Dahye Im (POSTECH)

Jongcheol Seo (POSTECH)

Dahye Im (POSTECH)

Jongcheol Seo (POSTECH)

It is well established that DNA and RNA hairpins play important regulatory roles in transcription and replication. The functional dynamics of hairpins are closely linked to their structural stability. This means that attempting to quantify their stability or elucidate their unzipping mechanisms is useful in improving our understanding of hairpin structures. One of the most widely used classical methods is measuring thermal stability, known as the melting temperature (T_m). Recent studies employing T-jump IR spectroscopy have suggested that DNA hairpins melt from the terminal end of the stem toward the loop. In this study, we propose a novel approach that utilises ion mobility spectrometry–mass spectrometry (IMS-MS) to investigate the structural transitions of hairpins, thereby complementing traditional approaches. During electrospray ionisation (ESI), hairpins acquire charges that result in charge-induced unzipping pathways. Three trends were identified: firstly, a native folded structure at low charge states; secondly, a stable intermediate between folded and unfolded structures; and thirdly, a linear unfolded structure at high charge states. We propose that this stable intermediate represents a partially opened structure at the bottom of the stem, consistent with previous studies. The arbitrary charge at which half of the hairpin becomes linear has been found to correlate closely with the melting temperature, extending the observable range below the boiling point. This approach can be expanded to encompass a range of secondary structures in DNA and RNA, including G-quadruplexes.