Use of circular permutation and end modification to position photoaffinityprobes for analysis of RNA structure

Photocrosslinking allows first-order structural analysis with relatively sm all amounts of biological material and can be applied in complex in vitro s ystems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modificatio n of circularly permuted RNAs. Application of this technique provided a lib rary of constraints that, together with biochemical and phylogenetic compar ative data, were used to develop a structure model of the bacterial ribonuc lease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted R NA transcripts can be modified with high efficiency at both the 5' and 3' t ermini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental co nditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure. (C) 1999 Academic Press.