New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA.

ABSTRACT: Nucleic acid-templated chemistry is a promising strategy for imaging genetic sequences in living cells. Here we describe the synthesis of two new nucleophiles for use in templated nucleophilic displacements with DNA probes. The nucleophilic groups are phosphorodithioate and phosphorotrithioate; we report on synthetic methods for introducing these groups at the 3'-terminus of oligonucleotides. Both new nucleophiles are found to be more highly reactive than earlier phosphoromonothioates. This increased nucleophilicity is shown to result in more rapid templated reactions with electrophilic DNA probes. The new probes were demonstrated in detection of specific genetic sequences in solution, with clear signal over background being generated in as little as 20 min. The probes were also tested for imaging ribosomal RNA sequences in live Escherichia coli; useful signal was generated in 20 min to 1h, approximately one quarter to one-half the time of earlier monothioate probes, and the signal-to-noise ratio was increased as well.

SUBMITTER: Miller GP 

PROVIDER: S-EPMC2265789 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature