similar to binding buffer B (20 mM Mops, pH 7.1, 5 mM MgCl2 , 1 mM spermine), lacking only the stabilizing polyamine spermine. In contrast, binding buffer A includes a physiological concentration of KCl (100 mM). Added monovalent cations have previously been shown to inhibit triplex formation in the presence of Mg 2/ and spermine4/, presumably by competing for participation in the population of condensed cations on DNA (7, 16). Because phosphate neutralization is less effective for monovalent cations (relative to Mg 2/ and spermine4/), added KCl shifts the equilibrium away from triplex formation. During the early stages of electrophoresis in EMSA, the reaction sample (containing 100 mM KCl in the case of buffer A) is surrounded by buffer lacking KCl. Diffusion of KCl into the gel buffer presumably causes a gradual depletion of this inhibitor as the sample enters the gel. This creates an opportunity for the
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triplex equilibrium to shift toward increased complex formation. We reasoned that if overestimation of triplex affinity for EMSA in the case of buffer A were due to depletion of inhibitory KCl early in the experiment, the effect might be resolved by a ‘‘quench’’ step prior to electrophoresis. To provide such a step, we assembled and incubated standard reactions involving oligonucleotide 2 binding to labeled duplex 5 / 6. However, reactions were supplemented with an excess (2 mM) of unlabeled duplex DNA target 5 / 6 just prior to loading the electrophoresis gel for EMSA. This excess duplex target was intended to act as a sink, driving all free oligonucleotide 2 into triplexes involving unlabeled duplex 5 / 6. In this way, if KCl depletion in the gel well enhanced the potential for triplex formation, no free oligonucleotide 2 would remain to bind labeled duplex 5 / 6. This quench procedure should be effective for all but the highest oligonucleotide concentrations studied (7.5 mM). In the latter case, the quench becomes irrelevant, as the labeled duplex is already fully converted to triplex form. The results of an EMSA experiment involving quenching are shown in Fig. 5. Samples in lanes 1–6 of Fig. 5A were assembled in buffer A and subjected to EMSA according to the standard procedure. Samples in lanes 7–12 were supplemented with 2 mM unlabeled duplex 5 / 6 just prior to electrophoresis. Careful examination of the data in Figs. 5A and 5B demonstrates an increase in the apparent Kd, EMSA (i.e., a decrease in apparent affinity) when the quench protocol was implemented. This quench effect was reproducible, causing an approximately twofold decrease in apparent affinity. Because the apparent affinities of oligonucleotides for their target in buffer A was approximately fourfold higher in EMSA than QDFA, we interpret Fig. 5 as evidence that about half of this effect can be attributed to changing buffer conditions during early stages of the EMSA experiment. After accounting for possible effects of KCl diffusion, the apparent Kd estimates for EMSA and QDFA are within approximately twofold under both experimental conditions, suggesting that EMSA can serve as a satisfactory substitute for the more rigorous QDFA procedure. It should be noted that the remaining differences in apparent Kd values might be attributed to other differences between the two assays. For example, QDFA involves supplemental 9 mM Ca2/ during the 2-min DNaseI reaction, and EMSA involves addition of glycerol to Ç7% prior to electrophoresis. In addition, the short duplex DNA target may be intrinsically more flexible than the 350-bp restriction fragment in its ability to accommodate triplex formation. In spite of these potentially important differences, binding data from these two assays are comparable.
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FIG. 5. Effect of quenching EMSA with excess cold competitor duplex prior to electrophoresis. (A) Gel result. Samples in lanes 1 and 7 contained no added binding oligomer. Lanes 2–6 and 8–12 contained 0.012, 0.06, 0.3, 1.5, or 7.5 mM concentrations of oligonucleotide 2. For lanes 7–12, excess unlabeled target duplex 5 / 6 (2 mM) was added to the samples just prior to electrophoresis. (B) Analysis of EMSA data. Binding data without quench (s). Binding data with quench by excess cold competitor duplex (j). The apparent value of Kd, EMSA was increased approximately twofold by quenching.
CONCLUSIONS
We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading.
8. Orson, F. M., Thomas, D. W., McShan, W. M., Kessler, D. J., and Hogan, M. E. (1991) Nucleic Acids Res. 19, 3435–3441. 9. Young, S. L., Krawczyk, S. H., Matteucci, M. D., and Toole, J. J. (1991) Proc. Natl. Acad. Sci. USA 88, 10023–10026. 10. Maher, L. J., Dervan, P. B., and Wold, B. (1992) Biochemistry 31, 70–81. 11. Maher, L. J. (1992) Biochemistry 31, 7587–7594. 12. Duval-Valentin, G., Thuong, N. T., and He´le`ne, C. (1992) Proc. Natl. Acad. Sci. USA 89, 504–508. 13. Beal, P. A., and Dervan, P. B. (1991) Science 251, 1360–1363. 14. Singleton, S. F., and Dervan, P. B. (1992) Biochemistry 31, 10995–11003. 15. Stilz, H. U., and Dervan, P. B. (1993) Biochemistry 32, 2177– 2185.
