Selective targeting of parallel G-quadruplex structure using L-RNA aptamer.

G-quadruplexes (G4) are special nucleic acid structures with diverse conformational polymorphisms. Selective targeting of G-quadruplex conformations and regulating their biological functions provide promising therapeutic intervention. Despite the large repertoire of G4-binding tools, only a limited number of them can specifically target a particular G4 conformation. Here, we introduce a novel method, G4-SELEX-Seq and report the development of the first L-RNA aptamer, L-Apt12-6, with high binding selectivity to parallel G4 over other nucleic acid structures. Using parallel dG4 c-kit 1 as an example, we demonstrate the strong binding affinity between L-Apt12-6 and c-kit 1 dG4 in vitro and in cells, and notably report the applications of L-Apt12-6 in controlling DNA replication and gene expression. Our results suggest that L-Apt12-6 is a valuable tool for targeting parallel G-quadruplex conformation and regulating G4-mediated biological processes. Furthermore, G4-SELEX-Seq can be used as a general platform for G4-targeting L-RNA aptamers selection and should be applicable to other nucleic acid structures.

Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA.

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA-protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.

Mapping In Vivo RNA Structures and Interactions.

RNA folds to form diverse secondary and tertiary structures and often interacts with other biomolecules to function in cells. The technologies developed to map in vivo RNA structures and interactions can be broadly classified into four categories.

Revealing lncRNA Structures and Interactions by Sequencing-Based Approaches.

Long noncoding RNAs (lncRNAs) have emerged as significant players in almost every level of gene function and regulation. Thus, characterizing the structures and interactions of lncRNAs is essential for understanding their mechanistic roles in cells. Through a combination of (bio)chemical approaches and automated capillary and high-throughput sequencing (HTS), the complexity and diversity of RNA structures and interactions has been revealed in the transcriptomes of multiple species. These methods have uncovered important biological insights into the mechanistic and functional roles of lncRNA in gene expression and RNA metabolism, as well as in development and disease. In this review, we summarize the latest sequencing strategies to reveal RNA structure, RNA-RNA, RNA-DNA, and RNA-protein interactions, and highlight the recent applications of these approaches to map functional lncRNAs. We discuss the advantages and limitations of these strategies, and provide recommendations to further advance methodologies capable of mapping RNA structure and interactions in order to discover new biology of lncRNAs and decipher their molecular mechanisms and implication in diseases.

Peptides Selected by G4-mRNA Display-Seq Enable RNA G-Quadruplex Recognition and Gene Regulation.

G-quadruplexes (G4s) are noncanonical secondary structures that play critical roles in both chemistry and biology. Although several approaches have been developed for G4 targeting, such as chemicals and antibodies, there is currently no general and efficient platform for G4-specific peptides. In this study, we developed a new platform, G4-mRNA display-Seq, for selecting peptides that specifically recognize the G4 target of interest. By using an RNA G4 (rG4) found in human telomerase RNA (hTERC) as the target, we have identified a novel short peptide, namely, peptide 11 (pep11), which displays high affinity and selectivity to hTERC rG4. Furthermore, we designed tandem and cyclic versions of pep11 and found that both modified versions exhibit stronger binding affinity with preferential rG4 selectivity. Notably, we have demonstrated that these peptides can negatively regulate gene expression by targeting rG4. Our results provide a universal platform for the discovery of G4-targeting peptides and demonstrate the ability of these peptides to regulate G4-mediated gene functions.

Tuning Material States and Functionalities of G-Quadruplex-Modulated RNA-Peptide Condensates.

RNA encodes sequence- and structure-dependent interactions to modulate the assembly and properties of biomolecular condensates. RNA G-quadruplexes (rG4s) formed by guanine-rich sequences can trigger the formation of liquid- or solid-like condensates that are involved in many aberrant phase transitions. However, exactly how rG4 motifs modulate different phase transitions and impart distinct material properties to condensates is unclear. Here, using RNA oligonucleotides and cationic peptides as model systems, we show that RNA-peptide condensates exhibit tunability in material properties over a wide spectrum via interactions arising from rG4 folding/unfolding kinetics. rG4-containing oligonucleotides formed strong pairwise attraction with peptides and tended to form solid-like condensates, while their less-structured non-G4 mutants formed liquid-like droplets. We find that the coupling between rG4 dissociation and RNA-peptide complex coacervation triggers solid-to-liquid transition of condensates prior to the complete unfolding of rG4s. This coupling points to a mechanism that material states of rG4-modulated condensates can be finely tuned from solid-like to liquid-like by the addition of less-structured RNA oligonucleotides, which have weak but dominant binding with peptides. We further show that the tunable material states of condensates can enhance RNA aptamer compartmentalization and RNA cleavage reactions. Our results suggest that condensates with complex properties can emerge from subtle changes in RNA oligonucleotides, contributing ways to treat dysfunctional condensates in diseases and insights into prebiotic compartmentalization.

Discovery of G-quadruplex-forming sequences in SARS-CoV-2.

The outbreak caused by the novel coronavirus SARS-CoV-2 has been declared a global health emergency. G-quadruplex structures in genomes have long been considered essential for regulating a number of biological processes in a plethora of organisms. We have analyzed and identified 25 four contiguous GG runs (G2NxG2NyG2NzG2) in the SARS-CoV-2 RNA genome, suggesting putative G-quadruplex-forming sequences (PQSs). Detailed analysis of SARS-CoV-2 PQSs revealed their locations in the open reading frames of ORF1 ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes. Identical PQSs were also found in the other members of the Coronaviridae family. The top-ranked PQSs at positions 13385 and 24268 were confirmed to form RNA G-quadruplex structures in vitro by multiple spectroscopic assays. Furthermore, their direct interactions with viral helicase (nsp13) were determined by microscale thermophoresis. Molecular docking model suggests that nsp13 distorts the G-quadruplex structure by allowing the guanine bases to be flipped away from the guanine quartet planes. Targeting viral helicase and G-quadruplex structure represents an attractive approach for potentially inhibiting the SARS-CoV-2 virus.

