2026-10-14T05:33:49Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34020449 2026-09-29T02:03:27Z category_1 category_4 category_915 category_12 category_13 category_14 category_21 category_873 category_734 category_931 category_133 portal_63 item_type_6 month_year_09_2026

Probing the Conformational Dynamics of VeCas9 Using High-Resolution Single-Molecule Junctions Shichao Zhong (25133744) Baitao Li (8904641) Zeyang Ji (1912864) Jingqi Zhang (7350785) Lihua Zhao (566464) Zhiheng Yang (7290278) Dan Wang (34472) Suhang He (6862745) Xuefeng Guo (444915) Biophysics Biochemistry Physical Sciences not elsewhere classified Cell Biology Genetics Molecular Biology Biotechnology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Information Systems not elsewhere classified Plant Biology transient binding modes situ mechanistic studies revolutionizing disease treatment base expansion events stranded dna substrates molecule junction platform directly observed vecas9 high temporal resolution vecas9 using high high temporal stranded dna working mechanism veillonella </ vecas9 function vecas9 activity time scale strong potential sensitivity depends real time potential applications molecule techniques molecule level mediated r loop formation kinetic parameters key steps interaction process integrating vecas9 guide rna fully characterized dynamic interactions dependent measurements crop improvement conformational dynamics cas9 function biotechnological innovation Gene editing enables precise genetic modifications, revolutionizing disease treatment, crop improvement, and biotechnological innovation. VeCas9, a recently identified CRISPR endonuclease from the <i>Veillonella</i> genus, exhibits a broader protospacer adjacent motif recognition range in comparison with the widely used SpCas9 nuclease. However, its working mechanism and potential applications in gene editing still need to be fully characterized. In this work, by integrating VeCas9 into a graphene-molecule-graphene single-molecule junction platform, we carried out label-free, in situ mechanistic studies of VeCas9 function at the single-molecule level with high temporal resolution. Using this setup, we monitored the dynamic interactions between VeCas9, single-guide RNA, and double-stranded DNA in real time, in both stable and transient binding modes. Through temperature-dependent measurements, we determined the kinetic parameters of key steps in the interaction process. Moreover, a series of double-stranded DNA substrates was employed to examine how various sequence mismatches affect the VeCas9 activity, revealing that its sensitivity depends on the position of the mismatch. More importantly, we directly observed VeCas9-mediated R-loop formation and quantified the time scale of single-base expansion events. These single-molecule observations provide mechanistic insights into the Cas9 function and demonstrate the strong potential of high temporal-resolution single-molecule techniques for studying dynamic biological processes. 2026-09-28T00:00:00Z Text Journal contribution 10.1021/jacs.6c11481.s001 https://figshare.com/articles/journal_contribution/Probing_the_Conformational_Dynamics_of_VeCas9_Using_High-Resolution_Single-Molecule_Junctions/34020449 CC BY-NC 4.0