Purine Tracts Research Articles

5-Aza-7-deazaguanine-Isoguanine and Guanine-Isoguanine Base Pairs in Watson-Crick DNA: The Impact of Purine Tracts, Clickable Dendritic Side Chains, and Pyrene Adducts.

The Watson–Crick coding system depends on the molecular recognition of complementary purine and pyrimidine bases. Now, the construction of hybrid DNAs with Watson–Crick and purine–purine base pairs decorated with dendritic side chains was performed. Oligonucleotides with single and multiple incorporations of 5‐aza‐7‐deaza‐2′‐deoxyguanosine, its tripropargylamine derivative, and 2′‐deoxyisoguanosine were synthesized. Duplex stability decreased if single modified purine–purine base pairs were inserted, but increased if pyrene residues were introduced by click chemistry. A growing number of consecutive 5‐aza‐7‐deazaguanine–isoguanine base pairs led to strong stepwise duplex stabilization, a phenomenon not observed for the guanine–isoguanine base pair. Spacious residues are well accommodated in the large groove of purine–purine DNA tracts. Changes to the global helical structure monitored by circular dichroism spectroscopy show the impact of functionalization to the global double‐helix structure. This study explores new areas of molecular recognition realized by purine base pairs that are complementary in hydrogen bonding, but not in size, relative to canonical pairs.

General Recognition of U-G, U-A, and C-G Pairs by Double-Stranded RNA-Binding PNAs Incorporated with an Artificial Nucleobase.

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA-RNA triplex formation. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dsRNA-binding PNAs (dbPNAs) can recognize internal Watson-Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases.

Non‐Canonical DNA in the c‐Myc Hotspot Region

G‐quadruplexes and H‐DNA are non‐canonical or non‐B form DNA structures. G‐quadruplexes are three dimensional structures formed by planar stacking of guanine quartets in the DNA. H‐DNA structures form at inverted repeats consisting of poly(purine)•poly(pyrimidine) sequences, in which the poly(purine) portion of one half of the repeat forms a Pu•Py‐Pu triplex by binding to the duplex form of the other half. Genetic “hotspots” are regions of DNA that are especially susceptible to double strand breaks. It has been shown that the structural conformation of DNA could be the cause for some of these breaks in “hotspot” regions. In particular, the well‐known transpositions involving the c‐Myc oncogene in Burkitt Lymphoma involves a “hotspot” region of the gene that is proposed to form two non‐canonical structures, a G‐quadruplex and H‐DNA structure, which are in competition which each other.Surface plasmon resonance (SPR) was used to detect H‐DNA or G‐quadruplex DNA formation in c‐MYC. An immobilized single‐stranded DNA construct encompassing the c‐MYC hotspot region was probed with short oligonucleotides complementary to different regions of the putative H‐DNA and G‐quadruplex DNA structures. These studies demonstrated that both the duplex portion of the putative H‐DNA as well as the poly(purine) track displayed limited hydridization to complement probes, commensurate with formation of an H‐DNA structure.Spectroscopic methods such as UV/vis melting, Förster resonance energy transfer (FRET), and circular dichroism (CD) have been used in previous studies to detect and characterize G‐quaduplexes. Using these spectroscopic methods, we were able to characterize the formation of non‐canonical DNA structure from an oligonucleotide comprised of the entire poly(purine) tract from the c‐MYC hotspot. These studies demonstrate that this poly(purine) sequence adopts a stable intermolecular G‐quadruplex in preference to the intramolecular G‐qaudruplex predicted earlier. This means that the H‐DNA in c‐MYC is likely not in competition for formation with the intramolecular G‐quadruplex suspected to form from this same region.Support or Funding InformationCancer Prevention and Research Institute of TexasThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA mutation motifs in the genes associated with inherited diseases.

Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs) rarely associated with mutations (coldspots) and frequently associated with mutations (hotspots) exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

Retrotransposon based TRAP marker displays diversity among onion (Allium cepa L.) genotypes

Ty1-copia like elements are distributed throughout the onion (Allium cepa) genome, and are actively transcribed. Sequence analysis by LTR finder identified that the copia element in onion is bound by long terminal repeats (LTR) of length 121bp. Primer binding site and poly purine tract are located on copia element at 193–211bp and 4593–4607bp respectively. To exploit the variation at the insertional locus and to explore its further use as a marker, a method using a primer located on the LTR and an arbitrary primer was developed for detecting variation in onion genotypes. This method resulted in the detection of polymorphism in 22 onion genotypes. Thirty-three polymorphic amplicons were detected in these genotypes. The genetic distance between the varieties varied from 0.83 to 0.99. Phylogenetic analysis revealed that the Indian short day onion clustered separately from the exotic varieties indicating that the origin of Indian varieties is distinct. Further, variation observed among Indian varieties was less than that of exotic varieties. The marker was detected across eight species of genus Allium and the genetic distance between the species varied from 0.34 to 0.88. Polymorphism detection was better across the species than within A. cepa. This retrotransposon based method is likely to supplement the marker resources for Allium spp.

A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation

Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

Abstract 6: A triple-helix forming oligonucleotide targeting a genomic locus is capable of sequence specific mutagenesis in human lymphoblastoid TK6 cells

Abstract It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming oligonucleotide (TFO) targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine. Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.

Generation of an improved foamy virus vector by dissection of cis-acting sequences

In contrast to other retroviruses, foamy viruses (FVs) generate their Pol protein precursor independently of the Gag protein from a spliced mRNA. The exact mechanism of Pol protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells.

Mass Spectrometry Studies of the Binding of the Minor Groove-Directed Alkylating Agent Alkamin to AT-Tract Oligonucleotides

Minor groove binding alkylating agents, which have potential as cancer drugs, generate cytotoxic DNA adducts that are relatively resistant to repair as a consequence of locating covalent attachment at purine N3 nitrogen atoms. Recently, we used electrospray and matrix-assisted laser desorption ionization mass spectrometry to study the binding of the minor groove-directed polybenzamide bis-half-mustard alkamin, and its monofunctional analogue alkamini, to the oligonucleotide d(CGCGAATTCGCG)(2), identifying a number of inter- and intrastrand alkamin cross-links involving the GAATTC sequence [ Abdul Majid , A. M. S. , Smythe , G. , Denny , W. A. , and Wakelin , L. P. G. ( 2007 ) Mol. Pharmacol. 71 , 1165 - 1178 ]. Here, we extend these studies to d(CGCAAATTTGCG)(2), A3T3, and d(CGCAAAAAAGCG).d(CGCTTTTTTGCG), A6/T6, in which the opportunity for both inter- and intrastrand cross-linking is enhanced. We find that both ligands alkylate all adenines in the longer AT-tracts, as well as the abutting guanines, whether they are in the same strand as the adenines or not, in a manner consistent with covalent attack on purine N3 atoms from the minor groove. Alkamin forms intrastrand cross-links involving A4 and A6 and A6 and G10 in A3T3 and all of the purines in the A6/T6 purine tract, including G10. In addition, it forms interstrand cross-links between A4, A5, A6 and A4', A5', A6', between G10 and the latter adenines in A3T3, and between G22 and adenines A5 and A6 in A6/T6. The reactivity of the abutting guanines provides unexpected opportunities for both inter- and intrastrand cross-linking by alkamin, such as the interstrand cross-link in the CAAAAAAG sequence. We conclude that positioning monofunctional mustard groups on either end of a minor groove-directed polybenzamide has the capacity to enhance interstrand cross-links at all manner of AT-tracts, including most in which the adenines are all in one strand.

Complex effects of foamy virus central purine-rich regions on viral replication

Similar to the lentiviruses family of retroviruses, foamy viruses (FVs) contain purine-rich sequences located in the center of the genome. Their function on viral replication or vector transfer remains elusive, although dual initiation of plus-strand reverse transcription has been suggested. To elucidate the physical nature of the central region of the prototype FV (PFV) genome, we performed 3′ and 5′ RACE experiments. Our results revealed that the PFV genome contains a centrally located gap in the DNA plus-strand with no definite termination and start point and of variable length. We did not find evidence for a DNA flap region. The PFV isolate harbors four centrally located purine-rich elements (A–D). Only the D element is identical in sequence to the 3′ poly purine tract (PPT). We mutated these elements while conserving or altering the overlapping pol reading frame and analyzed the mutants for transient replication in an infectious or for vector transfer in a replication-deficient background. In addition, we determined the protein composition of the respective viral particles. The A and B elements appeared to play a role in Pol protein encapsidation, the C element is likely involved in regulating gene expression, while mutation of the D element resulted in an insignificant reduction in transiently replicating virus and an approximately 50% reduction in vector titer. The reason for this deficit remains to be elucidated.

Interactions of platinum(II)-derivatized triplex-forming oligonucleotides with DNA

Polypyrimidine oligonucleotides can bind to tracts of contiguous purines in double-stranded DNA to form triple-stranded complexes. The stability of the triplex is reduced significantly if the target purine tract is interrupted by a single pyrimidine. Previous studies have shown that incorporation of an N4-aminoalkylcytosine into the triplex-forming oligonucleotide (TFO), opposite a single CG interruption, facilitates triplex formation. Examination of molecular models suggested that further modification of the amino group of the aminoalkyl arm might enable adduct formation with the N7 of the guanine of the CG interruption. To test this, we prepared 2′-deoxyribo-and 2′-O-methylribo-TFOs that contained cytosine (C), N4-(2-aminoethyl)cytosine (ae-C), or diethylenetriamineplatinum(II) (DPt-C) or cis-aquodiammineplatinum(II) (cPt-C) derivatives of N4-(2-aminoethyl)cytosine, positioned opposite a CG interruption of a polypurine tract found in the pol gene of HIV-1 proviral DNA. Although the C- and ae-C-derivatized deoxyribo-TFOs formed triplexes of modest stability and the DPt-C-modified TFO failed to form a triplex, the C- and ae-C-derivatized 2′-O-methylribo-TFOs formed remarkably stable triplexes (Tm = 57 °C). The DPt-C- and cPt-C-modified 2′-O-methylribo-TFOs also formed triplexes, although their stabilities were reduced (Tm = 33 °C), suggesting that the tethered platinum group may interfere sterically with TFO binding. Consistent with this hypothesis was the observation that triplex stability was restored (Tm = 57 °C) when the diethylenetriamineplatinum(II) group was tethered to the 5′-end of the 2′-O-methylribo-TFO via a 2-aminoethylcarbamate linkage. Taken together, these results suggest that 2′-O-methylribo-TFOs may be particularly useful in targeting purine tracts in DNA that have CG interruptions, and that further modification with platinum derivatives could lead to the design of TFOs that are capable of covalent binding to their target, thus increasing the effectiveness of the TFO.Key words: triplex-forming oligonucleotide, TFO, cisplatin, interrupted polypurine tract.

Z-DNA, an active element in the genome

Z-DNA is a left-handed helical form of DNA in which the double helix winds to the left in a zigzag pattern. DNA containing alternating purine and pyrimidine repeat tracts have the potential to adopt this non-B structure in vivo under physiological conditions, particularly in actively transcribed regions of the genome. Z-DNA is thought to play a role in the regulation of gene expression; Z-DNA is also thought to be involved in DNA processing events and/or genetic instability. For example, Z-DNA-forming sequences have the potential to enhance the frequencies of recombination, deletion, and translocation events in cellular systems. Although the biological function(s) of Z-DNA and related Z-DNA-binding proteins are not fully understood, accumulating experimental and clinical evidence support the idea that this non-B DNA conformation is involved in several important biological processes and may provide a target for the prevention and treatment of some human diseases. In this review, we discuss the properties of Z-DNA, proteins that are known to bind specifically to Z-DNA, and potential biological functions of this non-canonical DNA structure.

Solution structure of a purine rich hexaloop hairpin belonging to PGY/MDR1 mRNA and targeted by antisense oligonucleotides

A preferential target of antisense oligonucleotides directed against human PGY/MDR1 mRNA is a hairpin containing a stem with a G•U wobble pair, capped by the purine-rich 5′r(GGGAUG)3′ hexaloop. This hairpin is studied by multidimensional NMR and restrained molecular dynamics, with special emphasis on the conformation of south sugars and non-standard phosphate linkages evidenced in both the stem and the loop. The hairpin is found to be highly structured. The G•U wobble pair, a strong counterion binding site, displays structural particularities that are characteristic of this type of mismatch. The upper part of the stem undergoes distortions that optimize its interactions with the beginning of the loop. The loop adopts a new fold in which the single-stranded GGGA purine tract is structured in A-like conformation stacked in continuity of the stem and displays an extensive hydrogen bonding surface for recognition. The remarkable hairpin stability results from classical inter- and intra-strand interactions reinforced by numerous hydrogen bonds involving unusual backbone conformations and ribose 2′-hydroxyl groups. Overall, this work emphasizes numerous features that account for the well-ordered structure of the whole hairpin and highlights the loop properties that facilitate interaction with antisense oligonucleotides.

