2. Academic Validation
  3. Graft-seq precisely maps RNA modifications via site-specific chemical grafting strategy

Graft-seq precisely maps RNA modifications via site-specific chemical grafting strategy

  • Cell Rep Methods. 2025 Jul 21;5(7):101103. doi: 10.1016/j.crmeth.2025.101103.
Ting Li 1,  Ke Ding 2,  Ziming Bao 1,  Quan Ma 3,  Chenyang Huang 1,  Jie Cao 4,  Xiao Shu 1,  Minsong Gao 1,  Zisheng Luo 5,  Xushen Xiong 6,  Jianzhao Liu 7
Affiliations

Affiliations

  • 1 MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310058, China.
  • 2 State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; The Second Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou 311113, China.
  • 3 College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China.
  • 4 MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310058, China; Life Sciences Institute, Zhejiang University, Hangzhou 310058, China.
  • 5 College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China. Electronic address: luozisheng@zju.edu.cn.
  • 6 State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; The Second Affiliated Hospital & Liangzhu Laboratory, Zhejiang University School of Medicine, Hangzhou 311113, China. Electronic address: xiongxs@zju.edu.cn.
  • 7 MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310058, China; Life Sciences Institute, Zhejiang University, Hangzhou 310058, China; State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China; Center for RNA Medicine, International Institutes of Medicine, Zhejiang University, Yiwu 322000, China. Electronic address: liujz@zju.edu.cn.
PMID: 40645188 DOI: 10.1016/j.crmeth.2025.101103
Abstract

The current expansion of RNA epitranscriptomics calls for direct and high-precision mapping tools to characterize the intrinsically low abundant RNA modifications. Here, we developed a strategy, termed Graft-seq, which harnesses specific enzymatic and chemical reactions on an RNA modification site to covalently graft a known RNA branch and further utilizes the branch-to-main-chain or main-chain-to-branch landing/jumping site signal during reverse transcription to determine the locations of RNA modifications at single-base resolution. We developed a matched bioinformatics analysis pipeline for Graft-seq and successfully mapped internal N6-methyladenosine (m6A) and cap N6,2'-O-dimethyladenosine (m6Am), as well as nicotinamide adenine dinucleotide (NAD) on transcriptome-wide mRNAs and/or nuclear non-coding RNAs in different cell lines. Paralleled comparisons of Graft-seq with available techniques confirmed the effectiveness of the RNA grafting strategy. Graft-seq represents a direct and enrichment-free technique for characterizing RNA modifications at base resolution and offers the potential to discover new RNA modifications and RNA-RNA interactions.

Keywords

CP: biotechnology; CP: molecular biology; RNA m(6)A chemical grafting; direct detection method for RNA modification; m(6)Am and NAD cap detection; nuclear RNA methylation mapping; reverse transcriptase jumping in between templates.

Figures
Products