Consequences of short sequence polymorphisms in the formation of G-quadruplexes that control the expression of genes associated with human diseases.

CALCATERRA, N.B.; PIGA, E.J.; ARMAS, P.; PASCUTTI, F.; DAVID, A.P.

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

SOCIEDADES DE BIOCIENCIAS

Guanine quadruplexes (G-quadruplexes or G4) are non-canonical nucleic acid structures formed by the stacking of at least two guanine tetrads bonded by Hoogsteen hydrogen bonds. Intramolecular G4 are dynamic structures transiently formed in G-rich ssDNA during transcription and replication. Several genes related with human diseases (mainly proto-oncogenes) were reported to be transcriptionally regulated by G4 formed within their proximal promoter regions. G4 forming sequences (G4FS) are defined by the consensus G2-5N1-7G2-5N1-7G2-5N1-7G2-5. Subtle variations in G4FS, not only in the G-tracts but also in the connecting loops or proximal flanking sequences, may alter the G4 stability and/or topology, with putative consequences on gene expression control. The aim of this work was to find naturally occurring short sequence variations (SSV, including single nucleotide polymorphisms and short deletions and insertions) in G4FS that may affect the transcriptional regulation of genes related with human diseases. First, an in silico search was performed using the database of sequence variants available in the Ensembl genome browser (http://www.ensembl.org) to find SSV overlapping with the G4FS or in the proximal flanking sequences (5 nucleotides to each side) of G4 with reported functions in the transcriptional regulation of genes related with human diseases. Then, oligonucleotide sequences containing the G4FS, the polymorphic variants, and mutant versions that disrupt the G4 consensus were used to test G4 formation, stability and topology by in vitro assays (dot-blots with a single-chain anti-G4 antibody, circular dichroism spectroscopy, Thioflavin T fluorescence and intrinsic fluorescence spectroscopy). Results indicate that some SSV affect the stability and/or topology of the studied G4, defining several candidates to further test the effect of these variations on G4-mediated transcriptional regulation by in cellulo experiments.