Non-canonical DNA structures: Diversity and disease association.

A complete understanding of DNA double-helical structure discovered by James Watson and Francis Crick in 1953, unveil the importance and significance of DNA. For the last seven decades, this has been a leading light in the course of the development of modern biology and biomedical science. Apart from the predominant B-form, experimental shreds of evidence have revealed the existence of a sequence-dependent structural diversity, unusual non-canonical structures like hairpin, cruciform, Z-DNA, multistranded structures such as DNA triplex, G-quadruplex, i-motif forms, etc. The diversity in the DNA structure depends on various factors such as base sequence, ions, superhelical stress, and ligands. In response to these various factors, the polymorphism of DNA regulates various genes via different processes like replication, transcription, translation, and recombination. However, altered levels of gene expression are associated with many human genetic diseases including neurological disorders and cancer. These non-B-DNA structures are expected to play a key role in determining genetic stability, DNA damage and repair etc. The present review is a modest attempt to summarize the available literature, illustrating the occurrence of non-canonical structures at the molecular level in response to the environment and interaction with ligands and proteins. This would provide an insight to understand the biological functions of these unusual DNA structures and their recognition as potential therapeutic targets for diverse genetic diseases.

Correction: A novel G·G·T non-conventional intramolecular triplex formed by the double repeat sequence of Chlamydomonas telomeric DNA.

[This corrects the article DOI: 10.1039/D2RA00861K.].

A novel G·G·T non-conventional intramolecular triplex formed by the double repeat sequence of Chlamydomonas telomeric DNA.

Competition among DNA non-canonical structures has been widely studied in repetitive DNA sequences. The factors affecting DNA structural polymorphism have always been an important area of research. The Chlamydomonas reinhardtii telomere (TTTTAGGG) n is found to be an exception to the general idea of forming a folded G-quadruplex by a few repeats in any telomeric sequence. Herein, using gel electrophoresis, UV thermal melting, UV thermal difference spectra (TDS), circular dichroism, and fluorescence and NMR spectroscopy. We demonstrated that the double repeat of the C. reinhardtii telomere (TTTTAGGG)2 (Chlm2) adopts an intramolecular non-conventional triplex structure in Na+ solution. We report that the co-existence of reverse Hoogsteen (G·G) and Wobble base pairing (G·T) stabilizes the triplex structure. To the best of our knowledge, such a non-conventional triplex structure formed by any telomeric sequence has not been reported to date.

The four repeat Giardia lamblia telomere forms tetramolecular G-quadruplex with antiparallel topology.

Guanine rich DNA sequences of regulatory genomic regions form secondary structures known as G-quadruplexes usually stabilized by tetrads of Hoogsteen hydrogen bonded guanines. The in vivo existence of G-quadruplexes ascertains their biological roles. Human telomeric repeats are the most studied G-rich sequences. The four repeat Giardia telomeric sequence (TAGGG)4 differs from its human counterpart (TTAGGG)4, by deletion of one T at the G-tract intervening site of each repeat. We show here that whilst the two repeat Giardia telomeric sequence (TAGGG)2 forms parallel and antiparallel quadruplexes with tetramolecular topology exclusively, the four repeat version (TAGGG)4 forms a tetramolecular (antiparallel) and unimolecular (parallel) quadruplexes in Na+. The tetramolecular (antiparallel) G-quadruplex formed by four repeats of Giardia telomeric sequence is stabilized by the additional Watson-Crick bonding between its intervening TA bases aligned in antiparallel fashion. Four stranded antiparallel quadruplex for four repeats of any telomeric sequence have not been characterized till date. We hypothesize that telomeric association in antiparallel fashion, (via G-overhangs to form tetramolecular quadruplex) could be a biologically relevant molecular event. Further, coexistence of Hoogsteen as well as Watson-Crick base pairing might give insight for recognition of conformationally diverse DNA structures by ligands. Communicated by Ramaswamy H. Sarma.

A short GC-rich palindrome of human mannose receptor gene coding region displays a conformational switch.

Conformational switching in DNA is fundamental to biological processes. The structural status of a palindromic GC-rich dodecamer DNA sequence, integral part of human MRC2 coding region, and a related sequence of opposite polarity from human FDX1 gene were characterized and compared. UV-melting, circular dichroism, and gel electrophoresis experiments demonstrated the formation of intermolecular structures. Although stability and molecularity of both the oligomeric structures were found to be almost identical, their secondary structures differed remarkably as A1 MRC2 sequence showed A-like and B-like DNA conformation, whereas the A2 FDX1 sequence exhibited only the A-like signatures. The study is relevant for understanding structural polymorphism at genomic locations depending on DNA sequence and solution environment.

Structural polymorphism at LCR and its role in beta-globin gene regulation.

Information on the secondary structures and conformational manifestations of eukaryotic DNA and their biological significance with reference to gene regulation and expression is limited. The human beta-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression, is a contiguous piece of DNA with five tissue-specific DNase I-hypersensitive sites (HSs). Since these HSs have a high density of transcription factor binding sites, structural interdependencies between HSs and different promoters may directly or indirectly regulate LCR functions. Mutations and SNPs may stabilize or destabilize the local secondary structures, affecting the gene expression by changes in the protein-DNA recognition patterns. Various palindromic or quasi-palindromic segments within LCR, could cause structural polymorphism and geometrical switching of DNA. This emphasizes the importance of understanding of the sequence-dependent variations of the DNA structure. Such structural motifs might act as regulatory elements. The local conformational variability of a DNA segment or action of a DNA specific protein is key to create and maintain active chromatin domains and affect transcription of various tissue specific beta-globin genes. We, summarize here the current status of beta-globin LCR structure and function. Further structural studies at molecular level and functional genomics might solve the regulatory puzzles that control the beta-globin gene locus.

Structural polymorphism exhibited by a homopurine.homopyrimidine sequence found at the right end of human c-jun protooncogene.

Homopurine.homopyrimidine (Pu.Py) tracts are likely to play important biological role in eukaryotes. Using circular dichroism, UV-thermal denaturation and gel electrophoresis, we have analyzed the structural polymorphism of a 21-bp Pu.Py DNA segment within human c-jun protooncogene 3'-region, a potential target for triplex formation. Results show that below physiological pH and in the presence of Na+/K+ with Mg2+ the duplex is destabilized/disproportionated, resulting in strand mediated structural transitions to the self-associated structures of G- and C-rich strands separately, identified as G-quadruplex and i-motif species. A significant differential behavior of the monovalent cations was observed, accordingly the presence of Na+ in acidic as well as neutral pH facilitated the duplex formation, while K+ favored the formation of self-associated structures. In Na+ and Mg2+, under acidic and neutral pH conditions, the duplex displayed triphasic and biphasic melting profiles, respectively. This self-association property of oligonucleotides might limit their use as duplex targets in triplex formation. Study is also relevant for understanding structural and biological properties of DNA sequence containing homopurine tracts.

Possibility of an antiparallel (tetramer) quadruplex exhibited by the double repeat of the human telomere.

Under physiological concentrations of Na+ and K+, human telomeric DNA can self-associate into G-quadruplexes. On the basis of circular dichroism, gel electrophoresis, gel filtration, and UV-melting experiments, we report here that the double repeat of human telomere (d-TTAGGGTTAGGG; HUM2) forms parallel as well as antiparallel quadruplexes in the presence of K+, whereas Na+ facilitates only the antiparallel form. Here, the gel techniques and CD studies have proved to be complementary in detecting the molecularity and pattern of strand orientation. By correlating the gel and CD experiments, the antiparallel G-quadruplex was identified as a tetrameric species, whereas the parallel G-quadruplex was found to be dimeric. Both structural species were separated through gel filtration, which when run on native polyacrylamide gel electrphoresis (PAGE), confirmed their molecularity. UV-melting profiles also confirm the presence of two biphasic and one monophasic structural species in the presence of K+ and Na+, respectively. Though our observation is consistent with the recent NMR report (Phan, A. T., and Patel, D. J. (2003) J. Am. Chem. Soc. 125, 15021-15027), it seems to differ in terms of the molecularity of the antiparallel quadruplex. A model is proposed for an antiparallel tetrameric quadruplex, showing the possibility of Watson-Crick hydrogen bonds between intervening bases on antiparallel strands. This article expands the known structural motifs of DNA quadruplexes. To the best of our knowledge, four-stranded antiparallel quadruplexes have not been characterized to date. On the basis of the model, we hypothesize a possible mechanism for telomere-telomere association involving their G-overhangs, during certain stages of the cell cycle. The knowledge of peculiar geometries of the G-quadruplexes may also have implications for its specific recognition by ligands.