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

Ting Li; Ke Ding; Ziming Bao; Quan Ma; Chenyang Huang; Jie Cao; Xiao Shu; Minsong Gao; Zisheng Luo; Xushen Xiong; Jianzhao Liu

doi:10.1016/j.crmeth.2025.101103

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

Cell Rep Methods. 2025 Jul 3:101103. doi: 10.1016/j.crmeth.2025.101103. Online ahead of print.

Authors

Ting Li¹, Ke Ding², Ziming Bao¹, Quan Ma³, Chenyang Huang¹, Jie Cao⁴, Xiao Shu¹, Minsong Gao¹, Zisheng Luo⁵, Xushen Xiong⁶, Jianzhao Liu⁷

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.