Development of RNA G-quadruplex (rG4)-targeting L-RNA aptamers by rG4-SELEX.

RNA G-quadruplex (rG4)-SELEX is a method that generates L-RNA aptamers to target an rG4 structure of interest, which can be applied to inhibit G-quadruplex-mediated interactions that have important roles in gene regulation and function. Here we present a Protocol Extension substantially modifying an existing SELEX protocol to describe in detail the procedures involved in performing rG4-SELEX to identify rG4-specific binders that can effectively suppress rG4-peptide and rG4-protein associations. This Protocol Extension improves the speed of aptamer discovery and identification, offering a suite of techniques to characterize the aptamer secondary structure and monitor binding affinity and specificity, and demonstrating the utility of the L-RNA aptamer. The previous protocol mainly describes the identification of RNA aptamers against proteins of interest, whereas in this Protocol Extension we present the development of an unnatural RNA aptamer against an RNA structure of interest, with the potential to be applicable to other nucleic acid motifs or biomolecules. rG4-SELEX starts with a random D-RNA library incubated with the L-rG4 target of interest, followed by binding, washing and elution of the library. Enriched D-aptamer candidates are sequenced and structurally characterized. Then, the L-aptamer is synthesized and used for different applications. rG4-SELEX can be carried out by an experienced molecular biologist with a basic understanding of nucleic acids. The development of rG4-targeting L-RNA aptamers expands the current rG4 toolkit to explore innovative rG4-related applications, and opens new doors to discovering novel rG4 biology in the near future. The duration of each selection cycle as outlined in the protocol is ~2 d.

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum.

G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

Specific suppression of D-RNA G-quadruplex-protein interaction with an L-RNA aptamer.

G-quadruplexes (G4s) are nucleic acid structure motifs that are of significance in chemistry and biology. The function of G4s is often governed by their interaction with G4-binding proteins. Few categories of G4-specific tools have been developed to inhibit G4-protein interactions; however, until now there is no aptamer tool being developed to do so. Herein, we present a novel L-RNA aptamer that can generally bind to D-RNA G-quadruplex (rG4) structure, and interfere with rG4-protein interaction. Using hTERC rG4 as the target for in vitro selection, we report the shortest L-aptamer being developed so far, with only 25 nucleotides. Notably, this new aptamer, L-Apt.4-1c, adopts a stem-loop structure with the loop folding into an rG4 motif with two G-quartet, demonstrates preferential binding toward rG4s over non-G4s and DNA G-quadruplexes (dG4s), and suppresses hTERC rG4-nucleolin interactions. We also show that inhibition of rG4-protein interaction using L-RNA aptamer L-Apt.4-1c is comparable to or better than G4-specific ligands such as carboxypyridostatin and QUMA-1 respectively, highlighting that our approach and findings expand the current G4 toolbox, and open a new avenue for diverse applications.

G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications.

Guanine (G)-quadruplexes (G4s) are unique nucleic acid structures that are formed by stacked G-tetrads in G-rich DNA or RNA sequences. G4s have been reported to play significant roles in various cellular events in both macro- and micro-organisms. The identification and characterization of G4s can help to understand their different biological roles and potential applications in diagnosis and therapy. In addition to biophysical and biochemical methods to interrogate G4 formation, G4 fluorescent turn-on ligands can be used to target and visualize G4 formation both in vitro and in cells. Here, we review several representative classes of G4 fluorescent turn-on ligands in terms of their interaction mechanism and application perspectives. Interestingly, G4 structures are commonly identified in DNA and RNA aptamers against targets that include proteins and small molecules, which can be utilized as G4 tools for diverse applications. We therefore also summarize the recent development of G4-containing aptamers and highlight their applications in biosensing, bioimaging, and therapy. Moreover, we discuss the current challenges and future perspectives of G4 fluorescent turn-on ligands and G4-containing aptamers.

Spectroscopic analysis reveals the effect of a single nucleotide bulge on G-quadruplex structures.

Here we investigate and reveal the effect of bulge position and bulge identity on G-quadruplexes using label-free spectroscopic techniques. Notably, we report significant differences in the spectroscopic features of bulged DNA and RNA G-quadruplexes, and demonstrate that intrinsic fluorescence can be generally used to detect the formation of canonical and non-canonical G-quadruplexes.

Structural analysis reveals the formation and role of RNA G-quadruplex structures in human mature microRNAs.

Here we identify hundreds of RNA G-quadruplex (rG4) candidates in microRNAs (miRNAs), characterize the miRNA structure and miRNA-mRNA interactions on several mammalian-conserved miRNAs, and reveal the formation of rG4s in miRNAs. Notably, we study the effect of these rG4s in cells and uncover the role of rG4s in miRNA-mediated post-transcriptional regulation.