In Vitro Amplification and Selection of Engineered RNase P Ribozyme for Gene Targeting Applications.

Autor:	Trang P; School of Public Health, University of California, Berkeley, CA, USA., Zhang I; Program in Comparative Biochemistry, University of California, Berkeley, CA, USA., Liu F; School of Public Health, University of California, Berkeley, CA, USA. liu_fy@berkeley.edu.; Program in Comparative Biochemistry, University of California, Berkeley, CA, USA. liu_fy@berkeley.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2822, pp. 419-429.
DOI:	10.1007/978-1-0716-3918-4_26
Abstrakt:	Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu