Nucleic acid localization in diagnostics and therapeutics
Pai, Supriya Sudhakar
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Aptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier.