Zhang Chuyang, Nizal Hariz, Hennecker Christopher, Mittermaier Anthony
Department of Chemistry, McGill University, 801 Sherbrooke St. W., Montreal, QC H3A 0B8, Canada.
Nucleic Acids Res. 2025 Aug 27;53(16). doi: 10.1093/nar/gkaf815.
i-Motifs (iMs) are four-stranded non-canonical nucleic acid secondary structures that are formed by cytosine-rich sequences. Putative iM-forming sequences are concentrated in human promoter and telomeric regions, suggesting possible biological roles. However, many iMs do not readily fold at neutral pH, sparking interest in factors that may stabilize them. We performed a systematic study on how the nucleotides flanking iMs affect their stabilities and folding kinetics. We found that the mere presence of flanking nucleotides led to dramatically slower folding and lower stability compared to isolated iMs. Conversely, complementary flanking nucleotides that comprise an inverted repeat and form a hairpin with the iM in the loop led to greater stability and faster folding than the iM on its own. A bioinformatic analysis of human promoter regions showed that the flanking regions are more likely than average to be complementary to each other, suggesting that this stabilization might be biologically relevant. We analyzed several naturally occurring iM sequences and found that complementary flanking regions substantially stabilized the structures (up to 64-fold faster folding and 17.2°C more stable). Our results show that the regions of DNA flanking iMs are an important and hitherto overlooked factor in iM folding and stability.
i-基序（iMs）是由富含胞嘧啶的序列形成的四链非经典核酸二级结构。推测的iM形成序列集中在人类启动子和端粒区域，表明其可能具有生物学作用。然而，许多iM在中性pH值下不易折叠，这引发了人们对可能使其稳定的因素的兴趣。我们对iM两侧的核苷酸如何影响其稳定性和折叠动力学进行了系统研究。我们发现，与孤立的iM相比，仅仅是iM两侧存在核苷酸就会导致折叠速度大幅减慢和稳定性降低。相反，由反向重复序列组成并与环中的iM形成发夹结构的互补侧翼核苷酸，比单独的iM具有更高的稳定性和更快的折叠速度。对人类启动子区域的生物信息学分析表明，侧翼区域比平均水平更有可能相互互补，这表明这种稳定作用可能具有生物学相关性。我们分析了几个天然存在的iM序列，发现互补的侧翼区域极大地稳定了结构（折叠速度快达64倍，稳定性高17.2°C）。我们的结果表明，iM两侧的DNA区域是iM折叠和稳定性的一个重要但迄今被忽视的因素。
