| SELEX (Systematic Evolution of ligands by Exponential Enrichment) is a combinatorial chemistry technology for studying structure, function and molecular evolution of nucleic acid. It permits isolation of aptamers that bind a wide variety of targets with high affinity and specificity from complex libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. This technology has promising application in basic research, drug screening, and diagnosis. The objective of this research is to establish an in vitro selection method with a random RNA library and to screen RNA aptamers against bovine thrombin and gp!6( large subunit of the bacteriophage 029 teminase),which will provide basic data for drug screening and anti-virus therapeutic agents.As a large amount of T7RNA polymerase is necessary in the procedure of the SELEX with a random RNA library, we first successfully expressed T7RNA polymerase E. Coli. The purified T7RNA polymerase has good transcription activity. One milligram of purified T7RNA polymerase equals to 250Units of T7 enzyme bought from Promega.After obtaining enough purified T7RNA polymerase, RNA aptamers against to bovine thrombin were selected by the the SELEX method. Bovine thrombin was widely used as topical hemostastic agent in cardiovascular surgery, otolarynglolgic surgery andneurosurgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Consequently, life-threatening hemorrhage occurred in patients after several times of usage bovine thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. It may be of certain clinical significance for screening molecular specific recognizing bovine thrombin. Eighty one nucleotides RNA pool, containing 25 random nucleotides flanked by invariant primer was adopted in our experiment. After 7 rounds selection, two aptamers specific for bovine thrombin were identified with a Kd of 164nmol/L and 240nmol/L, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite.Then several trancated versions of aptamer T705 were further assayed. We found that removal of fixed flank sequences had no effect on the T705 inhibitory activity. However, lack of any sequences of random region leads to the elimination of its specific binding activity to bovine thrombin which may be caused by changing of the secondary structure. It demonstrated that the precise stem-loop structure was the basic structure of the aptamer exerted their inhibitory activity.Four antisense oligonucleotides complemertary to different portions of aptamer T705c were designed as potential antidotes. The clotting time assay indicated that the oligonucleotides c' and c'22 could completely reverse the anti-coagulant activity ofaptamer T705 in 15 min and shorten the clotting time in a dose-dependent manner. It's a very effective approach to get an antidote oligonucleotide rapidly by altering the shape of an aptamer from an active to an inactive conformation.After finishing the above research work on bovine thrombin, we also selected RNA aptamers specifically binding to gp16, a large subunit of phage 29 terminase. The RNA library was subjected to 9 successive rounds of selection with SELEXmethod, resulting in an increase of the percentage of the gp16-binding RNA from 14.4% for the first round pool to 37.8% for the ninth-round pool. After cloned and sequenced, the primary sequences could be classified into 5 families. The secondary structure of the aptamers analysis revealed that the stem-loop or bulge was the main motif in the i...
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