Crystallization and Structural Determination of 8-17 DNAzyme

Hehua Liu; Song Mao; Jia Sheng; Jianhua Gan

doi:10.1007/978-1-0716-2047-2_9

Crystallization and Structural Determination of 8-17 DNAzyme

Methods Mol Biol. 2022:2439:117-130. doi: 10.1007/978-1-0716-2047-2_9.

Authors

Hehua Liu¹, Song Mao², Jia Sheng³, Jianhua Gan⁴

Affiliations

¹ Shanghai Public Health Clinical Center, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, China.
² Department of Chemistry and The RNA Institute, University at Albany, State University of New York, Albany, NY, USA.
³ Department of Chemistry and The RNA Institute, University at Albany, State University of New York, Albany, NY, USA. jsheng@albany.edu.
⁴ Shanghai Public Health Clinical Center, State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, China. ganjhh@fudan.edu.cn.

PMID: 35226319
DOI: 10.1007/978-1-0716-2047-2_9

Abstract

DNAzymes are a group of DNA molecules that can catalyze various chemical reactions. Owing to their great application potentials, DNAzymes have received significant attention. However, due to their intrinsic difficulties in crystallization and structural determination, only very limited structural information of DNAzymes is available to date. Using co-crystallization with the African Swine Fever Virus Polymerase X (AsfvPolX) protein, we have recently solved a complex structure of the 8-17 DNAzyme, which represents the first structure of the catalytically active RNA-cleaving DNAzyme. In this chapter, we describe the detailed protocols including gene construction, AsfvPolX expression and purification, crystallization, structure determination, and in vitro cleavage assay. While the specific methods described herein were originally designed for the 8-17 DNAzyme, they can also be utilized to solve other DNAzyme structures.

Keywords: 8–17 DNAzyme; Crystallization; DNAzyme; RNA cleavage; Structure determination.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

African Swine Fever Virus* / genetics
African Swine Fever Virus* / metabolism
Animals
Crystallization
DNA / chemistry
DNA, Catalytic* / chemistry
RNA / genetics
Swine

Substances

DNA, Catalytic
RNA
DNA