DNA Recognition by Triple Helix Formation

Biochemistry, 1998

We have prepared a series of seven DNA fragments, based on the 160 base-pair tyrT sequence, which contain 12-14 base-pair oligopurine tracts at different positions, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. By using DNase I footprinting we find that in general, triplexes can only be formed at sites located toward the ends of nucleosomal DNA fragments. For the native fragment, bases 1-145 are in contact with the protein surface. Stable triplexes can be formed on these nucleosome-bound fragments for sites located before position 33 and beyond position 94. These are formed with both CT-containing oligonucleotides, generating parallel triplexes at pH 5.5, and GT-containing oligonucleotides forming antiparallel triplexes at pH 7.5. No antiparallel triplexes were formed at sites located between these positions. Parallel triplexes were also not formed at sites between positions 39-50 and 43-54 with oligonucleotide concentrations as high as 30 µM. However parallel triplex formation was evident at a site between positions 48 and 59, albeit with a reduced affinity compared to free DNA, suggesting that this oligopurine tract is less tightly associated with the nucleosome surface or that it has an altered translational position. The introduction of an oligopurine tract in the vicinity of the nucleosome dyad caused the fragment to adopt a different nucleosomal position, which could be targeted with parallel, but not antiparallel triplexes.

Chemistry & Biology, 2003

openers" for DNA duplexes [10-12], thus enabling hybridization of regular oligonucleotides and related probes with dsDNA via formation of so-called PD-loops [13][14][15]. One more approach, originally called "selective complementarity," is based on the pseudocomplementary strategy ) that makes it possible for a pair of mixed-base modified oligonucleotides to hybridize to dsDNA by strand invasion [16, 17]. Pseudocomplementarity means that two special derivatives of initially paired normal purine and pyrimidine are structurally ad-Summary justed in such a way that they (1) do not match each other but (2) are capable of a stable Watson-Crick-type The well-known Watson-Crick complementarity rules, pairing with the natural nucleobase complements (see which were discovered 50 years ago, elegantly direct for pseudocomplementary modified nucleothe specific pairing of two DNA single strands. On the bases we used). contrary, once formed, the double-stranded (ds) DNA Though robust, these approaches did not provide a lacks such a simple and sequence-universal recognifully satisfactory solution of the problem of dsDNA tartion principle, since most of the characteristic chemigeting by oligonucleotides. Indeed, mostly long oligocal groups of nucleobases are now buried deep inside purine tracts could presently be recognized via triplex the double helix, the major DNA form. We report a formation [6]. Although more general, the PD-looppromising versatile approach for highly selective recbased approach is still limited by purine-rich sequences ognition of designated sites within dsDNA featuring as well [11, 13]. On the other hand, the RecA-assisted considerable practical potential for a variety of molecsequence-unrestricted DNA recognition has much lower ular-biological, biotechnological, gene-therapeutic, specificity as compared to "pure" DNA-DNA (or DNAand diagnostic applications. It may also have implica-

Nucleic Acids Research, 2004

The parallel (recombination)`R-triplex' can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the¯uorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T´A) and 6MI*(C´G) triplets were monitored by their¯uorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. Thē uorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from uorescence of the single base analog. The similarity suggests that¯uorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative`branch migration' and strand exchange' structures and discuss why these are less likely than the R-type triplex.

Chemistry - A European Journal, 2011

Nucleic Acids Research, 1992

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.

Nucleic Acids Research, 1999

The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5′-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3′-ends of the 17-20 nt strands could form Watson-Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the singlestranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson-Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.

Biochemical Society Transactions, 2011

Triple-helical nucleic acids are formed by binding an oligonucleotide within the major groove of duplex DNA. These complexes offer the possibility of designing oligonucleotides which bind to duplex DNA with considerable sequence specificity. However, triple-helix formation with natural nucleotides is limited by (i) the requirement for low pH, (ii) the requirement for homopurine target sequences, and (iii) their relatively low affinity. We have prepared modified oligonucleotides to overcome these limitations, including the addition of positive charges to the sugar and/or base, the inclusion of cytosine analogues, the development of nucleosides for recognition of pyrimidine interruptions and the attachment of one or more cross-linking groups. By these means we are able to generate triplexes which have high affinities at physiological pH at sequences that contain pyrimidine interruptions.

Journal of Molecular Biology, 1995

A three-stranded DNA that is a putative intermediate of homologous recombination is a novel DNA triplex, R-form DNA. In R-form DNA the third strand includes both purines and pyrimidines and is parallel to the identical strand of the duplex. To test and refine our previously proposed R-form base triplets we have used two approaches: (1) dimethyl sulfate protection of R-form DNA; and (2) thermal dissociation of R-form DNAs in which the duplex strands were substituted in a strand-specific manner with either 7-deaza-guanine or 7-deaza-adenine. Together, the footprinting and isosteric substitution results demonstrate that the third strand in R-form DNA is in contact with the purines in the N7 position in the major groove of the Watson-Crick duplex in three ((GC):G, (AT):A and (TA):T) out of the four possible triplets. Furthermore, these results suggest that the N7 positions of the duplex play a significant role in stabilizing the DNA-DNA contacts during the homology recognition process.

2003

This year marks the 50th anniversary of the proposal of a double helical structure for DNA by James Watson and Francis Crick. The place of this proposal in the history and development of molecular biology is discussed. Several other discoveries that occurred in the middle of the twentieth century were perhaps equally important to our understanding of cellular processes; however, none of these captured the attention and imagination of the public to the same extent as the double helix. The existence of multiple forms of DNA and the uses of DNA in biological technologies is presented. DNA is also finding increasing use as a material due to its rather unusual structural and physical characteristics as well as its ready availability.

International Journal of Biological Macromolecules, 2001

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5%-(purine) m (pyrimidine) m -3%. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine-pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson-Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3%-3% phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P= phosphate and X= 1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.

ACS Omega, 2020

DNase I footprints of intermolecular DNA triplexes are often accompanied by enhanced cleavage at the 3′-end of the target site at the triplex-duplex junction. We have systematically studied the sequence dependence of this effect by examining oligonucleotide binding to sites flanked by each base in turn. For complexes with a terminal T.AT triplet, the greatest enhancement is seen with ApC, followed by ApG and ApT, with the weakest enhancement at ApA. Similar DNase I enhancements were observed for a triplex with a terminal C + .GC triplet, though with little difference between the different GpN sites. Enhanced reactivity to diethylpyrocarbonate was observed at As that flank the triplex-duplex junction at AAA or AAC but not AAG or AAT. Fluorescence melting experiments demonstrated that the flanking base affected the stability with a 4 °C difference in T m between a flanking C and G. Sequences that produced the strongest enhancement correlated with those having the lower thermal stability. These results are interpreted in terms of oligonucleotide-induced changes in DNA structure and/or flexibility.

Biochip Journal, 2011

High capability to distinguish single-nucleotide mismatches of genes using short oligonucleotide probes is essential in diagnostic methods for identification of point mutations and single nucleotide polymorphisms. To investigate the feasibility of using an aziridine-treated surface containing hyper-branched amine groups to discriminate single-nucleotide mismatches in a human gene, target probes for exons 5-8 of the p53 gene from liver cancer cells were hybridized with four types of surface-bound capture probes, one for perfect match and three for central single-nucleotide mismatches. The aziridine slide with high DNA-loading capacity exhibited greater ability to detect single-nucleotide mismatch than did the generic amine slide. When a T30 tether was linked to the capture probe, the mismatch discrimination capability increased when using a chemical cross-linker, but decreased when using UV irradiation for cross-linking. DNA duplexes had lower melting temperatures when the single-nucleotide mismatch was in the central region than when it was in the terminal region regardless of the type of mismatched nucleotide. Our results suggest that capture probes attached to the aziridine surface can effectively identify point mutations in a genomic sequence or and can estimate the affinity of gene-specific antisense oligonucleotide probes.

Biochemistry, 1995

The ligands form a series of quinoline derivatives with an alkylamine chain in the 4-position and different aryl substituents in the 2-position. By themselves these compounds do not alter DNase I digestion of the DNA duplexes at concentrations up to 100 p M. At a concentration of 10 p M they potentiate triplex formation, lowering the concentration of oligonucleotide required to produce a clear footprint by as much as 100-fold. As well as stabilizing triplexes which consist of well-characterized DNA triplets, they also promote the formation of complexes which contain central triplet mismatches. This reduction in the stringency of triple helix formation may be used to broaden the range of triplex target sequences and enable recognition at sites which contain short regions for which there are no good triplet matches.

Nucleic Acids Research, 1991

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6 -4) pyrimidlne photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH,

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: Protein domains constructed from tandem a-helical repeats have until recently been primarily associated with protein scaffolds or RNA recognition. Recent crystal structures of human mitochondrial termination factor MTERF1 and Bacillus cereus alkylpurine DNA glycosylase AlkD bound to DNA revealed two new superhelical tandem repeat architectures capable of wrapping around the double helix in unique ways. Unlike DNA sequence recognition motifs that rely mainly on major groove read-out, MTERF and ALK motifs locate target sequences and aberrant nucleotides within DNA by resculpting the double-helix through extensive backbone contacts. Comparisons between MTERF and ALK repeats, together with recent advances in ssRNA recognition by Pumilio/FBF (PUF) domains, provide new insights into the fundamental principles of protein–nucleic acid recognition.