G-quadruplexes (G4s) are non-classical DNA or RNA secondary structures that have been first observed decades ago. Over the years, these four-stranded structural motifs have been demonstrated to have significant regulatory roles in diverse biological processes, but challenges remain in detecting them globally and reliably. Compared to DNA G4s (dG4s), the study of RNA G4s (rG4s) has received less attention until recently. In this review, we will summarize the innovative high-throughput methods recently developed to detect rG4s on a transcriptome-wide scale, highlight the many novel and important functions of rG4 being discovered in vivo across the tree of life, and discuss the key biological questions to be addressed in the near future.

This work introduces a multidisciplinary strategy to characterize the structure, folding status, and function of the RNA G-quadruplex in APP 3′UTR.

We recently developed the rG4-seq method to detect and map in vitro RNA G-quadruplex (rG4s) structures on a transcriptome-wide scale. rG4-seq of purified human HeLa RNA has revealed many non-canonical rG4s and the effects adjacent sequences have on rG4 formation. In this study, we aimed to improve the outcomes and false-positive discrimination in rG4-seq experiments using a bioinformatic approach. By establishing connections between rG4-seq library preparation chemistry and the underlying properties of sequencing data, we identified how to mitigate indigenous sampling errors and background noise in rG4-seq. We applied these findings to develop a novel bioinformatics pipeline named rG4-seeker ( https://github.com/TF-Chan-Lab/rG4-seeker ), which uses tailored noise models to autonomously assess and optimize rG4 detections in a replicate-independent manner. Compared with previous methods, rG4-seeker exhibited better false-positive discrimination and improved sensitivity for non-canonical rG4s. Using rG4-seeker, we identified novel features in rG4 formation that were missed previously. rG4-seeker provides a reliable and sensitive approach for rG4-seq investigations, laying the foundations for further elucidation of rG4 biology.

Rational design of aptamers to incorporate unnatural nucleotides and special chemical moieties can expand their functional complexity and diversity. Spiegelmer (L-RNA aptamer) is a unique class of aptamer that is composed of unnatural L-RNA nucleotides, and so far there are limited L-RNA aptamer candidates and applications being reported. Moreover, the target binding properties of current L-RNA aptamers require significant improvement. Here, using L-Apt.4-1c as an example, we develop a simple and robust strategy to generate the first circular L-RNA aptamer, cycL-Apt.4-1c, quantitatively, demonstrate substantial enhancement in binding affinity and selectivity toward its target, and notably report novel applications of circular L-RNA aptamer in controlling RNA–protein interaction, and gene activity including telomerase activity and gene expression. Our approach and findings will be applicable to any L-RNA aptamers and open up a new avenue for diverse applications.

ABSTRACTWe recently developed the rG4-seq method to detect and map in vitro RNA G-quadruplex (rG4s) structures on a transcriptome-wide scale. rG4-seq of purified human HeLa RNA has revealed many non-canonical rG4s and the effects adjacent sequences have on rG4 formation. In this study, we aimed to improve the outcomes and false-positive discrimination in rG4-seq experiments using a bioinformatic approach. By establishing connections between rG4-seq library preparation chemistry and the underlying properties of sequencing data, we identified how to mitigate indigenous sampling errors and background noise in rG4-seq. We applied these findings to develop a novel bioinformatics pipeline named rG4-seeker(https://github.com/TF-Chan-Lab/rG4-seeker), which uses tailored noise models to autonomously assess and optimize rG4 detections in a replicate-independent manner. Compared with previous methods, rG4-seeker exhibited better false-positive discrimination and improved sensitivity for non-canonical rG4s. Using rG4-seeker, we identified novel features in rG4 formation that were missed previously. rG4-seeker provides a reliable and sensitive approach for rG4-seq investigations, laying the foundations for further elucidation of rG4 biology.