Imperfect G-quadruplex as an emerging candidate for transcriptional regulation.

G-quadruplexes (G4s) with continuous G-tracts are well-established regulators of gene expression and important therapeutic targets for various diseases. However, bioinformatics analyses have identified G4-like sequences containing interrupted G-tracts, incorporating non-G nucleotides as bulges (buG4s). Our findings show that the stability of buG4s is significantly influenced by the bulge position and size within the G-tract, with bulges at the 5' end exhibiting the highest stability. Moreover, a molecular crowding condition inducing by poly (ethylene glycol), providing a suitable intracellular environment, stabilizes buG4s, especially those with longer bulges, making their formation more pronounced. A transcription assay performed under crowding conditions revealed that the transcription arrested efficiency by buG4s is affected not only by stability but also by the position and size of the bulge. Based on these findings, we propose a model for the preliminary screening of buG4 sequences according to their stability, distinguishing functional sequences capable of transcriptional arrest (ΔG°37 ≤ -3.3 kcal·mol-1) from nonfunctional sequences (ΔG°37 > -3.3 kcal·mol-1). This provides valuable insight into estimating the efficiency of target buG4 sequences in either arresting or facilitating transcription, presenting a novel approach and emphasizing buG4s as emerging therapeutic targets.