5-Methylcytosine(5mC)is a crucial epigenetic modification which plays a significant role in the regulation of gene expression.Accurate and quantitative detection of 5mC at single-base resolution is essential for under...5-Methylcytosine(5mC)is a crucial epigenetic modification which plays a significant role in the regulation of gene expression.Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes.In this study,we develop a novel Naegleria TET-assisted deaminase sequencing(NTD-seq)method for the base-resolution and quantitative detection of 5mC in genomic DNA.The NTD-seq method utilizes a Naegleria TET-like dioxygenase(nTET)to oxidize 5mC,generating 5-methylcytosine oxidation products(5moC).We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A(A3A),creating an A3A mutant(A3Am).Treatment with A3Am results in the conversion of cytosine to uracil,while 5moC remains unchanged.Consequently,NTD-seq enables the direct deamination of cytosine to uracil by A3Am,which is sequenced as thymine,whereas 5mC,once oxidized to 5moC by nTET,resists deamination and is sequenced as cytosine.Therefore,the cytosines that persist in the sequencing data represent the original 5mC sites.We applied NTD-seq to HEK293T cells,generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq.NTD-seq emerges as a powerful,bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.展开更多
5-Hydroxymethylcytosine(5hmC)is a crucial DNA modification that participates in the regulation of various physiological processes.Although several methods have been developed to map 5hmC,there is still a high demand f...5-Hydroxymethylcytosine(5hmC)is a crucial DNA modification that participates in the regulation of various physiological processes.Although several methods have been developed to map 5hmC,there is still a high demand for genome-wide mapping at single-base resolution.Consequently,we developed a double-stranded DNA deamination sequencing(DDD-seq)approach for genome-wide mapping of 5hmC at single-base resolution.DDD-seq utilizes the double-stranded DNA deaminase SsdA_(cat)from Pseudomonas syringae,which efficiently deaminates cytosine(C),5-methylcytosine(5mC),5hmC,5-formylcytosine(5fC),and 5-carboxylcytosine(5caC)in duplex DNA,but not glycosylated 5hmC(5gmC).In DDD-seq,C,5mC,5fC,and 5caC in dsDNA are deaminated by SsdA_(cat)and read as T in sequencing,while 5gmC resists deamination,allowing its identification by detecting as C in sequencing.The map of 5hmC generated by DDD-seq inmouse cerebellum tissue closely aligns with that obtained from the ACE-seq method.Applying DDD-seq to mouse cerebellum tissue subjected to chronic sleep deprivation revealed significant changes of 5hmC distribution in genomic DNA.In contrast to previous single-stranded deaminase APOBEC3A-based mapping methods that require denaturation of dsDNA into ssDNA,DDD-seq eliminates this step,reducing risks associated with incomplete denaturation and simplifying sequencing library construction.Additionally,SsdA_(cat)demonstrates superior thermostability and activity across a wide range of pH values and temperatures,making DDD-seq applicable in broader scenarios with more accessible conditions.Collectively,the DDD-seq method is straightforward,bisulfite-free,and eliminates the need for DNA denaturation step,making it a valuable tool for mapping 5hmC in genomes at single-base resolution.展开更多
Organisms and cells,in response to environmental influences or during development,undergo considerable changes in DNA methylation on a genome-wide scale,which are linked to a variety of biological processes.Using Meth...Organisms and cells,in response to environmental influences or during development,undergo considerable changes in DNA methylation on a genome-wide scale,which are linked to a variety of biological processes.Using MethylC-seq to decipher DNA methylome at single-base resolution is prohibitively costly.In this study,we develop a novel approach,named MBRidge,to detect the methylation levels of repertoire CpGs,by innovatively introducing C-hydroxylmethylated adapters and bisulfate treatment into the MeDIP-seq protocol and employing ridge regression in data analysis.A systematic evaluation of DNA methylome in a human ovarian cell line T29 showed that MBRidge achieved high correlation(R>0.90)with much less cost(∼10%)in comparison with MethylC-seq.We further applied MBRidge to profiling DNA methylome in T29H,an oncogenic counterpart of T29’s.By comparing methylomes of T29H and T29,we identified 131790 differential methylation regions(DMRs),which are mainly enriched in carcinogenesis-related pathways.These are substantially different from7567 DMRs that were obtained by RRBS and related with cell development or differentiation.The integrated analysis ofDMRsin the promoterand expression of DMR-corresponding genes revealed thatDNAmethylation enforced reverse regulation of gene expression,depending on the distance fromthe proximalDMRto transcription starting sites in both mRNA and lncRNA.Taken together,our results demonstrate that MBRidge is an efficient and cost-effective method that can be widely applied to profiling DNA methylomes.展开更多
Circulating tumor DNA(ctDNA),carrying tumor-specific sequence mutations,is a promising biomarker for classification,diagnosis and prognosis of cancers.However,there is still a great challenge in discriminating single-...Circulating tumor DNA(ctDNA),carrying tumor-specific sequence mutations,is a promising biomarker for classification,diagnosis and prognosis of cancers.However,there is still a great challenge in discriminating single-base difference between ctDNA and its coexisting analogue(normal circulating DNA,ncDNA)at a serum sample.A locked nucleic acid(LNA)probe combined with a-HL nanopore sensor was designed,which achieved a high signal-to-background ratio(SBR)of^8.34 × 10^3,as well as a significant discrimination capability(~12.3 times)of single-base diffe rence.The accurate discrimination strategy is label-free,convenient,selective and sensitive,which has great potential in the early diagnosis of diseases and biomedical research fields.展开更多
We report a novel strategy to control and construct sequential assembly of DNA functionalized gold nanoparticles(AuNPs)from a single solution.We systematically investigated the temperature-dependent kinetics of DNA-li...We report a novel strategy to control and construct sequential assembly of DNA functionalized gold nanoparticles(AuNPs)from a single solution.We systematically investigated the temperature-dependent kinetics of DNA-linked AuNP assembly.A sharp kinetic transition in the assembly process,strongly dependent on temperature,was identified.As the temperature increased,the assembly rate rose continuously until it reached a critical kinetic temperature(T_(crit)).Beyond this point,the assembly rate sharply decreased to near zero within a narrow temperature window of 2–3℃.This sharp kinetic transition is advantageous for designing highly specific detection systems.We leveraged the transition to control the assembly of AuNPs functionalized with DNA strands that differ by single-base mismatches.Using AuNPs of different sizes,we demonstrated the sequential assembly of AuNPs functionalized with perfectly matched DNA strands,followed by assembly of AuNPs with DNA containing a single-base mismatch,and finally assembly of AuNPs with DNA containing two-base mismatches.We also first assembled AuNPs functionalized with DNA containing two-base mismatches at a lower temperature,followed by assembling AuNPs with DNA containing a single-base mismatch at a higher temperature.Both approaches of controlled sequential assembly are useful for bottom-up assembly applications to form desirable nanostructures.展开更多
Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA...Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA fragments （0.97 for 123 b/124 b） with UV detection. This quasi-IPN （interpenetrating network） sieving matrix showed low viscosity （23.5 mPa·s at 25 ℃） and decreased with increasing temperature. This polymer also exhibited dynamically coating capacity and could be used in the uncoated capillary. The effects of temperature and electric field strength on the DNA separation of quasi-IPN matrix were also investigated and found that the temperature and electric field strength could markedly affected the mobility behavior of DNA fragments. This polymer matrix has also applied to separate the bigger DNA fragments by capillary electrophoresis with UV detection. Under the denaturing conditions, this matrix separated the samples with last fragment of 1353 base in 40 rain, in which the doublet of 309/310 base was partial separated and the resolution was 0.88.展开更多
基金supported by the National Key Research and Development Program of China(2022YFA0806600,2022YFC3400700)the National Natural Science Foundation of China(22277093,22207090)+2 种基金the Key Research and Development Project of Hubei Province(2023BCB094)the Translational Medicine and Interdisciplinary Research Joint Fund of Zhongnan Hospital of Wuhan University(ZLJC2022001,ZNJC202208)the Natural Science Foundation of Shandong Province(ZR2024QB224)。
文摘5-Methylcytosine(5mC)is a crucial epigenetic modification which plays a significant role in the regulation of gene expression.Accurate and quantitative detection of 5mC at single-base resolution is essential for understanding its epigenetic functions within genomes.In this study,we develop a novel Naegleria TET-assisted deaminase sequencing(NTD-seq)method for the base-resolution and quantitative detection of 5mC in genomic DNA.The NTD-seq method utilizes a Naegleria TET-like dioxygenase(nTET)to oxidize 5mC,generating 5-methylcytosine oxidation products(5moC).We also engineered a variant of the human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A(A3A),creating an A3A mutant(A3Am).Treatment with A3Am results in the conversion of cytosine to uracil,while 5moC remains unchanged.Consequently,NTD-seq enables the direct deamination of cytosine to uracil by A3Am,which is sequenced as thymine,whereas 5mC,once oxidized to 5moC by nTET,resists deamination and is sequenced as cytosine.Therefore,the cytosines that persist in the sequencing data represent the original 5mC sites.We applied NTD-seq to HEK293T cells,generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq.NTD-seq emerges as a powerful,bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
基金supported by the National Key R&D Program of China(grant nos.2022YFC3400700 and 2022YFA0806600)the Fundamental Research Funds for the Central Universities(grant no.2042024kf0022)+1 种基金the National Natural Science Foundation of China(grant nos.22074110,22277093,and 22307099)the Key Research and Development Project of Hubei Province(grant no.2023BCB094).
文摘5-Hydroxymethylcytosine(5hmC)is a crucial DNA modification that participates in the regulation of various physiological processes.Although several methods have been developed to map 5hmC,there is still a high demand for genome-wide mapping at single-base resolution.Consequently,we developed a double-stranded DNA deamination sequencing(DDD-seq)approach for genome-wide mapping of 5hmC at single-base resolution.DDD-seq utilizes the double-stranded DNA deaminase SsdA_(cat)from Pseudomonas syringae,which efficiently deaminates cytosine(C),5-methylcytosine(5mC),5hmC,5-formylcytosine(5fC),and 5-carboxylcytosine(5caC)in duplex DNA,but not glycosylated 5hmC(5gmC).In DDD-seq,C,5mC,5fC,and 5caC in dsDNA are deaminated by SsdA_(cat)and read as T in sequencing,while 5gmC resists deamination,allowing its identification by detecting as C in sequencing.The map of 5hmC generated by DDD-seq inmouse cerebellum tissue closely aligns with that obtained from the ACE-seq method.Applying DDD-seq to mouse cerebellum tissue subjected to chronic sleep deprivation revealed significant changes of 5hmC distribution in genomic DNA.In contrast to previous single-stranded deaminase APOBEC3A-based mapping methods that require denaturation of dsDNA into ssDNA,DDD-seq eliminates this step,reducing risks associated with incomplete denaturation and simplifying sequencing library construction.Additionally,SsdA_(cat)demonstrates superior thermostability and activity across a wide range of pH values and temperatures,making DDD-seq applicable in broader scenarios with more accessible conditions.Collectively,the DDD-seq method is straightforward,bisulfite-free,and eliminates the need for DNA denaturation step,making it a valuable tool for mapping 5hmC in genomes at single-base resolution.
基金supported by grants from the NationalHigh Technology Research and Development Program of China(2012AA02A201,2012AA02A202)China-Canada Collaboration Project from the Ministry of Science and Technology of China(2011DFA30670)+1 种基金the National Natural Science Foundation of China(31171236/C060503)the Astra Zeneca Innovation Centre China.
文摘Organisms and cells,in response to environmental influences or during development,undergo considerable changes in DNA methylation on a genome-wide scale,which are linked to a variety of biological processes.Using MethylC-seq to decipher DNA methylome at single-base resolution is prohibitively costly.In this study,we develop a novel approach,named MBRidge,to detect the methylation levels of repertoire CpGs,by innovatively introducing C-hydroxylmethylated adapters and bisulfate treatment into the MeDIP-seq protocol and employing ridge regression in data analysis.A systematic evaluation of DNA methylome in a human ovarian cell line T29 showed that MBRidge achieved high correlation(R>0.90)with much less cost(∼10%)in comparison with MethylC-seq.We further applied MBRidge to profiling DNA methylome in T29H,an oncogenic counterpart of T29’s.By comparing methylomes of T29H and T29,we identified 131790 differential methylation regions(DMRs),which are mainly enriched in carcinogenesis-related pathways.These are substantially different from7567 DMRs that were obtained by RRBS and related with cell development or differentiation.The integrated analysis ofDMRsin the promoterand expression of DMR-corresponding genes revealed thatDNAmethylation enforced reverse regulation of gene expression,depending on the distance fromthe proximalDMRto transcription starting sites in both mRNA and lncRNA.Taken together,our results demonstrate that MBRidge is an efficient and cost-effective method that can be widely applied to profiling DNA methylomes.
基金financially supported by the National Natural Science Foundation of China (Nos.21475091,21775106)
文摘Circulating tumor DNA(ctDNA),carrying tumor-specific sequence mutations,is a promising biomarker for classification,diagnosis and prognosis of cancers.However,there is still a great challenge in discriminating single-base difference between ctDNA and its coexisting analogue(normal circulating DNA,ncDNA)at a serum sample.A locked nucleic acid(LNA)probe combined with a-HL nanopore sensor was designed,which achieved a high signal-to-background ratio(SBR)of^8.34 × 10^3,as well as a significant discrimination capability(~12.3 times)of single-base diffe rence.The accurate discrimination strategy is label-free,convenient,selective and sensitive,which has great potential in the early diagnosis of diseases and biomedical research fields.
基金supported by the Canadian Institutes for Health Research,the Natural Sciences and Engineering Research Council of Canada,and the Canada Research Chairs program.
文摘We report a novel strategy to control and construct sequential assembly of DNA functionalized gold nanoparticles(AuNPs)from a single solution.We systematically investigated the temperature-dependent kinetics of DNA-linked AuNP assembly.A sharp kinetic transition in the assembly process,strongly dependent on temperature,was identified.As the temperature increased,the assembly rate rose continuously until it reached a critical kinetic temperature(T_(crit)).Beyond this point,the assembly rate sharply decreased to near zero within a narrow temperature window of 2–3℃.This sharp kinetic transition is advantageous for designing highly specific detection systems.We leveraged the transition to control the assembly of AuNPs functionalized with DNA strands that differ by single-base mismatches.Using AuNPs of different sizes,we demonstrated the sequential assembly of AuNPs functionalized with perfectly matched DNA strands,followed by assembly of AuNPs with DNA containing a single-base mismatch,and finally assembly of AuNPs with DNA containing two-base mismatches.We also first assembled AuNPs functionalized with DNA containing two-base mismatches at a lower temperature,followed by assembling AuNPs with DNA containing a single-base mismatch at a higher temperature.Both approaches of controlled sequential assembly are useful for bottom-up assembly applications to form desirable nanostructures.
文摘Quasi-interpenetrating network of polyacrylamide （PAA） and polyvinylpyrrolidone （PVP） had been successfully used for single-base resolution of double-stranded DNA （0.76 for 123 bp/124 bp） and single-stranded DNA fragments （0.97 for 123 b/124 b） with UV detection. This quasi-IPN （interpenetrating network） sieving matrix showed low viscosity （23.5 mPa·s at 25 ℃） and decreased with increasing temperature. This polymer also exhibited dynamically coating capacity and could be used in the uncoated capillary. The effects of temperature and electric field strength on the DNA separation of quasi-IPN matrix were also investigated and found that the temperature and electric field strength could markedly affected the mobility behavior of DNA fragments. This polymer matrix has also applied to separate the bigger DNA fragments by capillary electrophoresis with UV detection. Under the denaturing conditions, this matrix separated the samples with last fragment of 1353 base in 40 rain, in which the doublet of 309/310 base was partial separated and the resolution was 0.88.
