Characterization of the G-quadruplexes in the transthyretin gene and its role in silencing transthyretin mRNA transcription.

ABSTRACT

Transthyretin Amyloidosis arises from the misfolding of monomers or oligomers of the normal transthyretin protein. Our investigation revealed that certain guanine-rich regions within the 5' UTR sequence of the transthyretin gene possess the ability to form G2-quadruplex structures, as determined through analysis with QGRS mapper. We demonstrated that small molecule ligands, including TMPyP4, Braco-19, NMM, and TO, have a significant impact on the stabilization of transthyretin G-quadruplexes. The objective of this study was to confirm the effect of ligands on transthyretin gene transcription through the stabilization of G-quadruplexes. To comprehend the interaction between ligands and transthyretin G-quadruplexes, a range of analytical techniques were employed, includingUV titration, fluorescence titration assays, circular dichroism, quantitative RT-PCR and cytotoxicity tests. The results revealed the presence of four putative G2-quadruplex sequences, which formed stable anti-parallel, parallel, and hybrid G2-quadruplex structures. Notably, Ttrg 3 (5'-GGAAGGAAGGGAGGGAGGG-3') exhibited the highest stability to form G-quadruplex. Furthermore, TmPyP4, Braco-19, NMM and TO were found to stabilize the parallel topology of Ttrg 3. After 48 h of incubation, the RT-PCR experiments revealed a significant reduction in transthyretin mRNA transcription in HepG2 cells when treated with 20 µM TmPyP4 and Braco-19, without inducing apoptosis. Our findings suggested that ligand-mediated stabilization of G-quadruplexes within the 5'-UTR can effectively silence transthyretin expression, highlighting the potential of G-quadruplex as a novel therapeutic target for Transthyretin Amyloidosis. This study might shed valuable lights for the development of innovative therapeutic approach against Transthyretin Amyloidosis.