A random-walk model for retardation of interacting species during gel electrophoresis: implications for gel-shift assays.

We recently showed that intermolecular DNA triplexes can form during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. We proposed a model to account for the resulting apparent comigration of triplexes with the duplex band when the lifetime of the triplex is much shorter than the time of electrophoresis. The model predicts that short-lived complexes can be detected by a gel-shift assay if the faster migrating component of the complex is labeled, a slower migrating component is in excess, and the complex itself migrates more slowly than either of the components. In this case the labeled component, after dissociation from the complex, overtakes a slower migrating band of the free, unlabeled second component and can be captured by the unlabeled component and again retarded; after dissociation of the newly formed complex the cycle is repeated. If the concentration of unlabeled component in the band is larger than some critical value (c(cr)), most of the labeled component becomes trapped in this band during the entire time of gel electrophoresis, thus effectively comigrating with the slower migrating unlabeled component. We call this mechanism of comigration "cyclic capture and dissociation" (CCD). Here we present a quantitative analysis of the model of CCD comigration which predicts that CCD comigration can be used not only for the detection of relatively short-lived complexes, but also for estimation of the specificity of complex formation.

Denaturation and association of DNA sequences by certain polypropylene surfaces.

We observed that DNA fragments in room temperature solution undergo low levels of denaturation in the presence of certain types of polypropylene tube surfaces. If the fragments contain (GT)n.(CA)n or (GA)n.(CT)n sequences, multimeric complexes are also formed. This surface activity is inhibited by addition of micromolar concentrations of an oligodeoxyribonucleotide of arbitrary sequence to the tube prior to adding the double-stranded DNA. The reaction was not observed in tubes made of borosilicate glass or in polypropylene-based tubes designed to have low-binding properties. In the case of the DNA fragments that form surfaced-induced multimers, similar complexes can be obtained by denaturation and renaturation of the fragment ("induced" association) without regard to the type of tube surface. However, induced association requires the presence of magnesium ions or polyethylene glycol (or concentration by evaporation) for efficient formation of complexes, whereas surface-dependent dissociation has no such requirements. This difference in buffer requirement suggests that association as well as denaturation takes place on the surface. We suggest models for the formation and structure of these complexes based on surface-dependent denaturation followed by misaligned renaturation of repeated sequences and intermolecular pairing of unpaired regions. This denaturation and complex formation may be important for the interpretation of protein-DNA binding experiments and might be related to hydrophobic interactions of DNA in vivo.

Alternate-strand triplex formation: modulation of binding to matched and mismatched duplexes by sequence choice in the Pu-Pu-Py block.

In double-stranded DNA, tandem blocks of purines (Pu) and pyrimidines (Py) can form triplexes by pairing with oligonucleotides which also consist of blocks of purines and pyrimidines, using both Py.Pu.Py (Y-type) and Pu.Pu.Py (R-type) pairing motifs in a scheme called "alternate-strand recognition," or ASR [Jayasena, S. D., & Johnston, B. H. (1992) Biochemistry 31, 320-327; Beal P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478]. We investigated the relative contributions of the Py.Pu.Py and Pu.Pu.Py blocks in the 16-bp duplex sequence 5'-AAGGAGAATTCCCTCT-3' paired with the third-strand oligonucleotides 5'-TTCCTCTTXXGGGZGZ-3' (XZ-16), where X and Z are either T or A and C is 5-methylcytosine, using chemical footprinting and get electrophoretic mobility shift measurements. We found that the left-hand, pyrimidine half (Y-block) of the third strand (TTCCTCTT, Y-8) forms a Py.Pu.Py triplex as detected by both dimethyl sulfate (DMS) probing and a gel-shift assay; in contrast, the triplex formed by the right-hand half alone (R-block) with X = T (TTGGGTGT, R-8) is not detectable under the conditions tested. However, when tethered to the Y-block (i.e., as XZ-16), the R-block contributes greatly increased specificity of target recognition and confers protection from DMS onto the duplex even under conditions unfavorable for Pu-Pu-Py triplexes (lack of divalent cations). In general, the 16-mer (XZ-16) can bind with apparent strength either greater or lesser than Y-8, depending on whether X and Z are A or T. The order of apparent binding strength, as measured by the target duplex concentration necessary to cause retardation of the third strand during gel electrophoresis, is TT-16 approximately AT-16 > Y-8 > AA-16 > TA-16. Chemical probing experiments showed that both halves of the triplex form even for AA-16, which binds with less apparent binding strength than the pyrimidine block alone (Y-8). The presence of the right half of the 16-mers, although detracting from affinity in cases of AA-16 and TA-16, provides strong specificity for the correct target compared to a target incapable of forming the Pu.Pu.Py part of the triplex. We discuss possible explanations for these observations in terms of alternate oligonucleotide conformations and suggest practical applications of affinity modulation by A-to-T replacements.

Capture in the gel: intermoleculare triplex formation during gel electrophoresis.