ACKNOWLEDGMENTS
16. Singleton, S. F., and Dervan, P. B. (1993) Biochemistry 32, 13171–13179.
We thank M. Fried for helpful discussions, C. Paya and C. HoltzHeppelmann for providing cloned DNA from the human FasL gene, and G. Soukup for comments on the manuscript. This work was supported by the Mayo Foundation and by NIH Grants GM47814 and GM54411, an American Cancer Society Junior Faculty Research Award, and the Harold W. Siebens Research Scholarship (L.J.M.).
17. Singleton, S. F., and Dervan, P. B. (1994) J. Am. Chem. Soc. 116, 10376–10382.
1. Moser, H. E., and Dervan, P. B. (1987) Science 238, 645–650. 2. Cooney, M., Czernuszewicz, G., Postel, E. H., Flint, S. J., and Hogan, M. E. (1988) Science 241, 456–459. 3. He´le`ne, C. (1991) Anti-Cancer Drug Design 6, 569–584. 4. Maher, L. J. (1992) BioEssays 14, 807–815. 5. Maher, L. J. (1996) Cancer Invest 14, 66–82. 6. Maher, L. J., Wold, B., and Dervan, P. B. (1989) Science 245, 725–730. 7. Maher, L. J., Dervan, P. B., and Wold, B. J. (1990) Biochemistry 29, 8820–8826.
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19. Priestley, E. S., and Dervan, P. B. (1995) J. Am. Chem. Soc. 117, 4761–4765. 20. Shindo, H., Torigoe, H., and Sarai, A. (1993) Biochemistry 32, 8963–8969.
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21. Fried, M. G., and Crothers, D. M. (1981) Nucleic Acids Res. 9, 6505–6525. 22. Lyamichev, V. I., Mirkin, S. M., Frank-Kamenetskii, M. D., and Cantor, C. R. (1988) Nucleic Acids Res. 16, 2165–2178. 23. Durland, R. H., Kessler, D. J., Gunnell, S., Duvic, M., Pettitt, B. M., and Hogan, M. E. (1991) Biochemistry 30, 9246–9255. 24. Roberts, R. W., and Crothers, D. M. (1992) Science 258, 1463– 1466. 25. Olivas, W. M., and Maher, L. J. (1994) Biochemistry 33, 983– 991.
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26. Semerad, C. L., and Maher, L. J. (1994) Nucleic Acids Res. 22, 5321–5325. 27. McDonald, C. D., and Maher, L. J. (1995) Nucleic Acids Res. 23, 500–506. 28. Olivas, W. M., and Maher, L. J. (1995) Biochemistry 34, 278– 284. 29. Olivas, W. M., and Maher, L. J. (1995) Nucleic Acids Res. 23, 1936–1941. 30. Olivas, W. M., and Maher, L. J. (1996) Nucleic Acids Res. 24, 1758–1764. 31. Soukup, G. A., Ellington, A. D., and Maher, L. J. (1996) J. Mol. Biol. 259, 216–228. 32. Manning, G. S. (1978) Q. Rev. Biophys. 2, 179–246. 33. Fried, M. G., and Crothers, D. M. (1984) J. Mol. Biol. 172, 241– 262. 34. Fried, M. G., and Liu, G. (1994) Nucleic Acids Res. 22, 5054– 5059.
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35. Hoopes, B., and McClure, W. (1981) Nucleic Acids Res. 9, 5493– 5504. 36. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 37. Cantor, C. R., and Schimmel, P. R. (1980) Biophysical Chemistry, p. 864, Freeman, San Francisco. 38. Takahashi, T., Tanaka, M., Inazawa, J., Abe, T., Suda, T., and Nagata, S. (1994) Int. Immunol. 6, 1567–1574. 39. Povsic, T. J., and Dervan, P. B. (1989) J. Am. Chem. Soc. 111, 3059–3061. 40. Shimizu, M., Konishi, A., Shimada, Y., Inoue, H., and Ohtsuka, E. (1992) FEBS Lett. 302, 155–158. 41. Griffin, L. C., and Dervan, P. B. (1989) Science 245, 967–971. 42. Skoog, D. A., and West, D. M. (1979) Analytical Chemistry, 3rd ed., pp. 74–77, Holt, Rinehart, and Winston, New York.
