Selective Chemical Labeling and Sequencing of 5-Carboxylcytosine in DNA at Single-Base Resolution

2020 ◽  
Vol 92 (18) ◽  
pp. 12710-12715
Author(s):  
Yalun Xie ◽  
Yafen Wang ◽  
Zhiyong He ◽  
Wei Yang ◽  
Boshi Fu ◽  
...  
Keyword(s):  
Chemical Labeling ◽  
Single Base ◽  
Single Base Resolution ◽  
Selective Chemical

scholarly journals Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaogang Li ◽  
Xinxin Shi ◽  
Yin Gong ◽  
Wenting Guo ◽  
Yuanrui Liu ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cost Effective ◽  
Chemical Labeling ◽  
Whole Genome Analysis ◽  
Single Base ◽  
Chemically Modified ◽  
Sequencing Method ◽  
Single Base Resolution ◽  
Selective Chemical ◽  
Apobec Family

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C) or 5mC. Herein, we developed a method based on embryonic stem cell (ESC) whole-genome analysis, which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single-base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

scholarly journals Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

2021 ◽  
Author(s):  
Xiaogang Li ◽  
Xinxin Shi ◽  
Wenting Guo ◽  
Yuanrui Liu ◽  
Chunwei Peng ◽  
...  
Keyword(s):  
Cost Effective ◽  
Chemical Labeling ◽  
Whole Genome Analysis ◽  
Single Base ◽  
Chemically Modified ◽  
Sequencing Method ◽  
Single Base Resolution ◽  
Selective Chemical ◽  
Tet Enzymes ◽  
Apobec Family

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation (TET) enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically-modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C)or 5-methylcytosine (5mC). Herein, we developed a method based on ESC whole-genome analysis which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme APOBEC-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

scholarly journals Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Science ◽  
2021 ◽  
pp. eabe7106
Author(s):  
Chunlei Jiao ◽  
Sahil Sharma ◽  
Gaurav Dugar ◽  
Natalia L. Peeck ◽  
Thorsten Bischler ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Genetic Material ◽  
Simultaneous Detection ◽  
Type Ii ◽  
Rna Detection ◽  
Single Base ◽  
Dna Targeting ◽  
Single Base Resolution ◽  
Rna Complexes

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In Type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA-of-interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished SARS-CoV-2 and its D614G variant with single-base resolution in patient samples.

Mapping messenger RNA methylations at single base resolution

2021 ◽  
Vol 63 ◽  
pp. 28-37
Author(s):  
Jie Cao ◽  
Xiao Shu ◽  
Xin-Hua Feng ◽  
Jianzhao Liu
Keyword(s):  
Messenger Rna ◽  
Single Base ◽  
Single Base Resolution

scholarly journals Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yubang Gao ◽  
Xuqing Liu ◽  
Bizhi Wu ◽  
Huihui Wang ◽  
Feihu Xi ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Populus Trichocarpa ◽  
Alternative Polyadenylation ◽  
High Accuracy ◽  
Single Base ◽  
Rna Immunoprecipitation ◽  
Single Base Resolution ◽  
Single Transcript ◽  
Quantitative Profiling ◽  
Identification And Quantification

AbstractThere are no comprehensive methods to identify N6-methyladenosine (m6A) at single-base resolution for every single transcript, which is necessary for the estimation of m6A abundance. We develop a new pipeline called Nanom6A for the identification and quantification of m6A modification at single-base resolution using Nanopore direct RNA sequencing based on an XGBoost model. We validate our method using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and m6A-sensitive RNA-endoribonuclease–facilitated sequencing (m6A-REF-seq), confirming high accuracy. Using this method, we provide a transcriptome-wide quantification of m6A modification in stem-differentiating xylem and reveal that different alternative polyadenylation (APA) usage shows a different ratio of m6A.

Detection of EndogenousK-rasmRNA in Living Cells at a Single Base Resolution by a PNA Molecular Beacon

2012 ◽  
Vol 9 (3) ◽  
pp. 685-693 ◽  
Author(s):  
Yossi Kam ◽  
Abraham Rubinstein ◽  
Aviram Nissan ◽  
David Halle ◽  
Eylon Yavin
Keyword(s):  
Molecular Beacon ◽  
Living Cells ◽  
Single Base ◽  
Single Base Resolution
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