Jones G D, Lesnik E A, Owens S R, Risen L M, Walker R T
The School of Chemistry, The University of Birmingham, UK.
Nucleic Acids Res. 1996 Nov 1;24(21):4117-22. doi: 10.1093/nar/24.21.4117.
The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides. A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide. However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex. A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity. The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide. However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1. It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.
测定了4'-硫代修饰的寡脱氧核苷酸与其DNA和RNA互补链形成的双链体的热稳定性，并与相应的未修饰寡脱氧核苷酸的热稳定性进行了比较。一个含有10个连续4'-硫代胸苷酸修饰的16聚体寡脱氧核苷酸与DNA靶标形成的双链体稳定性低于相应的未修饰寡脱氧核苷酸（ΔTm/修饰为-1.0℃）。然而，当相同的寡脱氧核苷酸与相应的RNA靶标结合时，与未修饰的双链体相比，观察到Tm有小幅升高（ΔTm/修饰为+0.16℃）。一项研究旨在确定含有4'-硫代胸苷酸修饰的寡脱氧核苷酸与在修饰位点对面含有单个错配的DNA或RNA形成双链体时的特异性，结果发现所得的Tm与野生型双链体几乎相同，这表明寡脱氧核苷酸中的4'-硫代修饰对特异性没有有害影响。使用蛇毒磷酸二酯酶（SVPD）和核酸酶S1检测了4'-硫代修饰的寡脱氧核苷酸的核酸酶稳定性。与相应的未修饰寡脱氧核苷酸相比，未观察到对外切核酸酶SVPD降解的显著抗性。然而，发现4'-硫代修饰的寡脱氧核苷酸对核酸酶S1的降解具有高度抗性。还证明了4'-硫代修饰的寡脱氧核苷酸仅在高酶浓度下才能引发大肠杆菌RNase H对RNA靶标的水解。
