Hammerhead ribozymes with long antisense flanks (>50 bases) have been used successfully to inhibit replication of human immunodeficiency virus type 1 (HIV-1) in living cells. To explain their increased efficacy versus antisense controls or catalytically inactive derivatives, one can consider dissociation of the ribozyme-product complex to allow a complete catalytic cycle. In this work we investigated the dissociation of a double-stranded RNA with 56 bp in vitro. Dissociation was observed in the presence of single-stranded RNA with sequence complementarity to one of the duplex strands. A displacement reaction between RNA single strands and the duplex, but not simple dissociation, was strongly suggested by the concentration dependence of this process, the influence of additional non-complementary sequences on the single strand and by the unusually low Arrhenius activation energy. The strand displacement reaction was slow in vitro at 37 degrees C and physiological ionic strength, but was increased to k approximately 10(3)-10(4)/M/s (approximately 10(4)-fold) at higher temperatures by cetyltrimethylammonium bromide. This compound is thought to enhance non-sequence-specific association of nucleic acids in a mechanistically similar way to that in which cellular hnRNP proteins are thought to act, indicating that strand displacement can be fast and, more importantly, could be tightly regulated in vivo.
The association rates of complementary nucleic acids can be increased by 2-3 orders of magnitude in vitro by cellular proteins and low molecular weight compounds including cetyltrimethylammonium bromide (CTAB). In this work, we provide experimental evidence that the CTAB-mediated enhancement of RNA-RNA annealing by approximately 3 orders of magnitude is due to a favorable activation entropy (DeltaS) and not due to a decrease of the Arrhenius activation energy (Ea) nor to major structural changes of the RNA. Two alternative models for the CTAB-facilitated RNA-RNA annealing will be discussed. First, CTAB could form a positively charged liquid matrix which could steer complementary RNA molecules and thereby increase their collision frequency and annealing rate. Second, increased annealing rates could be explained by stabilization of a non-base-specific precomplex of both complementary RNA molecules in solution.
The integrin v 3 plays a central role in angiogenesis. In this study, we used antisense oligodeoxyribonucleotides ( ONs ) directed against the v subunit of v 3 to inhibit integrin expression. Ten ON sequences, which were selected by systematic alignment of computer -predicted secondary structures of v mRNA, were transfected into human umbilical vein endothelial cells ( HUVECs ). Following stimulation by PMA, five antisense ONs significantly inhibited v mRNA and protein expression in activated HUVEC at a concentration of 0.05 M with complete prevention of PMA -induced v up -regulation by the most potent antisense ON. Inhibition of v expression was associated with significant inhibition of migration of HUVEC by 28% and had no effect on proliferation and apoptosis. Moreover, transfection of antisense ON inhibited the formation of tube -like structures of HUVEC in Matrigel by 44%. In a cell culture model of angiogenesis consisting of a co -culture of endothelial cells with fibroblasts, transfection of antisense ONs resulted in an inhibition of tube formation of 61%. In conclusion, v antisense ONs are potent inhibitors of angiogenesis in vitro. They might, therefore, be a therapeutic alternative to antagonists, which directly bind to v integrins, and might be useful for the treatment of malignant tumors and hematological malignancies.
Among the large number of possible antisense species against a given target RNA, only a small number shows effective suppression of the target gene in living cells. In the case of short-chain antisense oligonucleotides (asON) which usually comprise less than approximately 25 nucleotides, local structures of the target RNA seem to be of particular importance for the extent of gene suppression. Experimental approaches to identify promising local target sequences and, hence, complementary asON sequences, have provided tools to define asON that are biologically active at higher than statistical probability. However, experimental protocols are expensive, time consuming, and are associated with intrinsic basic and technical limitations. As insights into the structure-function relationship of asON as well as the role of sequence motifs increase, it becomes feasible to consider computer-based theoretical approaches for the design of effective asON. In the following we describe how individual steps of the theoretical design of asON may be automated by establishing and implementing suitable algorithms.
Periodontal diseases, such as gingivitis and periodontitis, are caused by a mixed infection by several types of bacteria in the dental plaque, causing a chronic inflammation of the gingival mucosa. Inflammatory processes in conjunction with immune responses to bacterial attacks are generally protective. In profound periodontitis, however, hyperresponsiveness and hypersensitivity of the immune system are counterproductive because of the destruction of the affected periodontal connective tissues. The intercellular adhesion molecule type 1 (ICAM-1) plays a key role in the onset and manifestation of inflammatory responses. Thus, inhibition of ICAM-1 expression could be of therapeutic relevance for the treatment of destructive periodontitis. Here, antisense oligonucleotides (AS-ON) directed against ICAM-1 suppress protein expression and mRNA levels specifically and effectively in primary human endothelial cells of different tissue origin. Moreover, downregulation of ICAM-1 expression is also observed in AS-ON-transfected inflamed gingival mucosal tissue of patients with periodontal diseases. This work strongly suggests exploiting the local topical application of ICAM-1-directed AS-ON as a therapeutic tool against inflammatory processes of the human gingiva.
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