Puri Nitin, Majumdar Alokes, Cuenoud Bernard, Miller Paul S, Seidman Michael M
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Biochemistry. 2004 Feb 10;43(5):1343-51. doi: 10.1021/bi035808l.
We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in living mammalian cells. We have described psoralen-linked TFOs with 2'-O-methyl and 2'-O-(2-aminoethyl) (2'-AE) substitutions that are active in a gene knockout assay in cultured cells. The assay is based on mutagenesis by psoralen, a photoactive DNA cross-linker. Previous work showed that TFOs with three or four 2'-AE residues were disproportionately more active than those with one or two substitutions. Here we demonstrate that for optimal bioactivity the 2'-AE residues must be clustered rather than dispersed. We have further characterized bioactive and inactive TFOs in an effort to identify biochemical and biophysical correlates of biological activity. While thermal stability is a standard monitor of TFO biophysical activity, we find that T(m) values do not distinguish bioactive and inactive TFOs. In contrast, measurements of TFO association rates appear to correlate well with bioactivity, in that triplex formation occurs disproportionately faster with the TFOs containing three or four 2'-AE residues. We asked if extending the incubation time prior to photoactivation would enhance the bioactivity of a TFO with a slow on rate relative to the TFO with a faster association rate. However, there was no change in bioactivity differential. These results are compatible with a model in which TFO binding in vivo is followed by relatively rapid elution by cellular functions, similar to that described for transcription factors. Under these circumstances, TFOs with faster on rates would be favored because they would be more likely to be in triplexes at the time of photoactivation.
我们正在研发三链螺旋形成寡核苷酸（TFO）作为活的哺乳动物细胞中的基因靶向试剂。我们已经描述了与补骨脂素连接的TFO，其具有2'-O-甲基和2'-O-（2-氨基乙基）（2'-AE）取代，这些TFO在培养细胞的基因敲除试验中具有活性。该试验基于补骨脂素（一种光活性DNA交联剂）的诱变作用。先前的工作表明，具有三个或四个2'-AE残基的TFO比具有一个或两个取代基的TFO活性更高。在这里，我们证明，为了获得最佳生物活性，2'-AE残基必须聚集而不是分散。我们进一步对生物活性和非活性TFO进行了表征，以努力确定生物活性的生化和生物物理相关性。虽然热稳定性是TFO生物物理活性的标准监测指标，但我们发现T(m)值无法区分生物活性和非活性TFO。相比之下，TFO结合速率的测量似乎与生物活性密切相关，因为含有三个或四个2'-AE残基的TFO形成三链体的速度明显更快。我们询问在光激活之前延长孵育时间是否会增强结合速率较慢的TFO相对于结合速率较快的TFO的生物活性。然而，生物活性差异没有变化。这些结果与一个模型相符，在该模型中，TFO在体内结合后会被细胞功能相对快速地洗脱，类似于转录因子的情况。在这些情况下，结合速率较快的TFO会更受青睐，因为它们在光激活时更有可能处于三链体状态。
