Improved methods for structure probing in large RNAs: a rapid ‘heterologous’ sequencing approach is coupled to the direct mapping of nudease accessible sites. Application to the 5′nal domain of eukaryotic 28S rRNA

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Abstract
He have developed a combined approach for probing native structures in large RNAs. In the first method, after digestion with a structure specific nuclease, accessible sites are mapped at sequence resolution along the entire RNA molecule which is used as a template for the reverse transcriptase elongation of a 5′end labelled selected primer (coding strand of a 3mall restriction fragment of the cloned gene). This method circumvents any prior end-labelling of RNA, a technique with major limitations for large RNAs. In the second approach, a rapid “heterologous” sequencing can be easily applied to definite domains of an RNA molecule in a variety of species (or individuals), without additional DNA cloning nor end-labelling of RNA. By talcing advantage of the presence of evolutionary conserved tracts within an RNA sequence, it allows a rapid analysis of RNA folding patterns in terms of phylogenetic comparisons : when located within such a conserved tract, selected restriction fragments from a cloned gene can be used as heterologous primers for sequencing the upstream divergent region in RNA3 of other species by currently available technology, i.e. reverse transcriptase elongation in the presence of chain terminator dideoxynucleotides.