TY - JOUR

T1 - Thermodynamic parameters obtained for the formation of the Cas12a-RNA/DNA complex

AU - Baranova, Svetlana V.

AU - Zhdanova, Polina V.

AU - Golyshev, Victor M.

AU - Lomzov, Alexander A.

AU - Pestryakov, Pavel E.

AU - Chernonosov, Alexander A.

AU - Koval, Vladimir V.

N1 - Финансирование: Финансирующий спонсор Номер финансирования Акроним Russian Science Foundation 20-14-00214 Ministry of Science and Higher Education of the Russian Federation 075-15-2022-263, 121031300056-8

PY - 2025/1

Y1 - 2025/1

N2 - The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a′s specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of −243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.

AB - The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a′s specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of −243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.

KW - CRISPR–Cas systems

KW - Cas12a

KW - Isothermal titration calorimetry

KW - Molecular dynamics

KW - Thermodynamics

KW - crRNA

UR - https://www.mendeley.com/catalogue/760ca259-c688-3cf7-80f8-135022db66a7/

UR - https://pubmed.ncbi.nlm.nih.gov/39693940/

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85212061675&origin=inward&txGid=cc073969b11a94dfbdabc58189dd192a

U2 - 10.1016/j.bbrc.2024.151176

DO - 10.1016/j.bbrc.2024.151176

M3 - Article

C2 - 39693940

VL - 743

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

M1 - 151176

ER -