Thioflavin T Monitoring of Guanine Quadruplex Formation in the rs689-Dependent INS Intron 1

Ana Lages, Christopher G. Proud, John W. Holloway, Igor Vorechovsky

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

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. The 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 the accessibility of the silencer. Here, we employed thioflavin T (ThT) to monitor G4 formation in synthetic DNAs and 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 a better characterization of antisense targets in INS primary transcripts for restorative strategies designed to improve the INS splicing defect associated with type 1 diabetes.

Original languageEnglish
Pages (from-to)770-777
Number of pages8
JournalMolecular Therapy - Nucleic Acids
Volume16
DOIs
Publication statusPublished or Issued - 7 Jun 2019

Keywords

  • 3′ splice site
  • G quadruplex
  • polymorphism
  • proinsulin gene
  • transcription
  • type 1 diabetes

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

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