ABSTRACT
RNA-RNA interactions are essential for biology, but they can be difficult to study due to their transient nature. While crosslinking strategies can in principle be used to trap such interactions, virtually all existing strategies for crosslinking are poorly reversible, chemically modifying the RNA and hindering molecular analysis. We describe a soluble crosslinker design (BINARI) that reacts with RNA through acylation. We show that it efficiently crosslinks noncovalent RNA complexes with mimimal sequence bias and establish that the crosslink can be reversed by phosphine reduction of azide trigger groups, thereby liberating the individual RNA components for further analysis. The utility of the new approach is demonstrated by reversible protection against nuclease degradation and trapping transient RNA complexes of E. coli DsrA-rpoS derived bulge-loop interactions, which underlines the potential of BINARI crosslinkers to probe RNA regulatory networks.
Subject(s)
Azides/metabolism , Cross-Linking Reagents/metabolism , Escherichia coli/chemistry , Phosphines/metabolism , RNA, Bacterial/metabolism , Acylation , Azides/chemistry , Cross-Linking Reagents/chemistry , Escherichia coli/metabolism , Phosphines/chemistry , RNA, Bacterial/chemistryABSTRACT
Human telomerase is a reverse-transcriptase enzyme that synthesizes the multikilobase repeating hexamer telomere sequence (TTAGGG)n at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification protocol assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromosomes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase-independent telomere elongation.
