PMID- 35904502 OWN - NLM STAT- MEDLINE DCOM- 20220817 LR - 20230118 IS - 1520-4898 (Electronic) IS - 0001-4842 (Linking) VI - 55 IP - 16 DP - 2022 Aug 16 TI - Lighting Up Nucleic Acid Modifications in Single Cells with DNA-Encoded Amplification. PG - 2248-2259 LID - 10.1021/acs.accounts.2c00269 [doi] AB - Nucleic acids are naturally decorated with various chemical modifications at nucleobases. Most nucleic acid modifications (NAMs) do not alter Watson-Crick base pairing but can regulate gene expression known as "epigenetics". Their abundances present a very wide range, approximately 10(-2) to 10(-6) of total bases. Different NAMs may coexist in spatial proximity (e.g., <20 nm) in the crowded intracellular environment. Considering the highly dynamic chromatin accessibility (physical access to DNA), the NAMs in inaccessible DNA probably plays different roles. These multilayered features of NAMs vary from cell to cell. Our understanding of the function and mechanism of NAMs in biological processes and disease states has largely been driven by the expanding array of sequencing-based methodologies. However, an underexplored aspect is the measurement of the subcellular distribution, spatial proximity, and inaccessibility of NAMs in single cells. In recent years, we have developed new approaches that light up single-cell NAMs with single-site sensitivity. These methods are mainly based on the integration of chemical or chemoenzymatic tools, DNA amplification and nanotechnology, and/or microfluidics. An overview of these methods together with conventional methods such as immunofluorescence (IF) and fluorescence in situ hybridization (FISH) is provided in this Account.Our laboratory has proposed DNA-encoded amplification (DEA) as the main strategy for developing a set of single-cell NAM imaging methods. In brief, DEA transforms the different features of NAMs into unique DNA primers for rolling circle amplification (RCA) followed by FISH imaging. The first method is base-encoded amplifying FISH (BEA-FISH), in which we convert individual NAMs into RCA primers via chemoselective labeling and click bioconjugation. It enables the in situ visualization of low-abundance NAMs (e.g., 5hmU), which is impracticable by conventional methods. We subsequently developed pairwise proximity-differentiated amplifying FISH (PPDA-FISH), which integrates BEA-FISH with DNA nanotechnology. PPDA-FISH utilizes proximity ligation and toehold strand displacement to label the adjacent site of two different NAMs (one-to-one proximity) and their respective residual sites with three unique RCA probes. It achieves simultaneous counting of the above-mentioned three types of modified sites in the same cells. The third method is cellular macromolecule-tethered DNA walking indexing (Cell-TALKING) to probe more than two NAMs within the same nanoenvironments. Cell-TALKING uses dynamic DNA proximity cleavage to continuously activate different preblocked RCA primers (for each NAM) near one walking probe (for one target molecule). We have explored three NAMs around one histone (one-to-many proximity) in different cancer cell lines and clinical specimens. Then, we describe a single-cell hydrogel encoding amplification (scHEA) method by integrating droplet microfluidics with BEA-FISH. This method generates hydrogel beads that encapsulate single cells and their genomic DNA after cell lysis. It realizes the analysis of global (accessible and inaccessible) DNA from the same cells. We find that the global levels of both 5hmC and 5hmU in single cells can distinguish different breast cancer cells. Finally, the current limitations of these strategies and the future development directions are also discussed. We hope that this Account can spark new ideas and invite new efforts from different disciplines for single-cell NAM analysis. FAU - Chen, Feng AU - Chen F AUID- ORCID: 0000-0003-0188-6256 AD - Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, P. R. China. FAU - Xue, Jing AU - Xue J AD - Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, P. R. China. FAU - Bai, Min AU - Bai M AD - Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, P. R. China. FAU - Fan, Chunhai AU - Fan C AUID- ORCID: 0000-0002-7171-7338 AD - School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules and National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai 200240, P. R. China. FAU - Zhao, Yongxi AU - Zhao Y AUID- ORCID: 0000-0002-1796-7651 AD - Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi 710049, P. R. China. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Review DEP - 20220729 PL - United States TA - Acc Chem Res JT - Accounts of chemical research JID - 0157313 RN - 0 (Hydrogels) RN - 0 (Nucleic Acids) RN - 9007-49-2 (DNA) SB - IM MH - DNA/analysis MH - Hydrogels MH - In Situ Hybridization, Fluorescence MH - *Nucleic Acid Amplification Techniques/methods MH - Nucleic Acid Hybridization MH - *Nucleic Acids EDAT- 2022/07/30 06:00 MHDA- 2022/08/18 06:00 CRDT- 2022/07/29 10:52 PHST- 2022/07/30 06:00 [pubmed] PHST- 2022/08/18 06:00 [medline] PHST- 2022/07/29 10:52 [entrez] AID - 10.1021/acs.accounts.2c00269 [doi] PST - ppublish SO - Acc Chem Res. 2022 Aug 16;55(16):2248-2259. doi: 10.1021/acs.accounts.2c00269. Epub 2022 Jul 29.