| 摘要 |
Nucleic acid hybridization under steady-state conditions is described by a kinetic model in which the intermediate state is assumed to be locally single stranded. An expression was derived that relates nucleic acid secondary structure to the rate of oligonucleotide-RNA hybridization. The model assumes that the hybridization of nucleic acids occurs through an intermediate state in which the region to be hybridized has a single-stranded conformation prior to binding the antisense oligonucleotide. In the derivation, a steady-state condition is assumed. The model is applicable to the steady-state condition in living cells and the initial stage of single-tube hybridization when full-length hybrid has not significantly accumulated and the concentration of nucleation complex is approximately constant. The model allows the calculation of a rate factor that is proportional to the rate constant for hybridization between complementary nucleic acids. When rate factors were calculated using a commercially available algorithm for estimating RNA secondary structure, they correlated well with rates for hybridization of antisense oligodeoxynucleotides (ODNs) to a 101-nucleotide artificial RNA that has been published and of molecular beacons to HIV-1 tat mRNA. For RNA-RNA annealing, the locations of 11-nucleotide long regions that have the maximum rate factor coincide with experimentally determined nucleation sites. Dependence of the maximum rate factor on the length of antisense RNA is in agreement with observed relationship between hybridization rates and antisense lengths. Rate factors calculated for 32-sites in HIV-1 integrase mRNA also correlated with hybridization of antisense ODN to each site when measured by ODN-mediated, ribonuclease H-dependent cleavage of RNA. The model identified sites that hybridized readily over a range of magnesium ion concentration expected to affect RNA tertiary structure, suggesting that tertiary structure was either absent or was not an important impediment to hybridization. Calculated target site rate factors also corresponded to published data for antisense oligonucleotide hybridization with mRNAs of human genes encoding multidrug resistance, angiotensin type-1 receptor, c-myb, acetylcholinesterase, and hepatitis C virus. These results support the general applicability of the kinetic model and its potential utility for rapid identification of sites for antisense attack of mRNA.
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