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AB969901
Quantitating Oligonucleotide Affinities for Duplex DNA: Footprinting vs Electrophoretic Mobility Shift Assays Matthew J. Ferber and L. James Maher, III1 Department of Biochemistry and Molecular Biology, Mayo Foundation, 200 First Street, SW, Rochester, Minnesota 55902
Received July 17, 1996
Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. Quantitative DNaseI footprinting assays (QDFA) offer a rigorous technique for this purpose. Electrophoretic mobility shift assays (EMSA) have proven to be simpler and more rapid. Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by QDFA. We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods. q 1997 Academic Press
Oligonucleotide-directed triple helix formation provides a strategy for designing potential artificial gene repressors (1–5). In vitro experiments suggest that triplexes can block DNA binding proteins (6, 7) and inhibit transcription initiation (2, 8–12). Recognition of homopurine/homopyrimidine sequences in duplex DNA involves hydrogen bonds between oligonucleotide 1 To whom correspondence should be addressed. Fax: 507-2842053. E-mail: [email protected].
bases and purine bases in the major groove. Triple helix formation occurs in two distinct patterns, termed the pyrimidine motif and purine motif (4, 5). In the pyrimidine motif, oligonucleotides bind parallel to the purine strand of the duplex by Hoogsteen hydrogen bonding to form TrArT and C/rGrC base triplets (1). In the purine motif, oligonucleotides bind antiparallel to the purine strand of the duplex by reverse Hoogsteen hydrogen bonding to form ArArT (or TrArT) and GrGrC base triplets (13). We are investigating triple helix formation as a possible approach to artificially regulate the expression of disease-related genes. Determining the affinities of oligonucleotides for duplex DNA is an important analytical problem that arises during the design of potential gene repressors based on triple helix recognition. It is reasonable to assume that maximizing triplex stability will be important for practical applications. Equilibrium dissociation constants (Kd values) for triple-helical complexes have been estimated using several techniques. The most rigorous approaches involve quantitative affinity cleavage (14–18) or quantitative DNaseI footprinting assays (QDFA)2 (19). These procedures involve the preparation of an equilibrium mixture containing a trace amount of radiolabeled target DNA duplex and known concentrations of unlabeled oligonucleotides for triplex formation. In affinity cleavage assays, the oligonucleotide ligands are covalently modified to carry cleaving functions [e.g., EDTArFe(II)] such that a fraction of oligonucleotide binding events results in scission of the target duplex backbone. Cleavage is detected by the appearance of labeled DNA fragments after electrophoresis. In QDFA, triplex formation is detected by 2 Abbreviations used: DMS, dimethyl sulfate; DNaseI, deoxyribonuclease I; EMSA, electrophoretic mobility shift assay(s); Mops, 3[N-morpholino]propanesulfonic acid; QDFA, quantitative DNaseI footprinting assay(s); TBE, Tris–borate EDTA buffer; tRNA, transfer ribonucleic acid.
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sequence-specific protection of the labeled DNA duplex from DNaseI cleavage. Though both of these analytical techniques can be made thermodynamically rigorous, each is relatively laborious. Both procedures require preparation of homogeneous DNA probes (typically hundreds of base pairs in length) with a single radiolabeled terminus. In addition, appropriate sequence standards must be created to allow interpretation of the results, and sequencing gels are typically required. Similar procedures are required for other footprinting agents such as DMS. Quantitative affinity cleavage assays further require the preparation of modified versions of the oligonucleotides of interest. Data analysis requires careful normalization of radioactive signals to internal standards. Simpler assays have been proposed for monitoring and quantitating triplex formation. Oligonucleotide binding can confer protection from DNA modification by restriction endonucleases and methylases that recognize sites that overlap the triplex (6). This observation provided an assay for thermodynamic and kinetic experiments with limited time resolution (7). Others have profitably exploited the differential affinity of single strands vs triplexes for nitrocellulose in filter binding assays (20), although the physical basis for this separation is not understood. Among the simplest and most convenient assays for triplex formation is the electrophoretic mobility shift assay (EMSA; 21), originally applied to triplex formation as a quantitative analytical tool by Hogan and coworkers (2) and subsequently applied to a variety of problems in triplex characterization (22–31). In this procedure, a trace amount of radiolabeled synthetic DNA duplex containing the target site of interest is incubated with known concentrations of oligonucleotides. Complexes are then separated by electrophoresis through a native polyacrylamide gel containing stabilizing cations such as Mg 2/. It is observed that triplehelical complexes migrate more slowly than the corresponding duplex. The mobility difference depends, in part, on the fraction of the length of the target duplex that is occupied by the third oligonucleotide strand at saturation. It should be noted that the physical basis for this electrophoretic retardation is unclear, as the formal charge/mass ratio of the triplex should be the same as for the duplex. Possible explanations involve the shape and flexibility of the resulting complexes and/ or the expectation that the high charge density of the triplex should enhance counterion condensation such that the fractional thermodynamic charge per phosphate is reduced (32). Although EMSA can separate triplex and duplex complexes, there is concern that this technique does not afford as rigorous an equilibrium measurement as is provided by footprinting methods such as QDFA.
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Whereas a footprinting reaction is terminated minutes after addition of the cleaving agent (providing a ‘‘snapshot’’ of the equilibrium mixture), EMSA continues the binding experiment from the initiation of the binding reaction to the completion of gel electrophoresis. During this time (which may be many hours), the samples experience a series of environments that differ from the conditions in the initial binding reaction. To apply EMSA as an alternative to more complex assays such as QDFA, one would like to assume that the electrophoresis portion of EMSA simply reports the extent of triplex formation in the sample that was initially loaded into the gel. To what extent is this true? In EMSA experiments with proteins and other ligands, complexes appear to persist during electrophoresis much longer than predicted by their anticipated lifetimes in free solution. This effect has been attributed to both caging and sequestration in the gel matrix. Caging maintains high local concentrations of dissociation products so that collisions between the dissociated partners are favored. Sequestration isolates each complex from interaction with other molecules, reducing exchange reactions catalyzed by collisions with other complexes or reagents (33, 34). Thus, by isolating and insulating complexes from one another, EMSA has the potential to preserve the distribution of labeled molecules between bound and free forms throughout the electrophoresis experiment. Because the EMSA technique has proven to be rapid and simple, we wished to compare Kd estimates from EMSA with estimates obtained in parallel using a more rigorous QDFA procedure. The data suggest that EMSA can provide results in reasonable agreement with QDFA and offer some insight into sources of deviation between the two methods. REAGENTS
Buffers. Reactions included either buffer A (20 mM Mops, pH 7.1, 100 mM KCl, 5 mM MgCl2 , 1 mM spermine) or buffer B (buffer A lacking KCl). Plasmid DNA. Plasmid pJ010 contains Ç500 bp of the human FasL gene, extending upstream from the translation initiation codon into the promoter region. The fragment is cloned into pCR-Script SK(/) (Stratagene). Supercoiled plasmid DNA was isolated from bacteria using the alkaline lysis method and purified using Qiagen columns as directed by the manufacturer (Qiagen). Oligonucleotides and analogs. Oligonucleotide sequences are shown in Fig. 1A. Synthetic oligonucleotides and oligonucleotide analogs were synthesized on ABI synthesizers using phosphoramidite reagents. Protected 5-methylcytosine phosphoramidite derivatives (2*-deoxy or 2*-O-methyl), protected thymidine phos-
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phoramidite derivatives (2*-deoxy or 2*-O-methyl), other 2*-O-methyl ribonucleoside phosphoramidites, and acridine phosphoramidite (hexamethylene tether) were obtained from Glen Research. Oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis, eluted from gel slices, and desalted by SepPak C18 cartridge chromatography (Waters). Oligonucleotides were quantitated by absorbance at 260 nm using molar extinction coefficients (M01 cm01) of 15,400 (dA), 11,700 (dG), 7300 (dC), 5700 (dMe5C), and 8800 (dT), assuming no hypochromicity. Enzymes and reagents. Restriction endonucleases and the Klenow fragment of DNA polymerase I were obtained from New England Biolabs. RQ1 DNaseI was purchased from Promega. Spermine tetrahydrochloride was obtained from Sigma. Formic acid and piperidine were purchased from Aldrich. METHODS
QDFA. A 350-bp StuI–EcoRI fragment was isolated from plasmid pJ010 (Fig. 1A) by native polyacrylamide gel electrophoresis, extracted from the gel slice by incubation overnight at 377C in extraction buffer (0.375 M ammonium acetate, 1 mM EDTA, 0.1% SDS), and purified by phenol/chloroform extraction and sequential precipitations from ethanol and spermine (35). The top strand of this fragment was uniquely labeled at the EcoRI site by end filling with the Klenow fragment of DNA polymerase I and [a-32P]dATP in the presence of 0.1 mM dTTP. The resulting labeled restriction fragment was purified by precipitation with spermine. To provide sequence markers, an A / G ladder was created by treatment of a sample of labeled restriction fragment with formic acid, followed by precipitation and cleavage with piperidine (36). Triplex binding reactions (25 mL) contained Ç4 1 104 cpm (Ç0.2 pmol) labeled FasL restriction fragment together with the indicated concentration of oligonucleotide. Reactions included either buffer A or buffer B. After incubation at 227C for at least 3 h, reactions were supplemented with 2.5 mL CaCl2 solution (100 mM) and treated with 0.3 units RQ1 DNase at 227C for 2 min. Reactions were terminated by the addition of 50 mL of a solution containing 0.2 M NaCl, 30 mM EDTA, 1% sodium sarcosinate, and 100 mg/mL yeast tRNA. Samples were precipitated from ethanol, resuspended in a denaturing buffer containing formamide and tracking dyes, heated to 907C for 4 min, cooled on ice, and loaded onto 5% denaturing polyacrylamide sequencing gels (19:1 acrylamide:bisacrylamide) prepared and electrophoresed in 0.51 TBE buffer (36). The resulting gels were dried and imaged. EMSA. Oligonucleotides comprising the target duplex were annealed as follows: 500 pmol each of oligo-
