Obtaining Crystals of Nucleic Acids in Complex with the Protein U1A Using the Soaking Method.
Methods Mol Biol
; 2439: 105-115, 2022.
Article
in En
| MEDLINE
| ID: mdl-35226318
X-ray crystallography is one of the most prominent techniques for determining high-resolution structures of nucleic acids. The major challenges are to obtain well-diffracting single crystals and to solve the phase problem. The absence of structural information impedes the elucidation of the molecular details of biological processes. A particularly intriguing example is the RNA-cleavage catalyzed by the 10-23 deoxyribozyme (DNAzyme). This DNAzyme consists of a catalytic core that is flanked by two substrate binding arms, which can be designed to bind any RNA of interest. Structure elucidation of the 10-23 DNAzyme in a biologically relevant conformation faces three major challenges: (1) stabilization of the RNA substrate to capture the DNA:RNA complex in the pre-catalytic conformation, (2) prevention of the formation of an artificial duplex conformation due to a self-complementary sequence in the catalytic core of the DNAzyme, and (3) the crystallization of nucleic acids with their uniform surfaces. Here, we provide a protocol for an innovative strategy facilitating the crystallization of protein:nucleic acid complexes using a soaking approach and discuss on how to apply this protocol for the structure elucidation of the 10-23 DNAzyme. For this purpose, we describe the purification procedure of an optimized variant of the RNA-binding protein U1A, the crystallization of this specific U1A variant, the soaking process with its specific RNA hairpin loop, and finally suggest a strategy for applying this procedure on the 10-23 DNAzyme in complex with its specific RNA target.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Nucleic Acids
/
Ribonucleoprotein, U1 Small Nuclear
Language:
En
Journal:
Methods Mol Biol
Journal subject:
BIOLOGIA MOLECULAR
Year:
2022
Document type:
Article
Affiliation country:
Germany
Country of publication:
United States