Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versus slow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitro selection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-abl sequence in comparison with either of the wild-type sequences bcr or abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abl transcript was specifically hydrolysed out of a mixture containing abl and bcr sequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either via helix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-abl fusion point. Target selectivity was found for a ribozyme that annealed fast via abl sequences and cleaved within the bcr portion of bcr-abl RNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-abl fusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-abl fusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-abl gene expression in Philadelphia chromosome-positive cells.
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PMID:Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes. 868 70

During the course of a study aimed at improving antisense oligodeoxynucleotide-mediated ex vivo bone marrow purging of patients suffering from chronic myeloid leukemia (CML), the properties of a number of antisense structures intended to reduce the expression of c-myc, mutant p53, and bcr-abl mRNAs and proteins were examined. The majority of the antisense oligodeoxynucleotides were designed to be capable of directing ribonuclease H (RNase H) cleavage of their target mRNAs. Streptolysin O (SLO) reversible permeabilization was used to deliver the oligodeoxynucleotides into the CML line KYO-1. We found that the efficiency and specificity of antisense oligonucleotide-induced reductions of target protein expression depended on target protein half-life, the oligonucleotide structure, and the specific sequence within the target mRNA. Transient reductions of c-myc mRNA and protein were achieved with a chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to the initiation codon, but cell proliferation was unaffected. In contrast, a chimeric oligodeoxynucleotide of similar structure targeted to an alternative site in the coding region of c-myc mRNA reduced target mRNA and protein levels for over 24 hours and halted cell proliferation. Chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to a point mutation in KYO-1 p53 mRNA efficiently reduced target mRNA expression, but only small, transient reductions in p53 protein expression were observed. However, a chimeric methylphosphonate-phosphorothioate oligodeoxynucleotide targeted to the same site reduced p53 protein to 30% of control levels over a 48-hour period. BCR-ABL protein expression was unaffected by chimeric oligodeoxynucleotides targeted to the breakpoint in bcr-abl mRNA, even when mRNA levels at early times were substantially reduced.
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PMID:The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog-mediated biologic responses. 974 66