Dear Editor,Mutations in genomic sequences exhibit a strong correlation with various pathological processes of cancers[1].Currently,the next-generation sequencing technique[2]and polymerase chain reaction(PCR)were the...Dear Editor,Mutations in genomic sequences exhibit a strong correlation with various pathological processes of cancers[1].Currently,the next-generation sequencing technique[2]and polymerase chain reaction(PCR)were the established benchmarks for analyzing DNA mutations.However,the two methods necessitate intricate experimental preparation,costly instrumentation,and skilled personnel,making them challenging for rapid mutations analysis.More importantly,these methods lack adequate accuracy for one base mutations analysis[3].Therefore,the development of a reliable and exceptionally sensitive mutation analysis approach holds immense importance in cancer diagnosis and treatment.展开更多
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 ge...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.展开更多
文摘Dear Editor,Mutations in genomic sequences exhibit a strong correlation with various pathological processes of cancers[1].Currently,the next-generation sequencing technique[2]and polymerase chain reaction(PCR)were the established benchmarks for analyzing DNA mutations.However,the two methods necessitate intricate experimental preparation,costly instrumentation,and skilled personnel,making them challenging for rapid mutations analysis.More importantly,these methods lack adequate accuracy for one base mutations analysis[3].Therefore,the development of a reliable and exceptionally sensitive mutation analysis approach holds immense importance in cancer diagnosis and treatment.
基金the National Key R&D Program of China(2022YFA1103702 and 2017YFA0506800)the National Natural Science Foundation of China(22022702 and 21977087)the Fundamental Research Funds for the Central Universities,and MOE Key Laboratory of Macromolecular Synthesis and Functionalization,Zhejiang University(2022MSF04).
文摘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.
