Effect of sequence on the folding and stability of DNA G-quadruplexes

Abstract

Oligonucleotide sequences rich in guanines are known to fold into four-stranded structures that are based on stacks of hydrogen-bonded G-quartets. Sequences with the potential to adopt these structures are found at the end of chromosomes in telomeric DNA, as well as in a number of biologically significant genomic locations, including gene promoter regions. There is considerable interest in establishing whether G- quadruplexes have a natural, regulatory role and also whether they could be targets for therapeutic intervention. G-rich sequences can form an extremely diverse range of quadruplex structures, which may vary in terms of the number of strands, the strand polarity and the conformation of the loop regions that join the G-tracts. Despite the frequency with which potential quadruplex-forming sequences occur within the genome, there is presently a limited understanding of the rules that govern the formation of these structures and their stability. This work has focused on investigating the effect of sequence on the formation and stability of DNA G-quadruplexes. Loop length is known to be an important criterion in determining quadruplex stability and topology. This work first examines the properties of a series of model quadruplex- forming sequences that contain short loops, long loops or combinations of the two, investigating the resultant effects on quadruplex folding, stability and kinetics. Utilising a variety of biophysical techniques, the results highlight the importance of single nucleotide loops in determining quadruplex topology. In the sequences studied, the presence of one single-thymidine loop was sufficient to promote the other loops into an identical conformation, resulting in the formation of parallel-stranded structures. This may be significant given the frequency with which single-nucleotide loops are observed amongst genomic quadruplex-forming sequences. Besides loop-length, loop sequence can also moderate quadruplex stability. The sequence effects of single nucleotide loops have been examined in both model and biologically relevant promoter sequences. The results show that quadruplex stability is sensitive to changes in single-nucleotide loop identity, with adenines significantly disfavoured over pyrimidine loops. Finally, the importance of the loop regions on quadruplex folding is well documented, however little is known regarding the length of the G-tract. The properties of intramolecular G-quadruplexes that are formed by sequences with increasing G-tract lengths have been examined. The results reveal that there is no simple relationship between quadruplex stability and the length of the G-tracts, and that sequences containing longer G-tracts are likely to form heterogeneous populations of folded structures. When challenged with their complementary strand, several G-rich sequences preferentially form quadruplex over duplex.

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