Adaptive role of increased frequency of polypurine tracts in mRNA sequences of thermophilic prokaryotes.

The mechanism of an organism's adaptation to high temperatures has been investigated intensively in recent years. It was suggested that the macromolecules of thermophilic microorganisms (especially proteins) have structural features that enhance their thermostability. We compared mRNA sequences of 72 fully sequenced prokaryotic proteomes (14 thermophilic and 58 mesophilic species). Although the differences between the percentage of adenine plus guanine content of whole mRNAs of different prokaryotic species are much lower than those of guanine plus cytosine content, the thermophile purine-pyrimidine (R/Y) ratio within their mRNAs is significantly higher than that of the mesophiles. The first and third codon positions of both thermophiles and mesophiles are purine-biased, with the bias more pronounced by the thermophiles. Thermophile mRNAs that display the highest R/Y ratio (1.43-1.69) are those of the ribosomal proteins, histone-like proteins, DNA-dependent RNA polymerase subunits, and heat-shock proteins. Within mesophilic prokaryotes and five eukaryotic species, the R/Y ratio of the mRNAs of heat-shock proteins is higher than their average over coding part of the genome. Polypurine tracts (R)(n) (with n > or = 5) are much more abundant within the thermophile mRNAs compared with mesophiles. Between two sequential pure-purinic codons of thermophile mRNAs, there is a rather strong tendency for the occurrence of adenine but not guanine tracts. The data suggest that mixed adenine.guanine and polyadenine tracts in mRNAs increase the thermostability beyond the contribution of amino acids encoded by purine tracts, which highlights the importance of ecological stress in the evolution of genome architecture.

An Unusual Sugar Conformation in the Structure of an RNA/DNA Decamer of the Polypurine Tract May Affect Recognition by RNase H

Retroviral conversion of single-stranded RNA into double-stranded DNA requires priming for each strand. While host cellular t-RNA serves as primer for the first strand, the viral polypurine tract (PPT) is primer for the second. Therefore, polypurine tracts of retroviruses are essential for viral replication by reverse transcriptase (RT). These purine tracts are resistant to cleavage during first strand synthesis. In obtaining the primer for second strand synthesis, the RNase H function of RT must cleave the PPT exactly for in vivo transcription to proceed efficiently and proper integration to occur. At the RNase H active site the protein makes contacts primarily along the backbone, with hydrogen bonds to the sugar–phosphate oxygen atoms. A high-resolution structure (1.10 Å) of the first ten base-pairs of the RNA/DNA hybrid PPT, r-(c-a-a-a-g-a-a-a-a-g)/d-(C-T-T-T-T-C-T-T-T-G), contains the highly deformable r-(a-g-a) steps found in retroviral polypurine tracts. This r-(a-g-a) motif is utilized in the “unzipping” or unpairing of bases that occurs when RT binds a malleable PPT. Another unusual feature found in our high-resolution PPT structure is the sugar switch at RNA adenine 2. All the RNA sugars are the expected C3′- endo, except sugar 2, which is C2′- endo, characteristic of B-form sugars. This local A-to- B conversion adversely affects the pattern of hydrogen bonds from protein to sugar–phosphate backbone, disrupting the catalytic site. Disruption could cause the enzyme to pause at the 5′-end of the PPT, leaving it intact. Pyrimidine–purine (YR) steps are most deformable and the T-A step especially can undergo A-to- B transitions readily.

Effect of DNA target sequence on triplex formation by oligo-2'-deoxy- and 2'-O-methylribonucleotides.