Circular L-RNA aptamer promotes target recognition and controls gene activity.

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.

RNA G-quadruplexes (rG4s): genomics and biological functions.

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.

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.

Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0.

BACKGROUND: RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue. RESULTS: We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance. CONCLUSIONS: rG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.

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.

An RNA G-Quadruplex Structure within the ADAR 5'UTR Interacts with DHX36 Helicase to Regulate Translation.

RNA G-quadruplex (rG4) structures in the 5' untranslated region (5'UTR) play crucial roles in fundamental cellular processes. ADAR is an important enzyme that binds to double-strand RNA and accounts for the conversion of Adenosine to Inosine in RNA editing. However, so far there is no report on the formation and regulatory role of rG4 on ADAR expression. Here, we identify and characterize a thermostable rG4 structure within the 5'UTR of the ADAR1 mRNA and demonstrate its formation and inhibitory role on translation in reporter gene and native gene constructs. We reveal rG4-specific helicase DHX36 interacts with this rG4 in vitro and in cells under knockdown and knockout conditions by GTFH (G-quadruplex-triggered fluorogenic hybridization) probes and modulates translation in an rG4-dependent manner. Our results further substantiate the rG4 structure-DHX36 protein interaction in cells and highlight rG4 to be a key player in controlling ADAR1 translation.

Proteomic and Transcriptome Profiling of G-Quadruplex Aptamers Developed for Cell Internalization.

Nucleic acid medicine is expected to be among the most promising next-generation therapies. Applications of nucleic acid in vivo are still challenging as a result of the difficulties in direct cell penetration without external assistance. To facilitate the cellular delivery of therapeutic nucleic acid, we developed cell-penetrating aptamers using cell-internalization Systematic Evolution of Ligands by EXponential enrichment (SELEX). Moreover, C20-4 min, a G-quadruplex-forming DNA aptamer, was discovered, showing a higher cell-penetrating capacity compared with other candidates, including AS1411. To verify the formation and understand the G-quadruplex folding topologies of enriched aptamer motifs, characteristic circular dichroism (CD) spectral features are analyzed. The CD spectra of C20-4 min strongly support the formation of parallel G-quadruplexes. Systematic analyses of the G-quadruplex regulation pathway have been performed by combining aptamer pull-down with mass spectrometry. We profiled G-quadruplex aptamers interacting with cellular proteins during internalization and identified helicases and GTPase proteins as cellular interacting partners. In addition, whole transcriptome analysis was performed to study the effects of G-quadruplex aptamers, revealing differentially expressed genes involved in the regulation of GTPase functions. Integrative analyses of transcriptome and proteomic have aided in understanding the functional hierarchy of molecular players in G-quadruplex nucleic acid mechanisms of internalization, which might facilitate developing a novel delivery system.

COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

Effect of RNA sequence context and stereochemistry on G-quadruplex-RHAU53 interaction.

RNA G-quadruplex (rG4) structure and its association with rG4-binding proteins/peptides are important for its function. However, there is very limited study that investigates what factors are involved in rG4 that drive the rG4-protein/peptide interaction. Here we study and uncover the effect of RNA sequence context and stereochemistry on G-quadruplex-peptide interaction. Using rG4-binding RHAU53 peptide as an example, we report that the number of G-quartet, thermostability, overhanging nucleotides, and RNA base chirality have an impact on rG4-RHAU53 binding. Notably, our data also demonstrate that RHAU53 preferentially binds to 5' G-quartet over 3' G-quartet, and showcase that RHAU53 interacts with unnatural L-rG4 for the first time. Our findings reported here offer unique insights to the potential development of targeting tools that recognize rG4 structure and rG4-binding peptide/protein.

rG4-seeker enables high-confidence identification of novel and non-canonical rG4 motifs from rG4-seq experiments.

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.

In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.

RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

Specific Binding of a d-RNA G-Quadruplex Structure with an l-RNA Aptamer.

G-quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4-binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l-RNA aptamer that targets a d-RNA G-quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l-RNA aptamer, Ap3-7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA-rG4-RHAU53 binding. Our approach and findings open a new door in further developing G4-specific tools for diverse applications.

The RNA structurome: transcriptome-wide structure probing with next-generation sequencing.

RNA folds into intricate structures that enable its pivotal roles in biology, ranging from regulation of gene expression to ligand sensing and enzymatic functions. Therefore, elucidating RNA structure can provide profound insights into living systems. A recent marriage between in vivo RNA structure probing and next-generation sequencing (NGS) has revolutionized the RNA field by enabling transcriptome-wide structure determination in vivo, which has been applied to date to human cells, yeast cells, and Arabidopsis seedlings. Analysis of resultant in vivo 'RNA structuromes' provides new and important information regarding myriad cellular processes, including control of translation, alternative splicing, alternative polyadenylation, energy-dependent unfolding of mRNA, and effects of proteins on RNA structure. An emerging view suggests potential links between RNA structure and stress and disease physiology across the tree of life. As we discuss here, these exciting findings open new frontiers into RNA biology, genome biology, and beyond.