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
  • 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: [email protected].
  • 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: [email protected].
  • 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: [email protected].
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.
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