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

Thioflavin T monitoring of guanine quadruplex formation in the rs689-dependent INS intron 1
Thioflavin T monitoring of guanine quadruplex formation in the rs689-dependent INS intron 1
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.
2162-2531
770-777
Lages, Ana
88542ce9-2e63-4900-8d1f-653cbea0d003
Proud, Christopher G.
5832db56-9069-4617-a80d-08b069093dba
Holloway, John
4bbd77e6-c095-445d-a36b-a50a72f6fe1a
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e
Lages, Ana
88542ce9-2e63-4900-8d1f-653cbea0d003
Proud, Christopher G.
5832db56-9069-4617-a80d-08b069093dba
Holloway, John
4bbd77e6-c095-445d-a36b-a50a72f6fe1a
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e

Lages, Ana, Proud, Christopher G., Holloway, John and Vorechovsky, Igor (2019) Thioflavin T monitoring of guanine quadruplex formation in the rs689-dependent INS intron 1. Molecular Therapy: Nucleic Acid, 16, 770-777. (doi:10.1016/j.omtn.2019.04.026).

Record type: Article

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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Accepted/In Press date: 27 April 2019
e-pub ahead of print date: 13 May 2019
Published date: 7 June 2019

Identifiers

Local EPrints ID: 431238
URI: http://eprints.soton.ac.uk/id/eprint/431238
ISSN: 2162-2531
PURE UUID: 63a114b8-a466-4d53-9405-e6351a671b5f
ORCID for John Holloway: ORCID iD orcid.org/0000-0001-9998-0464

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Date deposited: 28 May 2019 16:30
Last modified: 22 Nov 2021 02:41

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Contributors

Author: Ana Lages
Author: Christopher G. Proud
Author: John Holloway ORCID iD

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