A single molecule investigation of i-motif stability, folding intermediates, and potential as in-situ pH sensor

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for low pH levels (pH < 6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Forster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the intermediate states visited during i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor to establish the time scale for the pH drop in a commonly used oxygen scavenging system.

Automated summary

This paper presents a collection of single molecule studies on the i-motif structure formed by the human telomeric sequence. The i-motif structure has recently gained attention as a physiologically relevant structure and a pH sensor. The study establishes single molecule Forster resonance energy transfer (smFRET) as a tool to investigate the i-motif under different pH and ionic conditions, revealing the pH and salt dependence of i-motif formation and capturing the intermediate folding states in real time. Furthermore, the i-motif is demonstrated to be a potential in-situ pH sensor.