Metabolic Labeling and Imaging of Cellular RNA via Bioorthogonal Cyclopropene−Tetrazine Ligation

RNA labeling is vital for the study of an RNA structure,cellular distribution,localization,and metabolism.Herein,we report N6 cyclopropane-modified adenosine(cpA)as a new analog for metabolic RNA labeling.We successfully applied inverse electrondemand Diels–Alder(iEDDA)chemistry to label cellular RNA with cpA.This labeling technique is practical and provides a new platform to study RNA roles in cells in a metal-free manner.This simple and robust assay represents a significant advancement in the profiling methods of the nascent transcriptome using chemical approaches.

Systematic investigation of bioorthogonal cellular DNA metabolic labeling in a photo-controlled manner

Bioorthogonal cleavage and ligation reactions together form one more integrated system about the repertoire of bioorthogonal chemistry,capacitating an array of thrilling new biological applications.The bond-cleavage type and position of biomolecular remain a great challenge,which determines the metabolic pathway of the targets in living systems.Herein we designed two linkages of methylene and carbonyl group attached the N-3 position of the 5-ethynyl-2’-deoxyuridine(EdU)base or the oxygen atom at deoxyribose 3’position to a photocaging group,which would be cleaved by irradiation with 365 nm ultraviolet light.EdU derivatives linked by methylene at the N-3 position had better photodecage efficiency and stability in the absence of light.This paper provides a strategy for studying the nucleoside metabolic pathways in cells,which can easily and conveniently evaluate the effect of the position and type of the linkages.The developed strategy affords a reference for controlling spatial and temporal metabolism of small-molecule drugs,allowing direct manipulation of intact cells under physiological conditions.

Intracellular RNA Labeling Technologies for the Analysis of RNA Biology

As a cornerstone of the central dogma of molecular biology,RNA plays vital roles in living organisms.Over the past few decades,many RNA labeling technologies have been developed to elucidate the biological function of RNA.These technologies have signifi-cantly advanced our understanding of RNA secondary structure,localization,and turnover.Additionally,taking advantage of these innovative RNA labeling approaches,plenty of tool kits have been devised for the regulation of RNA-related biological process,such as gene expression and gene editing.In this review,we primarily focus on an array of intracellular RNA labeling methods,encom-passing chemical probes-based labeling,metabolic labeling,and proximity-dependent labeling.We also provide a brief overview of their applications in the research of RNA biology.Finally,the perspectives of RNA labeling are also discussed.

Combining Metabolic Alkyne Labeling and Click Chemistry for Secretome Analysis of Serum-Containing Conditioned Medium

Main observation and conclusion Bioorthogonal click chemistry has emerged as a powerful tool for the specific modification of proteins in complex mixtures.Metabolic labeling of proteins with azide followed by the copper-catalyzed azide−alkyne cycloaddition(CuAAC)with alkyne-based affinity probes/beads is widely applied to study protein turnover and post-translational modifications(PTMs).

a^(6)A-seq:N^(6)-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing

The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics.The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs.Here we developed an a^(6)A-seq method,which takes advantage of N^(6)-allyladenosine(a^(6)A)metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner.a^(6)A plays a role as a chemical sequencing tag in that the iodination of a^(6)A in mRNAs results in 1,N^(6)-cyclized adenosine(cyc-A),which induces base misincorporation during RNA reverse transcription,thus making a^(6)A-labelled mRNAs detectable by sequencing.A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine.a^(6)A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition.Compared with regular RNA-seq,a^(6)A-seq could more sensitively detect the change of mRNA production over a time scale.The experiment of a^(6)Acontaining mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a^(6)A-seq data.Together,our results show a^(6)A-seq is an effective tool to study RNA dynamics.

Selective inactivation of Gram-positive bacteria in vitro and in vivo through metabolic labelling

Bacterial infections are grave threats to human health,particularly those caused by the most common Grampositive bacteria.The massive administration of broad-spectrum antibiotics to treat various bacterial infections has led to the evolution and spread of drug resistance.As a universal antimicrobial technique unapt to induce drug resistance,photothermal therapy(PTT)is attracting extensive attention in recent years.However,its unspecific killing capability and side effects towards adjacent mammalian cells severely impede the practical applications.Herein,we proposed a metabolic engineering strategy to selectively inactivate Gram-positive bacteria by PTT.A bioorthogonal photothermal agent was prepared by the conjugation of IR-780 iodide and dibenzocyclooctyne(IR780-DBCO).Upon pre-metabolizing with 3-azido-D-alanine,Gram-positive bacteria rather than Gramnegative ones,such as Staphylococcus aureus and vancomycinresistant Enterococcus faecalis(VRE),could be specifically tied up by the explosive IR780-DBCO via copper-free click chemistry.Thereafter,they spontaneously detonated under 15 min near-infrared light irradiation and inactivated nearly 100% Gram-positive bacteria in vitro.Moreover,superbug VRE-induced infection was significantly inhibited by this approach in a mouse skin wound model.This metabolic labelling-based photothermal ablation strategy specific to Gram-positive microbes would stimulate the development of precise antibacterial candidates for preclinical applications.

Ac_(3)6deoGlcNAz could selectively label O-GlcNAc modified proteins with minimal S-glyco-modification

A novel metabolic chemical reporter of Ac_(3)6deo Glc NAz was developed and confirmed as an effective probe for O-Glc NAc modification. Ac_(3)6deo Glc NAz labeling predominantly occurs in intracellular OGlc NAcylated proteins rather than cell-surface glycoproteins. Of note, it could reduce the artificial S-glycomodification compared to Ac_(4)Gal NAz and Ac_(4)Glc NAz. This new reporter allows to be widely used in the field of proteomic identification of O-Glc NAcylation.

Amplified visualization and function exploration of exosomal protein-specific glycosylation using hybridization chain reaction from non-functional epitope

Exosomal glycoproteins play significant roles in many physiological and pathological procedures. However, the current methods for studying exosomal glycoproteins have low sensitivity or can affect exosomal biological function. Herein, we developed a proximity dual-tagging strategy using an induced hybridization chain reaction(HCR) from the target’s non-functional epitope for amplified visualization and functional exploration of exosomal protein-specific glycosylation. This strategy leverages dualtagging based on the aptamer with little influence on target function and metabolic glycan labelling, and the rigid product and high sensitivity of HCR. The method improves the signal of visualizing exosomal PD-L1(exo PD-L1) by 7.7-fold compared with the signal without HCR amplification without affecting the natural exo PD-L1/PD-1 interaction. As a result, we verified that the interaction between exo PD-L1 and PD-1 positive cells is positively correlated to the glycosylation level of exo PD-L1. Overall,we have developed a sensitive method with little functional influence to visualize exosomal protein-specific glycosylation in situ,offering a powerful tool for studying the biological implications of exosomal glycoproteins.