Systematic Evolution of Ligands by EXponential enrichment (SELEX) was used to isolate from a population of 10(13) RNA molecules two classes of high affinity RNAs that bind specifically to human alpha-thrombin. Class I RNAs are represented by a 24-nucleotide RNA (RNA 16.24), and class II RNAs are represented by a 33-nucleotide RNA (RNA 27.33). RNA 16.24 inhibits thrombin-catalyzed fibrin clot formation in vitro. Secondary structures are proposed for these RNAs, revealing a novel stem-loop structure for RNA 16.24, comprised of an unusually large 16-nucleotide loop. Mutants of RNA 16.24 were generated to investigate structural features critical to high-affinity binding. Phosphate modification with ethylnitrosourea identified regions of the RNAs necessary for electrostatic interactions. Competition with heparin suggests that these RNAs bind the electropositive heparin-binding site of thrombin. These ligands represent a novel class of thrombin inhibitors that may be suitable for therapeutic or diagnostic applications.
Twenty-three BK virus and JC virus DNA samples obtained from urine of pregnant women had almost exclusively archetypal transcriptional control regions. Rearrangements characteristic of laboratory strains are apparently not required for reactivation in humans. Unexpectedly, alignment shows that many elements identified previously in the BK virus enhancer are conserved in the JC virus archetype.
CD4+ Th cells produce cytokines that play a pivotal role in the induction and regulation of cell-mediated and humoral immunity. Th1 cells, characterized by their secretion of IFN-gamma, induce macrophage cytotoxicity, delayed hypersensitivity, and enhanced cellular immunity. Secretion of IFN-gamma may even suppress Th2-enhanced humoral immunity. A counterproductive Th1 response and concomitant secretion of IFN-gamma may result in inflammatory and autoimmune diseases. IFN-gamma regulation of T cell function has potential for therapeutic intervention. To isolate high affinity oligonucleotide inhibitors of IFN-gamma activity, combinatorial libraries of RNA molecules modified at the 2' position of pyrimidine nucleotides with fluoro (F), amino (NH2), or a mixture of F and NH2 (2'-F/NH2) were screened using the SELEX (systematic evolution of ligands by exponential enrichment) combinatorial chemistry process. Each modified library of RNA molecules provides an expanded repertoire of molecules with increased structural diversity and unique binding properties. This added diversity increases the possibility of isolating molecules with the desired functional properties. These RNAs modified at the 2' position have also been shown to be nuclease resistant. High affinity ligands to human IFN-gamma from each modified library were isolated and characterized. The K(d)s of these ligands were determined and their secondary structures were predicted. The specificity of these ligands for IFN-gamma binding was confirmed, and their ability to inhibit binding of IFN-gamma to its receptor on A549 human lung carcinoma cells was determined. A 2'-NH2-modified ligand (2'-NH2-30) is described that binds IFN-gamma with high affinity and inhibits IFN-gamma-induced expression of MHC class I and ICAM-1 by human myeloid leukemia cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.