Application of Nucleic Acid Aptamers to Viral Detection and Inhibition

Nucleic acid aptamers are small oligonucleotides that specifically bind to other molecules through noncovalent interactions that rely on complex tridimensional structural arrangements. Aptamers are generated through the iterative in vitro selection method called SELEX, resulting in specific binding against a wide variety of molecular targets including viruses. Because aptamers are obtained in vitro and can be synthetically produced, they have been envisioned as future diagnostic and therapeutic tools for human diseases including virus-borne pathologies. Aptamers have been isolated against a number of viruses including pandemic influenza virus, human papillomavirus and hepatitis C virus. Although aptamers have proven themselves as extremely sensitive detection tools triggering the development of affordable and highly diagnostic methods, their use as therapeutic moieties has been hampered by biostability, delivery and pharmacodynamical issues. Nevertheless, a new generation of chemically modified aptamers shows promise for the coming of age of protein-targeted noncatalytic oligonucleotides for the therapy of viral disease. The present review focuses on the most successful antiviral aptamers reported and includes a description of some of the novel methods developed for their use as diagnostic and therapeutic tool

A Triplex Ribozyme Expression System Based on a Single Hairpin Ribozyme

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with trans-cleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies