Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a Kd of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide "core" sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed.

Isolation and characterization of 2'-fluoro-, 2'-amino-, and 2'-fluoro-/amino-modified RNA ligands to human IFN-gamma that inhibit receptor binding.

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.

High-affinity RNA ligands to human alpha-thrombin.

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.

BK virus and JC virus shed during pregnancy have predominantly archetypal regulatory regions.

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.

Transactivation by the human T-cell leukemia virus Tax protein is mediated through enhanced binding of activating transcription factor-2 (ATF-2) ATF-2 response and cAMP element-binding protein (CREB).

The human T-cell leukemia virus type I (HTLV-I)-encoded transcriptional activator protein Tax is strongly implicated in HTLV-I pathogenesis. Tax regulates HTLV-I gene expression through three 21-base pair (bp) repeat enhancer elements located in the transcriptional control region of the virus. Tax does not bind these elements directly, but mediates transactivation through the cellular transcription factors that recognize a cAMP response element (CRE)-like sequence centered within each of the 21-bp repeats. In this report, we identify activating transcription factor-2 (ATF-2) and CRE-binding protein (CREB) as the principal T-cell proteins that bind the three 21-bp repeats in vitro. Purified Tax protein augments the level of RNA synthesis induced by ATF-2 and CREB in a cell-free transcription assay, providing evidence that Tax cooperates with these cellular proteins to activate HTLV-I transcription. Furthermore, Tax dramatically increases the binding of both the T-cell-derived and recombinant forms of ATF-2 and CREB to each of the 21-bp repeats. The target sequences for this enhancement reside within the DNA binding/dimerization domains of these proteins. These data suggest that Tax transactivates HTLV-I gene expression by increasing the number of bound ATF-2 and CREB molecules at the viral promoter.