Analysis of unusual gel mobility patterns formed by certain DNA triplexes has revealed that intermolecular triplex formation can occur during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. Control experiments showed that this capture of the third strand occurs by sequence-specific hybridization rather than some nonspecific retardation. This phenomenon can be used to detect triplexes by a gel-shift assay even if their lifetime is much shorter than the time of gel electrophoresis.

Complement-stabilized D-loop. RecA-catalyzed stable pairing of linear DNA molecules at internal sites.

The RecA protein (RecA) of Escherichia coli has the ability to pair a single-stranded DNA to a homologous sequence in a duplex DNA without requiring denaturation of the duplex. This ability has stimulated interest in the use of RecA for targeting probes to genomic DNA. However, because pairing generally requires that the double-stranded DNA either have a homologous end or be negatively supercoiled, the application of RecA to targeting has been very limited. Here, we show that if the sequence complementary to the probe is also included in the reaction, RecA can pair the two single strands to sites distant from any ends on linear DNA. The resulting structure, termed a complement-stabilized D-loop (csD-loop), is cleavable by restriction endonucleases and, upon removal of RecA, remains stable to temperatures up to the tm of the double-stranded probe. These results indicate that the csD-loop probably consists of two side-by-side Watson-Crick duplexes, much like a replication bubble. This novel reaction of RecA may be useful in gene mapping and isolation, as well as in sequence-specific cleavage of genomic DNA, and might have functions in vivo.

Sequence limitations of triple helix formation by alternate-strand recognition.

Until recently, oligonucleotide-directed triplex formation has been limited to oligopurine tracts of target DNA. Triplex formation by alternate-strand recognition relaxes this limitation by allowing triplexes to form at 5'-(Pu)m(Py)n-3' and 5'-(Py)m(Pu)n-3' sequences, with the third strand pairing first with purines on one strand and then switching to pair with purines on the other strand. In this study, the interaction of several oligonucleotides with the potential to form triplexes by alternate-strand recognition at the sequence 5'-A8C8A8-3' was studied by chemical probing and affinity cleaving. The results show that triplex formation can be readily accomplished at the 5'-A8C8-3' part of the sequence; however, base triplet formation is disrupted on either side of the strand switch and the Watson-Crick helix is distorted in such a way as to expose the N7 positions of purines adjoining the strand switch. Triplex formation is weak or nonexistent at the 3'-most A8 block, despite the opportunity for recruiting a spacer sequence for the second (C8-A8) strand switch by "slippage". This finding indicates that the C8-A8 strand switch is energetically unfavorable, although pairing at other 5'-(Py)n(Pu)n-3' sequences has been observed, with or without a spacer [Beal, P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478; Jayasena, S. D., & Johnston, B. H. (1992) Nucleic Acids Res. 20, 5279-5288]. Thus, alternate-strand recognition may not be feasible for certain sequences of 5'-(Py)m(Pu)n-3', at least under the conditions examined.

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.

Site-specific cleavage of the transactivation response site of human immunodeficiency virus RNA with a tat-based chemical nuclease.

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.

Intramolecular triple-helix formation at (PunPyn).(PunPyn) tracts: recognition of alternate strands via Pu.PuPy and Py.PuPy base triplets.

Triple-helical DNA shows increasing potential for applications in the control of gene expression (including therapeutics) and the development of sequence-specific DNA-cleaving agents. The major limitation in this technology has been the requirement of homopurine sequences for triplex formation. We describe a simple approach that relaxes this requirement, by utilizing both Pu.PuPy and Py.PuPy base triplets to form a continuous DNA triple helix at tandem oligopurine and oligopyrimidine tracts. [Triplex formation at such a sequence has been previously demonstrated only with the use of a special 3'-3' linkage in the third strand [Horne, D. A., & Dervan, P. B. (1990) J. Am. Chem. Soc. 112, 2435-2437].] Supporting evidence is from chemical probing experiments performed on several oligonucleotides designed to form 3-stranded fold-back structures. The third strand, consisting of both purine and pyrimidine blocks, pairs with purines in the Watson-Crick duplex, switching strands at the junction between the oligopurine and oligopyrimidine blocks but maintaining the required strand polarity without any special linkage. Although Mg2+ ions are not required for the formation of Pu.PuPy base triplets, they show enhanced stability in the presence of Mg2+. In the sequences observed. A.AT triplets appear to be more stable than G.GC triplets. As expected, triplex formation is largely independent of pH unless C+.GC base triplets are required.

The Z-Z junction: the boundary between two out-of-phase Z-DNA regions.

The boundary between two segments of Z-DNA that differ in the phase of their syn-anti alternation about the glycosidic bond is termed a Z-Z junction. Using chemical probes and two-dimensional gel electrophoresis, we examined a Z-Z junction consisting of the sequence d[(CG)8C(CG)8] inserted into a plasmid and used energy minimization techniques to devise a three-dimensional model that is consistent with the available data. We show that both alternating CG segments undergo the B-Z transition together to form a Z-Z junction. The junction is very compact, displaying a distinctive reactivity signature at the two base pairs at the junction. In particular, the 5' cytosine of the CC dinucleotide at the junction is hyperreactive toward hydroxylamine, and the two guanines of the GG dinucleotide on the complementary strand are less reactive toward diethyl pyrocarbonate than are the surrounding Z-DNA guanines. Statistical mechanical treatment of the 2-D gel data yields a delta G for forming the Z-Z junction equal to 3.5 kcal, significantly less than the cost of a B-Z junction and approximately equal to the cost of a base out of alternation (i.e., a Z-DNA pyrimidine in the syn conformation). The computer-generated model shows little distortion of the Z helix outside of the central two base pairs, and the energy of the structure and the steric accessibility of the reactive groups are consistent with the data.

