ABSTRACT
RNA G-quadruplex structures (rG4s) play important roles in the regulation of biological processes. So far, all the l-RNA aptamers developed to target rG4 of interest contain G4 motif itself, raising the question of whether non-G4-containing l-RNA aptamer can be developed to target rG4. Furthermore, it is unclear whether an l-Aptamer-based tool can be generated for G4 detection in vitro and imaging in cells. Herein, a new strategy is designed using a low GC content template library to develop a novel non-G4-containing l-RNA aptamer with strong binding affinity and improved binding specificity to rG4 of interest. The first non-G4-containing l-Aptamer, l-Apt.1-1, is identified with nanomolar binding affinity to amyloid precursor protein (APP) D-rG4. l-Apt.1-1 is applied to control APP gene expression in cells via targeting APP D-rG4 structure. Moreover, the first l-RNA-based fluorogenic bi-functional aptamer (FLAP) system is developed, and l-Apt.1-1_Pepper is engineered for in vitro detection and cellular imaging of APP D-rG4. This work provides an original approach for developing non-G4-containing l-RNA aptamer for rG4 targeting, and the novel l-Apt.1-1 developed for APP gene regulation, as well as the l-Apt.1-1_Pepper generated for imaging of APP rG4 structure can be further used in other applications in vitro and in cells.
ABSTRACT
G-quadruplexes (dG4 and rG4) are nucleic acid secondary structures formed by the self-assembly of certain G-rich sequences, and they have distinctive chemical properties and play crucial roles in fundamental biological processes. Small molecule G4 ligands were shown to be crucial in characterizing G4s and understanding their functions. Nevertheless, concerns regarding the specificity of these synthetic ligands for further investigation of G4s, especially for rG4 isolation purposes, have been raised. In comparison to G4 ligands, we propose a novel magnetic bead-based pulldown assay that enables the selective capture of general rG4s using functionalized l-Apt.4-1c from both simple buffer and complex media, including total RNA and the cell lysate. We found that our l-RNA aptamer can pulldown general rG4s with a higher efficiency and specificity than the G4 small molecule ligand BioTASQ v.1 in the presence of non-target competitors, including dG4 and non-G4 structures. Our findings reveal that biotinylated l-aptamers can serve as effective molecular tools for the affinity-based enrichment of rG4 of interest using this new assay, which was also verified by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) on endogenous transcripts. This work provides new and important insights into rG4 isolation using a functionalized l-aptamer, which can potentially be applied in a transcript-specific or transcriptome-wide manner in the future.
ABSTRACT
DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 103 to 3.7 × 107 CFU/mL, achieving a detection limit of 1.3 × 103 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella typhimurium. Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.
Subject(s)
Biosensing Techniques , DNA, Catalytic , DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , Biosensing Techniques/methods , Bacteria/isolation & purification , Bacteria/genetics , Limit of Detection , Nucleic Acid Amplification Techniques , Escherichia coli/isolation & purification , Escherichia coli/geneticsABSTRACT
RNA G-quadruplexes (rG4s) are non-canonical RNA secondary structures that were first reported several decades ago. Latest studies have suggested that they are widespread in the transcriptomes of diverse species, and they have been demonstrated to have key roles in various fundamental cellular processes. Among the RNA secondary structure probing assays developed recently, Reverse transcriptase stalling (RTS) and selective 2'-hydroxyl acylation analyzed by lithium ion-based primer extension (SHALiPE) enabled the identification and characterization of distinct structural features of an rG4 structure of interest. Herein, we present an experimental protocol describing in detail the procedures involved in the preparation of in vitro transcribed RNAs, buffers, and reagents for RTS and SHALiPE assays, as well as performing RTS and SHALiPE assays, to examine the formation of rG4 and reveal the rG4 structural conformation at nucleotide resolution in vitro. RTS and SHALiPE assays can be performed by an experienced molecular biologist or chemical biologist with a basic understanding of nucleic acids. The duration for the preparation of in vitro transcription and RNA preparation is around 2 days, and the duration for RTS and SHALiPE assays is approximately 5 h.
Subject(s)
G-Quadruplexes , RNA , RNA/chemistry , RNA-Directed DNA Polymerase , Transcriptome , NucleotidesABSTRACT
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.
Subject(s)
Aptamers, Nucleotide , G-Quadruplexes , Nucleic Acids , Aptamers, Nucleotide/chemistryABSTRACT
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.
Subject(s)
G-Quadruplexes , Humans , RNA/genetics , RNA/chemistry , Gene Expression Regulation , Peptides/genetics , RNA, Messenger/geneticsABSTRACT
We identify and characterize an RNA G-quadruplex (rG4) structure motif in the human microRNA 638 (hsa-miR-638). We investigate the formation and role of this rG4 in vitro and in cells, and reveal that it inhibits the miR-638 and MEF2C messenger RNA interaction and controls gene expression at the translational level.