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nucleotides 5 and 6 was mixed with 2 mL of 5 M NaCl and brought to a total volume of 42 mL with H2O. This annealing reaction mixture was incubated at 757C for 12 min and then gradually cooled to 257C. Thirty picomoles of the resulting oligonucleotide duplexes was radiolabeled using the Klenow fragment of DNA polymerase I and [a-32P]dATP in the presence of 0.1 mM each dGTP, dTTP, and dCTP. The resulting labeled duplex oligonucleotides were purified by Sephadex G-50 spin column chromatography. Triplex binding reactions (10 mL) contained 1 mg yeast tRNA and Ç6 1 104 cpm (Ç0.03 pmol) labeled FasL duplex 5 / 6 (Fig. 1B) together with the indicated concentration of oligonucleotide. Reactions included either buffer A or buffer B. After incubation at 227C for at least 3 h, 1 mL 80% (v/v) glycerol was added to each 10-mL reaction, and samples were loaded onto a 20% native polyacrylamide gel (19:1 acrylamide:bisacrylamide) prepared and electrophoresed in a buffer containing 50 mM Mops, pH 7, 5 mM MgCl2 . Electrophoresis was performed with recirculation at 47C for 15 h at 12 V/cm. Image analysis. Electrophoretic gels were imaged by storage phosphor technology using a Molecular Dynamics Storm 840 phosphorimager under the control of an Apple Macintosh 7100/80 microcomputer. Images were analyzed using ImageQuant software (Molecular Dynamics). Data analysis and estimation of Kd . To quantitate oligonucleotide binding to the target site in the FasL promoter using QDFA, radioactive signal was measured for two regions of each gel lane. One region (corresponding to box x in lane 16 of Fig. 2A) represents the extent of DNaseI cleavage within the promoter site targeted for triplex formation. A second region (corresponding to box y in lane 16 of Fig. 2A) represents the extent of DNaseI cleavage within an irrelevant sequence not targeted for triplex formation. The amount of DNaseI cleavage within the target sequence was first normalized using the signal in the nontargeted region of each gel lane: Snorm Å Sx /Sy ,
[1]
where Snorm is the normalized radioactive signal at the triplex target sequence of each lane and Sx and Sy are the radioactive signals in boxes x and y of each lane (see example in lane 16 of Fig. 2A). The footprinting data were then scaled relative to the maximum value of Snorm (obtained in the absence of added oligonucleotide) and the minimum value of Snorm (obtained for saturating amounts of added oligonucleotide) using the relation
u Å 1 0 [(Snorm 0 Snorm, sat)/(Snorm, 0 0 Snorm, sat), [2]
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where u is the fractional saturation of the DNA target site, Snorm, sat is the normalized radioactive signal within the triplex target site at saturating levels of oligonucleotide, and Snorm, 0 is the normalized radioactive signal within the triplex target site in the absence of added oligonucleotide. Values of the apparent triplex dissociation constant, Kd, QDFA , were then obtained by least-squares fitting of the experimental data to the binding isotherm
u Å ([O]n/K dn)/(1 / [O]n/K dn),
[3]
where [O] is the oligonucleotide concentration, and n is the Hill coefficient (37). Curve fitting was performed using Kaleidagraph (Abelbeck Software). To quantitate oligonucleotide binding to the target site in the FasL promoter using EMSA, radioactive signal was measured for two regions of each gel lane. One region (corresponding to box T in lane 8 of Fig. 3A) represents the radioactive signal due to triple-helical complex formation. A second region (corresponding to box D in lane 8 of Fig. 3A) represents the radioactive signal associated with free duplex probe 5 / 6. The fractional saturation, u, of the DNA duplex in each gel lane is then given by
u Å Striplex/(Striplex / Sduplex),
[4]
where Striplex and Sduplex are the radioactive signals associated with the triplex and duplex complexes, respectively (see example in lane 8 of Fig. 3A). Values of the apparent triplex dissociation constant, Kd, EMSA , were then obtained by least-squares fitting of the experimental data using Eq. [3]. RESULTS AND DISCUSSION
Model system. To compare QDFA and EMSA methods for estimating Kd values for triple-helical complexes, we selected a previously unexplored model system involving DNA sequences from the promoter of the human Fas ligand gene, FasL. The FasL promoter contains a consensus TATA box (Fig. 1A). Located 31 bp upstream from the TATA box is a 29-bp homopurine/ homopyrimidine sequence containing a single TrA interruption (Fig. 1A). This sequence is not highly conserved between human and mouse (38) and is of unknown regulatory significance. We are studying whether oligonucleotide binding to this sequence will alter expression of the FasL gene. In anticipation of these studies, we synthesized oligonucleotides 1–4 (Fig. 1A) to compare their binding affinities for the FasL target. These oligonucleotides contain several modifications intended to enhance their affinities for duplex DNA. All oligonucleotides contain 5-methylcy-
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FIG. 1. Experimental system. (A) Duplex DNA target used in QDFA. A 350-bp StuI–EcoRI restriction fragment containing a segment of the human FasL gene contains a nearly perfect homopurine/ homopyrimidine sequence (indicated by R and Y and boxed duplex sequence below), upstream of the TATA box and translation start codon (ATG). Synthetic oligonucleotides 1–4 were designed to bind to the homopurine target sequence in the pyrimidine triple helix motif. 5-Methylcytosine residues are indicated by italics. 2-O-Methyl backbones are indicated by underlining. An intercalating acridine residue is indicated by acr. (B) Synthetic duplex DNA target used for EMSA. Homopurine/homopyrimidine target sequence is boxed. The duplex was made blunt-ended during the labeling procedure.
