Work

RNAs synthesized using photocleavable biotinylated nucleotides have dramatically improved catalytic efficiency

Year: 2011

Type: article

Abstract: Obtaining homogeneous population of natively folded RNAs is a crippling problem encountered when preparing RNAs for structural or enzymatic studies. Most of the traditional methods that are employed to prepare large quantities of RNAs involve procedures that partially denature the RNA. Here, we present a simple strategy using 'click' chemistry to couple biotin to a 'caged' photocleavable (PC) guanosine monophosphate (GMP) in high yield. This biotin-PC GMP, accepted by T7 RNA polymerase, has been used to transcribe RNAs ranging in size from 27 to 527 nt. Furthermore we show, using an in-gel fluorescence assay, that natively prepared 160 and 175 kDa minimal group II intron ribozymes have enhanced catalytic activity over the same RNAs, purified via denaturing conditions and refolded. We conclude that large complex RNAs prepared by non-denaturing means form a homogeneous population and are catalytically more active than those prepared by denaturing methods and subsequent refolding; this facile approach for native RNA preparation should benefit synthesis of RNAs for biophysical and therapeutic applications.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkr464

Authors: Yiling Luo, Nadukkudy V. Eldho, Herman O. Sintim, T. Kwaku Dayie

Institution: University of Maryland, College Park

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,992

Funders: National Science Foundation, National Institutes of Health, National Institute of General Medical Sciences, Division of Chemistry