Subject(s)
G-Quadruplexes , MicroRNAs , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , MicroRNAs/genetics , RNA/chemistry , Gene ExpressionABSTRACT
Aptamers are single-stranded oligonucleotides that bind to their targets via specific structural interactions. To improve the properties and performance of aptamers, modified nucleotides are incorporated during or after a selection process such as systematic evolution of ligands by exponential enrichment (SELEX). We summarize the latest modified nucleotides and strategies used in modified (mod)-SELEX and post-SELEX to develop modified aptamers, highlight the methods used to characterize aptamer-target interactions, and present recent progress in modified aptamers that recognize different targets. We discuss the challenges and perspectives in further advancing the methodologies and toolsets to accelerate the discovery of modified aptamers, improve the throughput of aptamer-target characterization, and expand the functional diversity and complexity of modified aptamers.
ABSTRACT
Watson-Crick base-pairing of DNA allows the nanoscale fabrication of biocompatible synthetic nanostructures for diagnostic and therapeutic biomedical purposes. DNA nanostructure design elicits exquisite control of shape and conformation compared to other nanoparticles. Furthermore, nucleic acid aptamers can be coupled to DNA nanostructures to allow interaction and response to a plethora of biomolecules beyond nucleic acids. When compared to the better-known approach of using protein antibodies for molecular recognition, nucleic acid aptamers are bespoke with the underlying DNA nanostructure backbone and have various other stability, synthesis, and cost advantages. Here, we provide detailed methodologies to synthesize and characterize aptamer-enabled DNA nanostructures. The methods described can be generally applied to various designs of aptamer-enabled DNA nanostructures with a wide range of applications both within and beyond biomedical nanotechnology.
Subject(s)
Aptamers, Nucleotide , Nanostructures , Nucleic Acids , Aptamers, Nucleotide/chemistry , Nanostructures/chemistry , DNA/chemistry , Nanotechnology/methods , Nucleic Acids/chemistry , Nucleic Acid ConformationABSTRACT
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.
Subject(s)
Aptamers, Nucleotide , G-Quadruplexes , RNA/chemistry , Aptamers, Nucleotide/chemistry , GuanineABSTRACT
SARS-CoV-2, the causative agent of COVID-19, remains among the main causes of global mortality. Although antigen/antibody-based immunoassays and neutralizing antibodies targeting SARS-CoV-2 have been successfully developed over the past 2 years, they are often inefficient and unreliable for emerging SARS-CoV-2 variants. Novel approaches against SARS-CoV-2 and its variants are therefore urgently needed. Aptamers have been developed for the detection and inhibition of several different viruses such as HIV, influenza viruses, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV. Aptamers targeting SARS-CoV-2 represent a promising tool in the fight against COVID-19, which is of paramount importance for the current and any future pandemics. This review presents recent advances and future trends in the development of aptamer-based approaches for SARS-CoV-2 diagnosis and treatment.
Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Humans , COVID-19/diagnosis , SARS-CoV-2 , COVID-19 TestingABSTRACT
Small-molecule contaminants, such as antibiotics, pesticides, and plasticizers, have emerged as one of the substances most detrimental to human health and the environment. Therefore, it is crucial to develop low-cost, user-friendly, and portable biosensors capable of rapidly detecting these contaminants. Antibodies have traditionally been used as biorecognition elements. However, aptamers have recently been applied as biorecognition elements in aptamer-based biosensors, also known as aptasensors. The systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro technique used to generate aptamers that bind their targets with high affinity and specificity. Over the past decade, a modified SELEX method known as Capture-SELEX has been widely used to generate DNA or RNA aptamers that bind small molecules. In this review, we summarize the recent strategies used for Capture-SELEX, describe the methods commonly used for detecting and characterizing small-molecule-aptamer interactions, and discuss the development of aptamer-based biosensors for various applications. We also discuss the challenges of the Capture-SELEX platform and biosensor development and the possibilities for their future application.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Humans , SELEX Aptamer Technique/methods , Ligands , Biosensing Techniques/methods , AntibodiesABSTRACT
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.
Subject(s)
G-Quadruplexes , Humans , RNA/chemistry , Transcriptome , HEK293 Cells , Sequence Analysis, RNA/methodsABSTRACT
Nucleotide composition is suggested to infer gene functionality and ecological adaptation of species to distinct environments. However, the underlying biological function of nucleotide composition dictating environmental adaptations is largely unknown. Here, we systematically analyze the nucleotide composition of transcriptomes across 1000 plants (1KP) and their corresponding habitats. Intriguingly, we find that plants growing in cold climates have guanine (G)-enriched transcriptomes, which are prone to forming RNA G-quadruplex structures. Both immunofluorescence detection and in vivo structure profiling reveal that RNA G-quadruplex formation in plants is globally enhanced in response to cold. Cold-responsive RNA G-quadruplexes strongly enhanced mRNA stability, rather than affecting translation. Disruption of individual RNA G-quadruplex promotes mRNA decay in the cold, leading to impaired plant cold response. Therefore, we propose that plants adopted RNA G-quadruplex structure as a molecular signature to facilitate their adaptation to the cold during evolution.
Subject(s)
G-Quadruplexes , RNA/genetics , RNA/chemistry , Guanine/chemistry , RNA Stability , NucleotidesABSTRACT
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.
Subject(s)
G-Quadruplexes , 5' Untranslated Regions , RNA, Messenger/metabolismABSTRACT
Plasmodium falciparum, a protozoan parasite and causative agent of human malaria, has one of the most A/T-biased genomes sequenced to date. This may give the genome and the transcriptome unusual structural features. Recent progress in sequencing techniques has made it possible to study the secondary structures of RNA molecules at the transcriptomic level. Thus, in this study we produced the in vivo RNA structurome of a protozoan parasite with a highly A/U-biased transcriptome. We showed that it is possible to probe the secondary structures of P. falciparum RNA molecules in vivo using two different chemical probes, and obtained structures for more than half of all transcripts in the transcriptome. These showed greater stability (lower free energy) than the same structures modelled in silico, and structural features appeared to influence translation efficiency and RNA decay. Finally, we compared the P. falciparum RNA structurome with the predicted RNA structurome of an A/U-balanced species, P. knowlesi, finding a bias towards lower overall transcript stability and more hairpins and multi-stem loops in P. falciparum. This unusual protozoan RNA structurome will provide a basis for similar studies in other protozoans and also in other unusual genomes.
Subject(s)
Malaria, Falciparum , Malaria , Parasites , Animals , Genome, Protozoan , Humans , Malaria/genetics , Malaria, Falciparum/parasitology , Parasites/genetics , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , RNA , RNA, Protozoan/genetics , TranscriptomeABSTRACT
Adult muscle stem cells, also known as satellite cells (SCs), play pivotal roles in muscle regeneration, and long non-coding RNA (lncRNA) functions in SCs remain largely unknown. Here, we identify a lncRNA, Lockd, which is induced in activated SCs upon acute muscle injury. We demonstrate that Lockd promotes SC proliferation; deletion of Lockd leads to cell-cycle arrest, and in vivo repression of Lockd in mouse muscles hinders regeneration process. Mechanistically, we show that Lockd directly interacts with RNA helicase DHX36 and the 5'end of Lockd possesses the strongest binding with DHX36. Furthermore, we demonstrate that Lockd stabilizes the interaction between DHX36 and EIF3B proteins; synergistically, this complex unwinds the RNA G-quadruplex (rG4) structure formed at Anp32e mRNA 5' UTR and promotes the translation of ANP32E protein, which is required for myoblast proliferation. Altogether, our findings identify a regulatory Lockd/DHX36/Anp32e axis that promotes myoblast proliferation and acute-injury-induced muscle regeneration.
Subject(s)
DEAD-box RNA Helicases , G-Quadruplexes , Molecular Chaperones , Muscle Development , Myoblasts , RNA, Long Noncoding , 5' Untranslated Regions , Animals , Cell Proliferation , DEAD-box RNA Helicases/metabolism , Mice , Molecular Chaperones/metabolism , Muscles/metabolism , Myoblasts/cytology , RNA, Long Noncoding/metabolism , RegenerationABSTRACT
Guanine quadruplex (G4) structure is a four-stranded nucleic acid secondary structure motif with unique chemical properties and important biological roles. Amyloid precursor protein (APP) is an Alzheimer's disease (AD)-related gene, and recently, we reported the formation of RNA G4 (rG4) at the 3'UTR of APP mRNA and demonstrated its repressive role in translation. Herein, we apply rG4-SELEX to develop a novel L-RNA aptamer, L-Apt.8f, which binds to APP 3'UTR D-rG4 strongly with subnanomolar affinity. We structurally characterize the aptamer and find that it contains a thermostable and parallel G4 motif, and mutagenesis analysis identifies the key nucleotides that are involved in the target recognition. We also reveal that the L-Apt.8f-APP D-rG4 interaction is enantiomeric-, magnesium ion-, and potassium ion-dependent. Notably, L-Apt.8f preferentially recognizes APP rG4 over other structural motifs, and it can control the APP reporter gene and native transcript translation in cells. Our work introduces a novel strategy and reports a new L-aptamer candidate to target APP 3'UTR rG4 structure, which laid the foundation for further applying L-RNA as an important class of biomolecule for practical L-aptamer-based targeting and controlling of gene expression in cells.
Subject(s)
Aptamers, Nucleotide , G-Quadruplexes , 3' Untranslated Regions , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Aptamers, Nucleotide/chemistry , Gene ExpressionABSTRACT
RNA structures are essential to support RNA functions and regulation in various biological processes. Recently, a range of novel technologies have been developed to decode genome-wide RNA structures and novel modes of functionality across a wide range of species. In this review, we summarize key strategies for probing the RNA structurome and discuss the pros and cons of representative technologies. In particular, these new technologies have been applied to dissect the structural landscape of the SARS-CoV-2 RNA genome. We also summarize the functionalities of RNA structures discovered in different regulatory layers-including RNA processing, transport, localization, and mRNA translation-across viruses, bacteria, animals, and plants. We review many versatile RNA structural elements in the context of different physiological and pathological processes (e.g., cell differentiation, stress response, and viral replication). Finally, we discuss future prospects for RNA structural studies to map the RNA structurome at higher resolution and at the single-molecule and single-cell level, and to decipher novel modes of RNA structures and functions for innovative applications.