RESUMEN
G-quadruplexes (G4s) are reported to present on the SARS-CoV-2 RNA genome and control various viral activities. Specific ligands targeting those viral nucleic acid structures could be investigated as promising detection methods or antiviral reagents to suppress this menacing virus. Herein, we demonstrate the binding between a G4 structure in the RNA of SARS-CoV-2 and a fluorescent probe created by fusing a parallel-G4 specific RHAU53 and a cyan fluorescent protein. The specific binding of G4 in SARS-CoV-2 by RHAU peptide was easily detected under the fluorescence spectrometer. The drawbacks of this approach and potential solutions are also discussed.
RESUMEN
G-quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich sequences, which are ubiquitously found in the human genome and transcriptome. Targeting G4s by specific ligands provides a powerful tool to monitor and regulate G4s-associated biological processes. RHAU peptides, derived from the G4-binding motif of "RNA Helicase associated with AU-rich element" (RHAU), have emerged as extraordinary ligands for specific recognition of parallel G4s. This review highlights the significances of recent studies investigating potential applications of the engineered RHAU peptides incorporated to different functional moieties.
Asunto(s)
G-Cuádruplex , Humanos , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Péptidos/genética , BiologíaRESUMEN
Protein dimerization plays a key role in many biological processes. Most cellular events such as enzyme activation, transcriptional cofactor recruitment, signal transduction, and even pathogenic pathways are significantly regulated via protein-protein interactions. Understanding and controlling the molecular mechanisms that regulate protein dimerization is crucial for biomedical applications. The limitations of engineered protein dimerization provide an opportunity for molecular chemistry to induce dimerization of protein in biological events. In this review, molecular control over dimerization of protein and activation in this respect are discussed. The well known molecule glue-based approaches to induced protein dimerization provide powerful tools to modulate the functionality of dimerized proteins and are shortly highlighted. Subsequently metal ion, nucleic acid and host-guest chemistry are brought forward as novel approaches for orthogonal control over dimerization of protein. The specific focus of the review will be on host-guest systems as novel, robust and versatile supramolecular approaches to modulate the dimerization of proteins, using functional proteins as model systems.
RESUMEN
We generated a novel G-quadruplex (G4)-specific endonuclease by fusing a G4 recognition domain of the RHAU helicase with a cleavage domain of the Fok1 nuclease. The fusion protein can specifically bind a parallel G4 and cleave a double-stranded DNA (dsDNA) next to it. The new endonuclease could be used to detect a G4 in a long dsDNA, providing a useful tool for mapping the formation of G4s in the genome.
Asunto(s)
Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , G-CuádruplexRESUMEN
We studied parallel G4-mediated protein dimerization and activation by incorporating a RHAU peptide with a fluorescent protein FRET pair CFP/YFP and an apoptotic casp9. Occurrence of energy tranfer (from donor CFP to acceptor YFP) and enhancement of 60-fold cleavage efficiency of casp9 were observed in the presence of parallel G4, which indicated that parallel G4 can induce dimerization and activation of proteins. This novel approach holds a great promise for studying G4-targeting functional dimeric proteins in celllular biology.
RESUMEN
We developed a ribonuclease for site-specific targeting and cleavage of single-stranded RNA. The engineered RNase protein was constructed by incorporating two independent functional domains, an RNase HI domain that could cleave the RNA strand in a DNA-RNA hybrid, and a domain of the RHAU protein that could selectively recognize a parallel DNA G-quadruplex (G4). The newly designed RNase first recruits a DNA guide oligonucleotide containing both a parallel G4 motif and a template sequence complementary to the target RNA. This RNase:DNA complex targets and efficiently cleaves the single-stranded RNA in a site-specific manner. A major cleavage site occurs at the RNA region that is complementary to the DNA template sequence. The newly designed RNase can serve as a simple tool for RNA manipulation and probing RNA structure.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , División del ARN/fisiología , Ribonucleasa H/metabolismo , ARN Helicasas DEAD-box/fisiología , ADN/metabolismo , G-Cuádruplex , Oligonucleótidos/genética , Ingeniería de Proteínas/métodos , ARN/metabolismo , División del ARN/genética , Ribonucleasa H/fisiología , Ribonucleasas/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.
Asunto(s)
ADN/análisis , Colorantes Fluorescentes/química , G-Cuádruplex , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/química , Péptidos/química , ARN/análisis , Colorantes Fluorescentes/análisis , Péptidos/análisisRESUMEN
The low solubility of sugars has hampered the lipase-catalyzed synthesis of fatty acid sugar esters in organic solvents and ionic liquids (ILs), because several solvents that are able to effectively dissolve sugars are detrimental to enzymes. In this work, in order to prepare a high concentration of sugars in ILs, we have developed a new procedure that entails mixing an aqueous sugar solution into ILs followed by removal of the water from the solution. The glucose concentrations in the supersaturated [Emim][TfO] and [Bmim][TfO] were 19 and 10 times higher, respectively, than the solubilities (6.1 and 4.8 g/L) of glucose in the ILs at 25 degrees C. Furthermore, the supersaturated glucose solutions in ILs were maintained over a long period of time without any significant loss of glucose. In ILs that were extremely supersaturated with glucose, lipase-catalyzed esterifications of glucose with vinyl laurate, and lauric acid were successfully carried out. The conversion increased from 8% to 96% at 1 day of reaction by using supersaturated solution in [Bmim][TfO] which had dissolved glucose concentration of 400% higher than its solubility, compared with the reaction using saturated glucose solution. By making the glucose concentration in ILs much higher than the solubility through our novel and simple method, the initial rate and conversion of the lipase-catalyzed reaction were significantly improved.