The interactions of pyrimidine deoxyribo- or 2'-O-methylribo-psoralen-conjugated, triplex-forming oligonucleotides, psTFOs, with a 17-bp env-DNA whose purine tract is 5'-AGAGAGAAAAAAGAG-3', or an 18-bp gag-DNA whose purine tract is 5'-AGG GGGAAAGAAAAAA-3', were studied over the pH range 6.0-7.5. The stability of the triplex formed by a deoxy-env-psTFO containing 5-methylcytosines and thymines decreased with increasing pH (T(m) = 56 degrees C at pH 6.0; 27 degrees C at pH 7.5). Replacement of 5-methylcytosines with 8-oxo-adenines reduced the pH dependence, but lowered triplex stability. A 2'-O-methyl-env-psTFO containing uracil and cytosine did not form a triplex at pH 7.5. Surprisingly, replacement of the cytosines in this oligomer with 5-methylcytosines dramatically increased triplex stability (T(m) = 25 degrees C at pH 7.5), and even greater stability was achieved by selective replacement of uracils with thymines (T(m) = 37 degrees C at pH 7.5). Substitution of the contiguous 5-methylcytosines of the deoxy-gag-psTFO with 8-oxo-adenines significantly reduced pH dependence and increased triplex stability. In contrast to the behavior of env-specific TFOs, triplexes formed by 2'-O-methyl-gag-psTFOs did not show enhanced stability. Replacement of the 3'-terminal phosphodiester of the TFO with a methylphosphonate group significantly increased the resistance of both deoxy- and 2'-O-methyl-TFOs to degradation by 3'-exonucleases, while maintaining triplex stability.

Structure of a β-Alanine-linked Polyamide Bound to a Full Helical Turn of Purine Tract DNA in the 1:1 Motif

Polyamides composed of N-methylpyrrole (Py), N-methylimidazole (Im) and N-methylhydroxypyrrole (Hp) amino acids linked by β-alanine (β) bind the minor groove of DNA in 1:1 and 2:1 ligand to DNA stoichiometries. Although the energetics and structure of the 2:1 complex has been explored extensively, there is remarkably less understood about 1:1 recognition beyond the initial studies on netropsin and distamycin. We present here the 1:1 solution structure of ImPy–β–Im–β–ImPy–β-Dp bound in a single orientation to its match site within the DNA duplex 5′-CC AAAGAGAAGCG-3′·5′-CG CTTCTCTTTGG-3′ (match site in bold), as determined by 2D 1H NMR methods. The representative ensemble of 12 conformers has no distance constraint violations greater than 0.13 Å and a pairwise RMSD over the binding site of 0.80 Å. Intermolecular NOEs place the polyamide deep inside the minor groove, and oriented N–C with the 3′–5′ direction of the purine-rich strand. Analysis of the high-resolution structure reveals the ligand bound 1:1 completely within the minor groove for a full turn of the DNA helix. The DNA is B-form (average rise=3.3 Å, twist=38°) with a narrow minor groove closing down to 3.0–4.5 Å in the binding site. The ligand and DNA are aligned in register, with each polyamide NH group forming bifurcated hydrogen bonds of similar length to purine N3 and pyrimidine O2 atoms on the floor of the minor groove. Each imidazole group is hydrogen bonded via its N3 atom to its proximal guanine's exocyclic amino group. The important roles of β-alanine and imidazole for 1:1 binding are discussed.

Bias of purine stretches in sequenced chromosomes

We examined more than 700 DNA sequences (full length chromosomes and plasmids) for stretches of purines (R) or pyrimidines (Y) and alternating YR stretches; such regions will likely adopt structures which are different from the canonical B-form. Since one turn of the DNA helix is roughly 10 bp, we measured the fraction of each genome which contains purine (or pyrimidine) tracts of lengths of 10 bp or longer (hereafter referred to as ‘purine tracts’), as well as stretches of alternating pyrimidines/purine (‘pyr/pur tracts’) of the same length. Using this criteria, a random sequence would be expected to contain 1.0% of purine tracts and also 1.0% of the alternating pyr/pur tracts. In the vast majority of cases, there are more purine tracts than would be expected from a random sequence, with an average of 3.5%, significantly larger than the expectation value. The fraction of the chromosomes containing pyr/pur tracts was slightly less than expected, with an average of 0.8%. One of the most surprising findings is a clear difference in the length distributions of the regions studied between prokaryotes and eukaryotes. Whereas short-range correlations can explain the length distributions in prokaryotes, in eukaryotes there is an abundance of long stretches of purines or alternating purine/pyrimidine tracts, which cannot be explained in this way; these sequences are likely to play an important role in eukaryotic chromosome organisation.

Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker

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.

5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates.

Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) "pseudo-origin" (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cis-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin.