Arti Pothukuchy

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance.

Coupling Sensitive Nucleic Acid Amplification with Commercial Pregnancy Test Strips.

The detection of nucleic acid biomarkers for point-of-care (POC) diagnostics is currently limited by technical complexity, cost, and time constraints. To overcome these shortcomings, we have combined loop-mediated isothermal amplification (LAMP), programmable toehold-mediated strand-exchange signal transduction, and standard pregnancy test strips. The incorporation of an engineered hCG-SNAP fusion reporter protein (human chorionic gonadotropin-O6 -alkylguanine-DNA alkyltransferase) led to LAMP-to-hCG signal transduction on low-cost, commercially available pregnancy test strips. Our assay reliably detected as few as 20 copies of Ebola virus templates in both human serum and saliva and could be adapted to distinguish a common melanoma-associated SNP allele (BRAF V600E) from the wild-type sequence. The methods described are completely generalizable to many nucleic acid biomarkers, and could be adapted to provide POC diagnostics for a range of pathogens.

Identifying Protein Variants with Cross-Reactive Aptamer Arrays

Much of the work directed at the creation of arrays for protein analysis relies on the use of highly selective molecules as receptors[1]. In these arrays, the receptors are immobilized and exposed to a mixture of target molecules[1d] or to heterogeneous biological samples such as serum from cancer patients[2]. However, such arrays generally only detect the molecules they were programmed to detect, and are ill-suited for the detection of protein variants, such as sequence changes, deletions, and insertions.

Supercharging enables organized assembly of synthetic biomolecules

There are few methods for the assembly of defined protein oligomers and higher order structures that could serve as novel biomaterials. Using fluorescent proteins as a model system, we have engineered novel oligomerization states by combining oppositely supercharged variants. A well-defined, highly symmetrical 16-mer (two stacked, circular octamers) can be formed from alternating charged proteins; higher order structures then form in a hierarchical fashion from this discrete protomer. During SUpercharged PRotein Assembly (SuPrA), electrostatic attraction between oppositely charged variants drives interaction, while shape and patchy physicochemical interactions lead to spatial organization along specific interfaces, ultimately resulting in protein assemblies never before seen in nature.

Ribozymes as Molecular Biology Reagents

Catalytic RNA molecules (ribozymes) can catalyze a number of biochemical processes, including tRNA processing, mRNA splicing and regulation, and of course peptide bond formation. While many of these reactions involve phosphodiester bond rearrangement, catalysts selected by directed evolution further expand the range of reactions available to ribozymes to include the formation of carbon–carbon bonds and redox reactions. This chapter reviews the adaptation of ribozymes into unique as well as alternative tools for (1) developing biosensors and reporters, (2) manipulation of target RNA, and (3) biocatalysis of non-phosphoryltransfer reactions.