Jayasena S D, Johnston B H
Cell and Molecular Biology Laboratory, SRI International, Menlo Park, California 94025.
Biochemistry. 1993 Mar 23;32(11):2800-7. doi: 10.1021/bi00062a010.
Until recently, oligonucleotide-directed triplex formation has been limited to oligopurine tracts of target DNA. Triplex formation by alternate-strand recognition relaxes this limitation by allowing triplexes to form at 5'-(Pu)m(Py)n-3' and 5'-(Py)m(Pu)n-3' sequences, with the third strand pairing first with purines on one strand and then switching to pair with purines on the other strand. In this study, the interaction of several oligonucleotides with the potential to form triplexes by alternate-strand recognition at the sequence 5'-A8C8A8-3' was studied by chemical probing and affinity cleaving. The results show that triplex formation can be readily accomplished at the 5'-A8C8-3' part of the sequence; however, base triplet formation is disrupted on either side of the strand switch and the Watson-Crick helix is distorted in such a way as to expose the N7 positions of purines adjoining the strand switch. Triplex formation is weak or nonexistent at the 3'-most A8 block, despite the opportunity for recruiting a spacer sequence for the second (C8-A8) strand switch by "slippage". This finding indicates that the C8-A8 strand switch is energetically unfavorable, although pairing at other 5'-(Py)n(Pu)n-3' sequences has been observed, with or without a spacer [Beal, P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478; Jayasena, S. D., & Johnston, B. H. (1992) Nucleic Acids Res. 20, 5279-5288]. Thus, alternate-strand recognition may not be feasible for certain sequences of 5'-(Py)m(Pu)n-3', at least under the conditions examined.
直到最近，寡核苷酸导向的三链体形成还仅限于靶DNA的寡嘌呤区段。通过交替链识别形成三链体放宽了这一限制，允许三链体在5'-(Pu)m(Py)n-3'和5'-(Py)m(Pu)n-3'序列处形成，第三条链首先与一条链上的嘌呤配对，然后切换与另一条链上的嘌呤配对。在本研究中，通过化学探针和亲和切割研究了几种寡核苷酸在序列5'-A8C8A8-3'处通过交替链识别形成三链体的相互作用。结果表明，在序列的5'-A8C8-3'部分可以很容易地形成三链体；然而，在链切换的两侧碱基三联体形成受到破坏，并且沃森-克里克螺旋发生扭曲，使得与链切换相邻的嘌呤的N7位置暴露。尽管有机会通过“滑动”为第二条（C8-A8）链切换招募一个间隔序列，但在最末端的3'-A8区段三链体形成较弱或不存在。这一发现表明，C8-A8链切换在能量上是不利的，尽管在其他5'-(Py)n(Pu)n-3'序列处已观察到配对，无论有无间隔序列[比尔，P.A.，&德凡，P.B.（1992年）《美国化学会志》114，1470 - 1478；贾亚塞纳，S.D.，&约翰斯顿，B.H.（1992年）《核酸研究》20，5279 - 5288]。因此，至少在所研究的条件下，对于某些5'-(Py)m(Pu)n-3'序列，交替链识别可能不可行。
