Synthesis of an amplifiable reporter RNA for bioassays

Abstract

The replacement of reporter groups, such as fluorescent molecules or enzymes, by an amplifiable reporter should lead to bioassays of greatly increased sensitivity, since a very large number of copies of the reporter can be accumulated in a short time. Midivariant RNA is an appropriate reporter, since it is autocatalytically replicated by Q beta RNA polymerase in vitro. This RNA can be amplified exponentially, with a population doubling time of 36 seconds, resulting in the synthesis of 10(6) copies of each molecule in 12 minutes. We have used chemical methods to attach biotin to the 5' terminus of midivariant RNA via a disulfide linker. This biotinylated RNA combines with avidin to give a product that is readily purified by gel electrophoresis. The RNA-biotin-avidin adduct, and the RNA released from it by reductive cleavage of the linker arm, replicate normally. The RNA-biotin-avidin adduct should be a suitable reporter for a variety of replication-assisted bioassays involving biotinylated antibodies or biotinylated nucleic acid probes.

Chemical cross‐linking and mass spectrometry to map three‐dimensional protein structures and protein–protein interactions

“…Carbodiimides are so-called 'zero-length' cross-linking reagents because they do not introduce a spacer chain into the protein. They are used to mediate amide bond formation between spatially close (<3 Å ) groups; for example, a carboxylate and an amine group or a phosphate and an amine group (Hoare & Koshland, 1966;Chu, Kramer, & Orgel, 1986;Ghosh et al, 1990). A second reagent such as sulfo-Nhydroxysuccinimide could be added to modify the reaction (Scheme 1C).…”

Fluorescence Spectroscopy: Applications in Chemical Biology

“…Perhaps the biggest liability of these dyes is their toxicity, which necessitates careful handling and disposal. The 5 -backbone phosphates can be targets for modification through carbodiimide-mediated formation of a phosphoramidate (120,121). Such reactions, however, are not easy to control, and the expertise required makes this type of approach not very common.…”

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

“…In forming these bonds no additional chemical structure is introduced between the conjugating molecules. Carbodiimides have been used as coupling agents for nucleotide-protein conjugates [23,30,31]. The carbodiimide activates the 5 0 -phosphate group of Fig.…”