The CIAP – Aptamer Complex: A Potential System for cDNA Detection
Current detection methods for gene expression include the Northern blot, qPCR, and microarrays, which all come with a unique set of disadvantages. Thus the creation of an alternative gene expression detection system is necessary to address some of the shortcomings of these assays. The novel detection system we propose for this purpose is an enzyme linked oligonucleotide assay (ELONA) that utilizes aptamers, which replace the antibody in the traditional antibody-reporter protein complex of an enzyme linked immunosorbent assay (ELISA). Aptamers are randomized sequences of single stranded RNA or DNA nucleic acid strands that can bind to pre-selected targets such as proteins, nucleic acids, and small organic compounds with high affinity and specificity.
The ELONA is focused specifically upon displacement methodologies, in which binding between an already discovered aptamer and the reporter protein calf intestinal alkaline phosphatase (CIAP) is prevented by a DNA inhibitor flanked by sequences complementary to a target cDNA sequence. A colorimetric signal is then generated depending on whether or not CIAP is able to bind onto this aptamer, thus allowing for the quantification of target cDNA and subsequently, gene expression.
The particular benefits of utilizing this aptamer-based system are significant cost reductions and the removal of sample strand labeling prior to running the assay. As of now, inhibition tests involving inhibitor with target flanks and random target cDNAs have shown that this system is capable of quantifying target cDNA, albeit at a reduced sensitivity due to approximately a 10:1 ratio of ITF to aptamer required for full inhibition. In the future, optimization of the incubation and hybridization of target to ITF will be performed to further flesh out the capabilities of this prototype.