ABSTRACT
Aptamers are short oligonucleotides that fold into well-defined three-dimensional architectures thereby enabling specific binding to molecular targets such as proteins. To be successful as a novel therapeutic modality, it is important for aptamers to not only bind their targets with high specificity and affinity, but also to exhibit favorable properties with respect to in vivo stability, cost-effective synthesis, and tolerability (i.e., safety). We describe methods for generating aptamers comprising 2 - deoxy purines and 2 -O-methyl pyrimidines (dRmY) that broadly satisfy many of these additional constraints. Conditions under which dRmY transcripts can be efficiently synthesized using mutant T7 RNA polymerases have been identified and used to generate large libraries from which dRmY aptamers to multiple target proteins, including interleukin (IL)-23 and thrombin, have been successfully discovered using the SELEX process. dRmY aptamers are shown to be highly nuclease-resistant, long-lived in vivo, efficiently synthesized, and capable of binding protein targets in a manner that inhibits their biologic activity with K(D) values in the low nM range. We believe that dRmY aptamers have considerable potential as a new class of therapeutic aptamers.
Subject(s)
Aptamers, Nucleotide/therapeutic use , Animals , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Base Sequence , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Drug Stability , Humans , Mice , Molecular Structure , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SELEX Aptamer Technique , Transcription, Genetic , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
We have utilized in vitro selection technology to develop allosteric ribozyme sensors that are specific for the small molecule analytes caffeine or aspartame. Caffeine- or aspartame-responsive ribozymes were converted into fluorescence-based RiboReporter trade mark sensor systems that were able to detect caffeine or aspartame in solution over a concentration range from 0.5 to 5 mM. With read-times as short as 5 min, these caffeine- or aspartame-dependent ribozymes function as highly specific and facile molecular sensors. Interestingly, successful isolation of allosteric ribozymes for the analytes described here was enabled by a novel selection strategy that incorporated elements of both modular design and activity-based selection methods typically used for generation of catalytic nucleic acids.