The S1-sensitive form of d(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids.

Homopurine-homopyrimidine sequences that flank certain actively transcribed genes are hypersensitive to single strand-specific nucleases such as S1. This has raised the possibility that an unusual structure exists in these regions that might be involved in recognition or regulation. Several of these sequences, including d(C-T)n.d(A-G)n, are known to undergo a transition in plasmids to an underwound state that is hypersensitive to single strand-specific nucleases; this transition occurs under conditions of moderately acid pH and negative supercoiling. Chemical probes were used to examine the reactivity of a restriction fragment from a human U1 gene containing the sequence d(C-T)18.d(A-G)18 as a function of supercoiling and pH, and thus analyze the structure in this region. Hyperreactivity was seen in the center and at one end of the (C-T)n tract, and continuously from the center to the same end of the (A-G)n tract, in the presence of supercoiling and pH less than or equal to 6.0. These results provide strong support for a triple-helical model recently proposed for these sequences and are inconsistent with other proposed structures.

Chemical probing of the B-Z transition in negatively supercoiled DNA.

An analysis of the B-to-Z transition as a function of supercoiling for a natural Z-DNA-forming sequence found in plasmid pBR322 is presented at nucleotide resolution. The analysis is based on reactivity to four chemical probes which exhibit hyperreactivity in the presence of Z-DNA: hydroxylamine, osmium tetroxide, diethyl pyrocarbonate and dimethyl sulfate. We find that the initial transition occurs largely within a 14 base pair region which is mostly alternating purines and pyrimidines. With increasing negative supercoiling. Z-DNA extends into flanking regions having less and less alternating character, first one direction and then in the other. Evidence of B-Z junctions is seen at four sites bracketing these three adjacent regions. One of these Z-forming regions contains the non-alternating sequence CTCCT, suggesting that such sequences can form Z-DNA without great difficulty if they are adjacent to alternating sequences. A plasmid containing three copies of a 61 base pair fragment bearing the entire Z-forming region shows equal reactivity of all three copies at any given superhelical density, implying that they compete equally and independently for the torsional strain energy which promotes the B-Z transition, and are unaffected by adjacent sequences more than 20-30 base pairs away.

Stochastic distribution of a short region of Z-DNA within a long repeated sequence in negatively supercoiled plasmids.

We have analyzed, at nucleotide resolution, the progress of the B-to-Z transition as a function of superhelical density in a 2.2-kilobase plasmid containing the sequence d(C-A)31.d(T-G)31. The transition was monitored by means of reactivity to two chemical probes: diethyl pyrocarbonate, which is sensitive to the presence of Z-DNA, and hydroxylamine, which detects B-Z junctions. At a threshold negative superhelical density between about 0.048 and 0.056, hyper-reactivity to diethyl pyrocarbonate appears throughout the CA/TG repeat and remains as the superhelical density is further increased. However, there is no reactivity characteristic of B-Z junctions until the superhelical density reaches 0.084, when single cytosines at each end of the repeat become hyper-reactive to hydroxylamine. A two-dimensional gel analysis of this system by others (Haniford, D. B., and Pulleyblank, D. E. (1983) Nature 302, 632-634) indicates that only about half of the 62 base pairs of the CA/TG repeat undergo the initial transition at omega = 0.056. Our results indicate that this region of Z-DNA is free to exist anywhere along the CA/TG repeat and is probably constantly in motion. Well defined B-Z junctions are seen only when there is sufficient supercoiling to convert the entire CA/TG sequence to Z-DNA. The implications for possible B-Z transitions in chromosomal domains of different sizes are discussed.

Chemical probes of DNA conformation: detection of Z-DNA at nucleotide resolution.

Chemical probes sensitive to alterations in DNA conformation, especially Z-DNA, have been identified. These permit cleavage of DNA at sites of unusual structure, the results of which can be displayed on a sequencing gel. Using supercoiled plasmids containing inserts of d(C-G)16 and d(C-A)31 X d(T-G)31, it was found that hydroxylamine and osmium tetraoxide react preferentially with cytosines and thymines, respectively, near B-DNA-Z-DNA junctions; diethylpyrocarbonate reacts more strongly with purines within Z-DNA regions; and dimethylsulfate and diethylsulfate react more strongly with guanines in Z-DNA that are out of phase with the usual pattern of purine-pyrimidine alternation. Our results show that B-Z boundaries are mobile and that with increasing torsional strain, the Z-DNA regions can expand to include nonalternating nucleotide sequences.