Nielsen P E, Egholm M, Buchardt O
Department of Medical Biochemistry and Genetics, Panum Institute, Copenhagen, Denmark.
Gene. 1994 Nov 4;149(1):139-45. doi: 10.1016/0378-1119(94)90422-7.
The effects of PNA (peptide nucleic acid) bound to double-stranded (ds) DNA targets positioned downstream from phage T3 or T7 promoters in pBluescriptKS+ derived plasmids on transcription by RNA polymerases T3 or T7 have been studied. The dsDNA targets A10, 5'-A5GA4 or 5'-A2GA2GA4, and the corresponding PNAs T10, T5CT4 and T2CT2CT4 were used and the target-PNA strand displacement complexes were performed in low-salt buffer, since PNA does not bind efficiently to ds DNA in higher salt than 50 mM. It is shown that transcription elongation is arrested at the target site with PNA bound to the template strand, whereas only a marginal effect is observed with PNA bound to the non-template strand. With PNA T10, transcription arrest occurs at the first base of the PNA-binding site, while the arrest with the PNA T5CT4 takes place 2-3 nt inside the PNA binding site. In the case of PNA T2CT2CT4 the arrest is less efficient and occurs at the last 1-3 nt of the binding site. Transcription arrest was also shown for PNAs T6 and T8, although with a much lower efficiency. These results show that efficient transcription elongation arrest can be obtained by PNA targeting of the template DNA strand.
研究了与源自pBluescriptKS+质粒中噬菌体T3或T7启动子下游的双链（ds）DNA靶标结合的肽核酸（PNA）对RNA聚合酶T3或T7转录的影响。使用了dsDNA靶标A10、5'-A5GA4或5'-A2GA2GA4以及相应的PNA T10、T5CT4和T2CT2CT4，并且靶标-PNA链置换复合物在低盐缓冲液中进行，因为PNA在高于50 mM的盐浓度下不能有效地与dsDNA结合。结果表明，当PNA与模板链结合时，转录延伸在靶位点处被阻断，而当PNA与非模板链结合时，仅观察到微小的影响。对于PNA T10，转录阻断发生在PNA结合位点的第一个碱基处，而对于PNA T5CT4，阻断发生在PNA结合位点内2-3个核苷酸处。对于PNA T2CT2CT4，阻断效率较低，发生在结合位点的最后1-3个核苷酸处。对于PNA T6和T8也显示出转录阻断，尽管效率要低得多。这些结果表明，通过PNA靶向模板DNA链可以实现有效的转录延伸阻断。