tosine substituted for cytosine (39). Oligonucleotides 1, 3, and 4 were synthesized with 2*-O-methylated RNA backbones with the goal of greater triplex stability (40). The single TrA interruption in the FasL homopurine target sequence is accommodated by a G residue in oligonucleotide 1, with the goal of forming a GrTA base triplet at this position (41). Oligonucleotide 4 contains a 5*-acridine modification, intended to stabilize triplex formation by intercalation (3). For QDFA, a 350-bp FasL promoter fragment was isolated and labeled on the purine-rich strand, 3* to the homopurine element (Fig. 1A). This labeled DNA duplex was incubated with various concentrations of oligonucleotides 1–4 in buffer A or buffer B (buffer A lacking KCl), treated with DNaseI, and electrophoresed on sequencing gels as described under Methods. For EMSA, synthetic DNA duplex 5 / 6 (Fig 1B) was radiolabeled, incubated with various concentrations of oligonucleotides 1–4 in buffer A or buffer B, and loaded onto native polyacrylamide gels in the presence of Mg 2/ as described under Methods. QDFA data. An example of a footprinting result in buffer B is shown in Fig. 2A. Lane 1 displays an A
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FIG. 2. Example of results of QDFA using a 350-bp fragment of the human FasL gene. (A) Gel result. This example depicts oligonucleotide binding in buffer B. Lane 1 contains an A / G sequence ladder. The sample in lane 2 was not exposed to DNaseI. Samples in lanes 3–23 were exposed to DNaseI after incubation with 0.012, 0.06, 0.3, 1.5, or 7.5 mM concentrations of oligonucleotides 1–4. Bracket at left indicates position of homopurine/homopyrimidine target sequence. Boxes x and y in lane 16 exemplify regions quantitated in each gel lane to calculate normalized radioactive signal within the triplex target site, as described under Methods. (B) Estimation of Kd, QDFA . Analysis of QDFA data was performed as described under Methods.
/ G sequence ladder that identifies the triplex target sequence in the FasL promoter fragment. DNaseI was added to samples in lanes 3–23. No oligonucleotide was added to the samples in lanes 1–3. Increasing concentrations of the indicated oligonucleotides were added to samples in the remaining gel lanes. Footprints for oligonucleotide 1 (Fig. 2A, lanes 4–8) involve the entire homopurine target sequence, while complexes with the shorter oligonucleotides 2–4 protect only the 3* half of the site, as expected (Fig. 2A, lanes 9–23). Careful examination of the footprints also reveals that the triplex formation is accompanied by enhanced DNaseI cleavage at the 3* end of the homopurine target sequence. In all cases, the highest oligonucleotide concentration tested (7.5 mM) is sufficient to saturate the homopurine target site. The electrophoretic data from multiple QDFA experiments of this type were quantitated as described under Methods. As an example, the data from the gel in Fig. 2A are shown in Fig. 2B. Leastsquares curve fitting suggests similar Kd, QDFA values in the 1007 M range, with some variation in the optimum value of the Hill coefficient. The entire Kd, QDFA data set is included in the fifth column of Table 1. The QDFA data may be summarized as follows. Binding affinities for oligonucleotides 1–4 are between 60 and 300 nM for experiments performed in buffer B.
