G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.

Alex Kreig,Sua Myong,Jacob Calvert,Ramreddy Tipanna,Emily Cullum,Janet Sanoica

doi:10.1093/nar/gkv749

Abstract

G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson–Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

Highlights

The G-quadruplex (GQ) is a four-stranded secondary structure of DNA that arises from a Guanine (G)-rich sequence
SmFRET was used in conjunction with circular dichroism (CD) to quantitatively measure and verify the GQ conformations
The results clearly indicated that N-methyl mesoporphyrin IX (NMM) binding and concomitant quenching is specific to parallel GQs [10]

Summary

Introduction

The G-quadruplex (GQ) is a four-stranded secondary structure of DNA that arises from a Guanine (G)-rich sequence. The G-rich, single stranded DNA sequences have been shown to fold into stable GQ structures following the basic algorithm of [G3N1–7G3N1–7G3N1–7 G3] where triplet G bases are separated by a loop sequence, N [1]. This structure is stabilized by Hoogsteen base pairing between guanine bases as well as monovalent cations such as potassium or sodium [2,3]. Stable GQs may arise from the hexanucleotide repeat expansion (HRE), (GGGGCC)n, which is the most prevalent genetic cause of neuro-degenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [22,23]

Methods

Results

Conclusion