A Screening Protocol for Exploring Loop Length Requirements for the Formation of a Three Cytosine-Cytosine⁺...

Area of Science:

• Molecular Biology
• Biophysics
• Structural DNA Research

Background:

• DNA i-Motifs (iMs) are tetra-helical structures formed by cytosine-rich sequences.
• Increasing interest in iMs stems from their therapeutic and technological applications.
• Understanding iM folding requirements is limited, hindering their practical use.

Purpose of the Study:

• To systematically screen sequences for i-Motif (iM) formation.
• To determine the minimal loop lengths for intramolecular iM formation.
• To investigate the influence of cytosine distribution on iM stability.

Main Methods:

• Development of a novel step-by-step pipeline for sequence screening.
• Focus on model sequences with three C-C+ base pairs.
• Analysis of loop lengths connecting DNA strands through iM grooves.

Main Results:

• Two nucleotides are required for minor groove strand connection; three for major groove.
• Intramolecular iMs require specific cytosine distribution for stability.
• Sequences with two cytosines in the first and third runs form more stable iMs.

Conclusions:

• Established minimal loop length requirements for intramolecular iM formation.
• Identified asymmetric cytosine distribution as critical for iM stability.
• Findings advance the development of iM prediction tools and rational design for technological applications.