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Oligonucleotide 4 displays the highest affinity under these conditions. In the presence of physiological concentrations of KCl (buffer A), triplex formation is inhibited by 7- to 43-fold, consistent with previous observations (7). Binding affinities in buffer A range between 0.39 and 5.2 mM, with oligonucleotide 4 again forming the tightest complex. For both buffers A and B, the range of estimated Kd, QDFA values for oligonucleotides 1–4 spans less than one order of magnitude. EMSA data. An example of an electrophoretic gel mobility shift result in buffer A is shown in Fig. 3A. No oligonucleotide was added to the sample in lane 1. Increasing concentrations of the indicated oligonucleotides were added to samples in the remaining gel lanes. Triple-helical complexes for oligonucleotide 1 (Fig. 3A, lanes 2–5) have migrated more slowly than complexes with the shorter oligonucleotides 2–4 (Fig. 3A, lanes 6–17). In buffer A, the highest oligonucleotide concentration tested (7.5 mM) is sufficient to saturate the homopurine target site for oligonucleotides 2–4 (Fig. 3A, lanes 9, 13, and 17) and nearly saturates in the case of oligonucleotide 1 (Fig. 3A, lane 5). The electrophoretic data from multiple EMSA experiments of this type were quantitated as described under Methods. As an example, the data from the gel in Fig. 3A are shown in Fig. 3B. As for the QDFA example in Fig. 2, least-
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Kd Values for Triple-Helical Complexes Oligomer
Buffer
1 2 3 4 1 2 3 4
A A A A B B B B
Kd,EMSA (mM) 1.2 0.18 0.29 0.10 0.22 0.025 0.097 0.061
{ { { { { { { {
0.16 0.018 0.050 0.009 0.090 0.010 0.050 0.024
n
Kd,QDFA (mM)
4 4 2 2 4 3 2 2
5.2 0.82 3.3 0.390 0.12 0.096 0.30 0.060
{ { { { { { { {
0.90 0.049 0.10 0.16 0.080 0.017 0.26 0.022
n 4 4 2 2 2 2 2 2
Kd,QDFA/Kd,EMSA 4.3 4.3 11.4 3.9 0.6 3.8 3.1 1.0
{ { { { { { { {
0.95 0.48 2.0 0.39 0.43 1.7 3.1 0.53
Note. Buffer compositions are given under Materials and Methods. Data are presented as Kd { standard deviation from the indicated number of experimental replications (n). Standard deviations for the final data column were calculated by conventional error propogation methods (42).
squares curve fitting of the EMSA data in Fig. 3A suggests similar Kd, EMSA values. The entire Kd, EMSA data set is also included in the third column of Table 1. The EMSA data may be summarized as follows. Binding affinities for oligonucleotides 1–4 are between 0.1 and 1.2 mM for experiments performed in buffer A (containing 100 mM KCl). As observed in QDFA, oligonucleotide 4 displays the highest affinity under these conditions. In the absence of KCl (buffer B), triplex formation is enhanced by 1.6- to 7.6-fold. Binding affinities in buffer B range between 25 and 220 nM, with oligonucleotide 2 forming the tightest complex. As observed for the QDFA data, the range of estimated Kd, EMSA values for oligonucleotides 1–4 spans approximately one order of magnitude in both buffers A and
B. As previously observed by QDFA, it is noteworthy that the 2*-O-methylated RNA backbone of oligonucleotide 3 did not stabilize triplexes. Complexes involving oligonucleotide 3 were two- to threefold less stable than those involving DNA oligonucleotide 2 (Table 1). This unexpected result suggests that triplex stabilization by RNA-like backbones may be sequence-dependent. Data comparison. The main goal of this work was to compare Kd estimates obtained by QDFA and EMSA. This comparison is presented in the last column of Table 1 and in Fig. 4. The data demonstrate that QDFA and EMSA techniques consistently provide Kd estimates that differ by less than one order of magnitude (final column of Table 1). In addition, the degree of agreement between techniques depends on buffer con-
FIG. 3. Example of results of EMSA using a radiolabeled 42-bp duplex carrying the homopurine/homopyrimidine target sequence. (A) Gel result. This example depicts oligonucleotide binding in buffer A. Lane 1 contained no added binding oligomer. Samples in lanes 2–17 were electrophoresed after incubation with 0.012, 0.06, 0.3, or 1.5 mM concentrations of oligonucleotides 1–4. Mobilities of duplex (D) and triplex (T) species are indicated at left. Rectangles T and D in lane 8 exemplify regions quantitated in each gel lane to calculate the fractional saturation of the radiolabeled duplex DNA probe by added oligonucleotide, as described under Methods. (B) Estimation of Kd, EMSA . Analysis of EMSA data was performed as described under Methods.
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FIG. 4. Comparison of Kd, QDFA and Kd, EMSA data. Binding data from QDFA (stippled bars) and EMSA (open bars) as a function of oligonucleotide (indicated below chart) and binding buffer condition (indicated above chart). Error bars indicate standard deviations.
ditions. Kd estimates differ by 4- to 11-fold in Buffer A (containing 100 mM KCl), but are identical (within experimental error) for oligonucleotides 1, 3, and 4 in buffer B (Fig. 4). The overall conclusion is that Kd, EMSA estimates are in reasonable agreement with Kd, QDFA . Despite the reasonable agreement between Kd values obtained from QDFA and EMSA, two questions were raised by these data. First, why is the agreement between Kd estimates closer for experiments in buffer B? Second, why might EMSA underestimate Kd values (i.e., overestimate oligonucleotide affinities) relative to QDFA in buffer A? We hypothesize that answers to these questions are related to the fact that QDFA involves constant reaction buffer conditions throughout the experiment, while EMSA samples experience a buffer change between the binding and electrophoresis phases of the experiment. In particular, the EMSA gel buffer (50 mM Mops, pH 7, 5 mM MgCl2) is very similar to binding buffer B (20 mM Mops, pH 7.1, 5 mM MgCl2 , 1 mM spermine), lacking only the stabilizing polyamine spermine. In contrast, binding buffer A includes a physiological concentration of KCl (100 mM). Added monovalent cations have previously been shown to inhibit triplex formation in the presence of Mg 2/ and spermine4/, presumably by competing for participation in the population of condensed cations on DNA (7, 16). Because phosphate neutralization is less effective for monovalent cations (relative to Mg 2/ and spermine4/), added KCl shifts the equilibrium away from triplex formation. During the early stages of electrophoresis in EMSA, the reaction sample (containing 100 mM KCl in the case of buffer A) is surrounded by buffer lacking KCl. Diffusion of KCl into the gel buffer presumably causes a gradual depletion of this inhibitor as the sample enters the gel. This creates an opportunity for the
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triplex equilibrium to shift toward increased complex formation. We reasoned that if overestimation of triplex affinity for EMSA in the case of buffer A were due to depletion of inhibitory KCl early in the experiment, the effect might be resolved by a ‘‘quench’’ step prior to electrophoresis. To provide such a step, we assembled and incubated standard reactions involving oligonucleotide 2 binding to labeled duplex 5 / 6. However, reactions were supplemented with an excess (2 mM) of unlabeled duplex DNA target 5 / 6 just prior to loading the electrophoresis gel for EMSA. This excess duplex target was intended to act as a sink, driving all free oligonucleotide 2 into triplexes involving unlabeled duplex 5 / 6. In this way, if KCl depletion in the gel well enhanced the potential for triplex formation, no free oligonucleotide 2 would remain to bind labeled duplex 5 / 6. This quench procedure should be effective for all but the highest oligonucleotide concentrations studied (7.5 mM). In the latter case, the quench becomes irrelevant, as the labeled duplex is already fully converted to triplex form. The results of an EMSA experiment involving quenching are shown in Fig. 5. Samples in lanes 1–6 of Fig. 5A were assembled in buffer A and subjected to EMSA according to the standard procedure. Samples in lanes 7–12 were supplemented with 2 mM unlabeled duplex 5 / 6 just prior to electrophoresis. Careful examination of the data in Figs. 5A and 5B demonstrates an increase in the apparent Kd, EMSA (i.e., a decrease in apparent affinity) when the quench protocol was implemented. This quench effect was reproducible, causing an approximately twofold decrease in apparent affinity. Because the apparent affinities of oligonucleotides for their target in buffer A was approximately fourfold higher in EMSA than QDFA, we interpret Fig. 5 as evidence that about half of this effect can be attributed to changing buffer conditions during early stages of the EMSA experiment. After accounting for possible effects of KCl diffusion, the apparent Kd estimates for EMSA and QDFA are within approximately twofold under both experimental conditions, suggesting that EMSA can serve as a satisfactory substitute for the more rigorous QDFA procedure. It should be noted that the remaining differences in apparent Kd values might be attributed to other differences between the two assays. For example, QDFA involves supplemental 9 mM Ca2/ during the 2-min DNaseI reaction, and EMSA involves addition of glycerol to Ç7% prior to electrophoresis. In addition, the short duplex DNA target may be intrinsically more flexible than the 350-bp restriction fragment in its ability to accommodate triplex formation. In spite of these potentially important differences, binding data from these two assays are comparable.
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FIG. 5. Effect of quenching EMSA with excess cold competitor duplex prior to electrophoresis. (A) Gel result. Samples in lanes 1 and 7 contained no added binding oligomer. Lanes 2–6 and 8–12 contained 0.012, 0.06, 0.3, 1.5, or 7.5 mM concentrations of oligonucleotide 2. For lanes 7–12, excess unlabeled target duplex 5 / 6 (2 mM) was added to the samples just prior to electrophoresis. (B) Analysis of EMSA data. Binding data without quench (s). Binding data with quench by excess cold competitor duplex (j). The apparent value of Kd, EMSA was increased approximately twofold by quenching.
CONCLUSIONS
We show that QDFA and EMSA techniques provide Kd estimates that agree within one order of magnitude under common experimental conditions. Agreement is best in buffers with low concentrations of monovalent cations. Surprisingly, EMSA appears to slightly overestimate triplex stabilities relative to QDFA in the presence of physiological concentrations of monovalent cations (100 mM). Under these conditions, agreement between the techniques can be improved by quenching EMSA samples with excess unlabeled competitor duplex just prior to gel loading.
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ACKNOWLEDGMENTS
16. Singleton, S. F., and Dervan, P. B. (1993) Biochemistry 32, 13171–13179.
We thank M. Fried for helpful discussions, C. Paya and C. HoltzHeppelmann for providing cloned DNA from the human FasL gene, and G. Soukup for comments on the manuscript. This work was supported by the Mayo Foundation and by NIH Grants GM47814 and GM54411, an American Cancer Society Junior Faculty Research Award, and the Harold W. Siebens Research Scholarship (L.J.M.).
17. Singleton, S. F., and Dervan, P. B. (1994) J. Am. Chem. Soc. 116, 10376–10382.
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