L., Minx P. microbicide candidate. As a first step we resolved aptamer stability in the milieu of proteins CP 376395 present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2-F-modified RNA. We demonstrate that this aptamer can be guarded from your nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of important positions with 2-O-Me-modified nucleotides. Finally, we show that this aptamer can be guarded from all nucleases present in both vaginal and rectal compartments using Zn2+ cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials. to distinguish diseased from wild-type prion protein conformations(7), and IFITM2 in the medical center, to treat age-related macular degeneration (Macugen?). Additionally, our laboratory has recently been developing a clinically relevant RNA-based aptamer to prevent HIV-1 contamination (8, 9). The RNA world hypothesis says that life originated using RNA as CP 376395 the inherited genetic molecule, which was superseded by DNA due to its greater stability. The difference in stability between DNA and RNA is due to the presence of the 2-OH group in the ribose ring of the latter (observe Fig. 4RNase A) and certain metal ions. The absence of a 2-OH group in DNA renders it stable to basic conditions and resistant to RNases. It is this theory that motivated the incorporation of chemically altered nucleotides in siRNAs and aptamers for use in both the laboratory and the clinic. In general, the 2-OH group of all pyrimidines is usually substituted by 2-F, affording a high degree of stability and longevity to the RNA molecules. The purine ribonucleosides are often left unmodified as they are less subject to attack by RNases, such as those of the abundant RNase A superfamily. Open in a separate window Physique 4. Protection of the aptamer through targeted chemical modifications. studies. Thus, in this article we assessed the stability of the aptamer in both vaginal and rectal lavages and we demonstrate that multiple, unusual, and potent nucleases exist at these sites that could dampen the effectiveness of this type of therapeutic. However, we also present evidence that degradation can be circumvented by empirically recognized chemical modifications and through product formulation design. EXPERIMENTAL PROCEDURES Lavage Acquisition Lavage fluid (PBS wash) was recovered from your rectum (10 ml) or vagina (5 ml) and clarified through a 0.2-m filter. The recovered liquid was aliquoted and stored at ?80 C. In all cases informed CP 376395 consent was obtained in writing from participants for biological sample collection, and the study experienced ethics committee approval. Aptamer Synthesis The 2-F pyrimidine-modified, ribopurine aptamer UCLA005 is usually a derivative of the previously published synthetic aptamer UCLA1 (8). It differs from UCLA1 in that the 5-end carries a terminal Cy5 dye followed by three locked nucleic acid thymidines instead of the 5-DMTr-C6-SS-C6 moiety. UCLA005 was synthesized by Integrated DNA Technologies, BVBA, Leuven, Belgium, by solid phase -cyanoethylphosphoramidite chemistry and purified by HPLC. The synthetic protocol has been previously published (8). A derivative of UCLA005, called UCLA005v1, was synthesized as before except that this three LNA thymidines were replaced with three 2-= 0 time point, was produced by adding 30 l of PBS to the aptamer in a total of 50 l and immediately adding a 1:1 ratio of formamide loading buffer (labeled in all figures). A size marker ladder of the UCLA005 aptamer was created by partial alkaline hydrolysis (incubation of 2 g of aptamer in 50 mm NaHCO3, pH 9.2, at 95 C for 13 min). Products of the degradation assay were separated by electrophoresis on an 18% polyacrylamide, 8 m urea gel. Bands were visualized using an Odyssey? (LI-COR) fluorescence scanner, and were quantified using the integrated intensities.