Thioflavin T monitoring of guanine quadruplex formation in the rs689-dependent INS intron 1

Abstract

The human proinsulin gene (INS) contains a thymine-to-adenine variant (rs689) located in the 3’ splice site (3’ss) recognition motif of the first intron. Adenine at rs689 is strongly associated with type 1 diabetes. By weakening the polypyrimidine tract, the adenine allele reduces the efficiency of intron 1 splicing, which can be ameliorated by antisense oligonucleotides blocking a splicing silencer located upstream of the 3’ss. The silencer is surrounded by guanine-rich tracts that may form guanine quadruplexes (G4s) and modulate accessibility of the silencer. Here, we employed thioflavin T (ThT) to monitor G4 formation in synthetic DNAs/RNAs derived from INS intron 1. We show that the antisense target is surrounded by ThT-positive segments in each direction, with oligoribonucleotides exhibiting consistently higher fluorescence than their DNA counterparts. The signal was reduced for ThT-positive oligonucleotides that were extended into the silencer, indicating that flanking G4s have a potential to mask target accessibility. Real-time monitoring of ThT fluorescence during INS transcription in vitro revealed a negative correlation with ex vivo splicing activities of corresponding INS constructs. Together, these results provide better characterization of antisense targets in INS primary transcripts for restorative strategies designed to improve the INS splicing defect associated with type 1 diabetes.

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Identifiers

ORCID for John Holloway: orcid.org/0000-0001-9998-0464

ORCID for Igor Vorechovsky: orcid.org/0000-0002-6740-6502

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