Non-canonical DNA/RNA g-quadruplex and i-motif structures as therapeutic targets for neuroinflammation in multiple sclerosis

Overview

This research program investigates non-canonical DNA and RNA secondary structures—specifically G-quadruplex (GQ) and i-motif structures—as novel therapeutic targets for neuroinflammation in multiple sclerosis (MS). The work focuses on four genes involved directly or indirectly in DNA methylation whose expression is mis-regulated in MS: DNMT3a (DNA methyltransferase), TET3 (DNA demethylase), MBD2 (methyl-CpG binding protein), and SHMT1 (serine hydroxymethyltransferase, a regulator of methyl donor availability). By targeting the GQ and i-motif structures formed in the promoter and 5'-untranslated regions of these genes, the program aims to develop new molecular interventions that modulate methylation-related gene expression, addressing key MS pathologies including blood-brain barrier breakdown, inflammation, demyelination, and neurodegeneration.

Technical specifications

Core approach:

• Biophysical identification and characterization of G-quadruplex and i-motif structures in regulatory regions of DNMT3a, TET3, MBD2, and SHMT1
• Analysis of both DNA G-rich/C-rich sequences and corresponding mRNA G-rich sequences for GQ-forming potential
• Use of 1H NMR spectroscopy, circular dichroism (CD), UV spectroscopy, and native polyacrylamide gel electrophoresis (PAGE) for structural validation
• Examination of equilibrium between GQ/i-motif conformations and canonical duplex structures

Functional validation platform:

• Dual-luciferase reporter assays (firefly luciferase with Renilla control) using wild-type and mutant 5'-UTR sequences to assess transcription and translation effects
• qRT-PCR for transcriptional analysis and luciferase activity measurement for translational readout

Targeting strategy:

• Evaluation of 2'-Fluoro-arabinonucleic Acid (FANA) oligonucleotides as structure-selective ligands to disrupt or stabilize GQ/i-motif conformations in vitro and in cells

Technology readiness level

The program has completed initial proof-of-concept studies on SHMT1, with biophysical methods confirming GQ and i-motif formation by the gene's 5'-UTR sequences and demonstrating their coexistence with duplex DNA. Current and planned work extends biophysical characterization to DNMT3a, TET3, and MBD2, followed by functional reporter assays to link structure formation to gene expression outcomes. The FANA oligonucleotide targeting approach is at an early in vitro and cellular evaluation stage. Overall, the technology is at an early-to-mid discovery phase (TRL 2–3), with foundational evidence supporting the biological relevance of the targets but requiring further validation before preclinical development.