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. 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.

Keywords: 3′ splice site; G quadruplex; polymorphism; proinsulin gene; transcription; type 1 diabetes.

Figure 1

ThT Monitoring of INS Intron-1-Derived DNA Oligonucleotides (A) Tested (upper panel) and control (lower panel) oligonucleotides. Each tested oligonucleotide is shown as a black rectangle representing the indicated portion of the wild-type (WT) partial sequence of INS intron 1 (GenBank: AH002844.2). SSO21, antisense oligonucleotide that reduced INS intron 1 retention ex vivo. Int7 folds into parallel G4, as shown by far-UV circular dichroism and NMR. CD3 forms a hairpin/G4 equilibrium in vitro; two C>T mutations (CD4) shifted the equilibrium toward G4 formation. The number of Gs in each oligonucleotides, QGRS Mapper scores, and G4 Hunter scores are shown in the last three columns. (B) Mean relative fluorescence intensity (relative fluorescence units [RFU]) of 80 μM ThT at 508 nm in the presence of the indicated DNAs. Tested oligonucleotides are in black, positive G4 controls are in green, negative controls are in blue, and additional controls (see the main text) are in orange and purple. Error bars denote the SD of three independent ThT assays. *p < 0.05, **p < 0.01, or ***p < 0.001 for comparisons with negative controls (in blue). The mean for CLASP1 is denoted by a dashed red line as an arbitrary threshold. ThT-only controls are not shown. (C) NMM spectra for a subset of INS-derived DNA oligonucleotides and controls.

Figure 2

ThT Fluorescence of the INS Intron-1-Derived Int1 and -7 that Extended into the Antisense Target Region (A) Schematics of tested DNA oligonucleotides (black rectangles) and their G scores. The antisense target is underlined. (B and C) Mean fluorescence intensity of ThT at 508 nm in the presence of oligonucleotides extended from Int1 (B) and Int7 (C) into the target region. Error bars denote SD from three independent fluorescence assays. Asterisks denote significant p values < 0.05, when comparing fluorescence intensity of the extended nucleotide with the non-extended one. (D and E) Correlation between ThT fluorescence trace and G4H (D) or QGRS Mapper (E) scores.

Figure 3

ThT G4 Screening in INS DNA and RNA Oligonucleotides Oligoribonucleotide concentrations were 2 (A), 10 (B), and 20 (C) μM. AV3 and dT22 were used as negative controls. Error bars denote SDs from three independent assays. *p < 0.05 or **p < 0.01, determined by Tukey’s multiple-comparisons test. NS, corrected p values > 0.05.

Figure 4

ThT Monitoring of Oligoribonucleotides Derived from the Antisense Target Region (A) Schematics of tested oligoribonucleotides (horizontal black bars below and above the primary transcript). Mutations (in red) altered G4/hairpin equilibrium (CD4) and/or intron retention (M3, M5, and M6). (B) Mean fluorescence intensity of 80 μM ThT at 508 nm in the presence of the WT oligoribonucleotides (black), their mutants (red), and positive (green) and negative (blue) controls. Error bars represent SDs from three independent fluorescence assays. Asterisks denote significant p values < 0.01.

Figure 5

Real-Time ThT Monitoring of RNA G4 Formation during INS Transcription (A–E) The fluorescence intensity of ThT was measured during transcription in vitro for 10,800 s and, after DNase addition, for an additional 900 s: (A), WT, (B) del5, (C) M3, (D) M5, and (E) positive control. Orange lines denote INS-derived sequences and a positive control (PC), yellow lines represent their 7-deazaG counterparts, and green lines denote no-template controls (NC). PC (Ampliscribe T7-Flash Kit) contained three putative G4s predicted by the QGRS Mapper. (F) Pearson correlation coefficients between intron retention levels (%) and fluorescence intensity at the indicated time points. (G) Resolution of INS-derived pre-mRNAs (shown at the bottom) on a native polyacrylamide gel.