We propose to adapt novel, entropy-driven DNA circuitry that has only recently been developed to pressing needs in point-of-care diagnostics. The metastable DNA circuits will be activated by hybridization to a specific mRNA allele, and will undergo a series of strand exchange amplification reactions, either linear or exponential, culminating in the production of a deoxyribozyme peroxidase that can generate an easy-to-read colorimetric signal. We will use a novel optimization technique, sequential injection analysis, to find reaction conditions that give robust signal-to-noise ratios under a variety of sample and environmental conditions. We will apply the optimized DNA circuitry to the detection of rifampicin resistant RNA polymerase alleles in M. tuberculosis in sputum samples obtained from Afghanistan. These experiments should set the stage for the development of a one-tube, colorimetric assay for point-of-care diagnosis of drug resistant M. tuberculosis infection.
The impact of marrying novel assay techniques, novel optimization methods, and realistic applications in the field cannot be underestimated for advancing options for public health monitoring and maintenance. The selfsame DNA circuitry can be readily redesigned for the detection of other mRNAs and alleles, and should therefore find great use in first world clinical settings, as well.
| Jung, Cheulhee; Ellington, Andrew D (2016) A primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). Anal Bioanal Chem 408:8583-8591 |
| Milligan, J N; Ellington, A D (2015) Using RecA protein to enhance kinetic rates of DNA circuits. Chem Commun (Camb) 51:9503-6 |
| Meyer, Adam J; Ellefson, Jared W; Ellington, Andrew D (2014) Library generation by gene shuffling. Curr Protoc Mol Biol 105:Unit 15.12. |
| Jiang, Yu Sherry; Bhadra, Sanchita; Li, Bingling et al. (2014) Mismatches improve the performance of strand-displacement nucleic Acid circuits. Angew Chem Int Ed Engl 53:1845-8 |
| Chen, Xi; Briggs, Neima; McLain, Jeremy R et al. (2013) Stacking nonenzymatic circuits for high signal gain. Proc Natl Acad Sci U S A 110:5386-91 |
| Jiang, Yu Sherry; Li, Bingling; Milligan, John N et al. (2013) Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly. J Am Chem Soc 135:7430-3 |
| Li, Bingling; Jiang, Yu; Chen, Xi et al. (2012) Probing spatial organization of DNA strands using enzyme-free hairpin assembly circuits. J Am Chem Soc 134:13918-21 |
| Magalhães, Maria L B; Byrom, Michelle; Yan, Amy et al. (2012) A general RNA motif for cellular transfection. Mol Ther 20:616-24 |
| Li, Bingling; Chen, Xi; Ellington, Andrew D (2012) Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions. Anal Chem 84:8371-7 |
| Allen, Peter B; Arshad, Seyed A; Li, Bingling et al. (2012) DNA circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform. Lab Chip 12:2951-8 |
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