Debin A, Malvy C, Svinarchuk F
Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institute Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif cedex, France.
Nucleic Acids Res. 1997 May 15;25(10):1965-74. doi: 10.1093/nar/25.10.1965.
In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro . Moreover, this triplex was stable inside cells when preformed in vitro . However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine.(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine.(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro . In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.
在之前的一项研究中，我们发现，靶向小鼠c-pim-1原癌基因启动子的短三链螺旋形成寡核苷酸（TFO）在体外生理条件下能形成非常稳定的三链螺旋。此外，这种三链体在体外预先形成后，在细胞内也很稳定。然而，在二甲基亚砜足迹分析研究中，我们未能在细胞内检测到该序列形成三链体。在本研究中，为了确定我们之前的体内实验结果是仅限于这种特定的短三链体，还是可以推广到其他嘌呤·（嘌呤/嘧啶）三链体，我们测试了文献中已描述的其他三个DNA靶点。所有这些嘌呤·（嘌呤/嘧啶）三链体在体外高温下都是特异性且稳定的。体内研究表明，预先形成的三链体在细胞内至少能稳定存在3天。这清楚地表明细胞内环境有利于嘌呤·（嘌呤/嘧啶）三链体的存在。三链体也能在细胞核中形成。然而，对于所有测试的序列，我们在完整细胞的体内实验中，通过二甲基亚砜足迹分析均未能检测到任何三链螺旋的形成。我们的结果表明，DNA靶点的（i）染色质化、（ii）细胞内钾离子浓度以及（iii）TFO与DNA靶点在细胞质与细胞核中的分隔，均不是细胞内三链体形成受阻的原因。我们推测存在一种基于TFOs的区室化和/或TFO捕获的细胞机制，该机制将寡核苷酸与DNA靶点分隔开。需要进一步开展工作来寻找寡核苷酸衍生物及其递送方法，以克服细胞内三链体形成的问题。
