Probing the competition between duplex and G-quadruplex/i-motif structures using a conformation-sensitive fluorescent...

Area of Science:

• Genomics
• Molecular Biology
• Biophysical Chemistry

Background:

• Genomic DNA contains segments capable of forming G-quadruplex (GQ) and i-motif (iM) structures.
• These non-canonical structures are implicated in crucial gene regulatory functions.
• Distinguishing between duplex, GQ, and iM DNA structures is vital for understanding genomic regulation.

Purpose of the Study:

• To develop a single fluorescent probe capable of detecting both GQ and iM structures.
• To differentiate these four-stranded structures from standard duplex DNA.
• To assess the utility of a conformation-sensitive fluorescent nucleoside analog for iM detection.

Main Methods:

• Incorporation of a 5-(benzofuran-2-yl)uracil-based fluorescent nucleoside analog into C-rich DNA oligonucleotides.
• Utilized steady-state and time-resolved fluorescence spectroscopy to analyze structural transitions.
• Correlated fluorescence data with Circular Dichroism (CD) and thermal melting studies.

Main Results:

• The fluorescent analog successfully detected iM structures in C-rich DNA oligonucleotides.
• The probe distinguished iM structures from random coil and duplex DNA conformations.
• Determined the transition pH for random coil to iM structure formation using fluorescence techniques.

Conclusions:

• The developed sensor provides a versatile platform for profiling DNA structural preferences.
• Enables the assessment of double-stranded promoter regions for their propensity to form four-stranded structures.
• Supports the discovery of novel binders for functional GQ and iM structures.