Distance and Affinity Dependence of Triplex-Induced Recombination
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- 作者：Melissa P. Knauert ; Janice A. Lloyd ; Faye A. Rogers ; Hirock J. Datta ; Michael L. Bennett ; Daniel L. Weeks ; and Peter M. Glazer
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：March 15, 2005
- 年：2005
- 卷：44
- 期：10
- 页码：3856 - 3864
- 全文大小：398K
- 年卷期：v.44,no.10(March 15, 2005)
- ISSN：1520-4995
文摘
Triplex-forming oligonucleotides (TFOs) have the potential to serve as gene therapeutic agentson the basis of their ability to mediate site-specific genome modification via induced recombination.However, high-affinity triplex formation is limited to polypurine/polypyrimidine sites in duplex DNA.Because of this sequence restriction, careful analysis is needed to identify suitable TFO target sites withinor near genes of interest. We report here an examination of two key parameters which influence theefficiency of TFO-induced recombination: (1) binding affinity of the TFO for the target site and (2) thedistance between the target site and the mutation to be corrected. To test the influence of binding affinity,we compared induced recombination in human cell-free extracts by a series of G-rich oligonucleotideswith an identical base composition and an increasing number of mismatches in the third strand bindingcode. As the number of mismatches increased and, therefore, binding affinity decreased, inducedrecombination frequency also dropped. There was an apparent threshold at an equilibrium dissociationconstant (Kd) of 1 × 10-7 M. In addition, TFO chemical modification with N,N-diethylethylenediamine(DEED) internucleoside linkages to confer improved binding was found to yield increased levels of inducedrecombination. To test the ability of triplex formation to induce recombination at a distance, episomaltargets with informative reporter genes were constructed to contain polypurine TFO target sites at varyingdistances from the mutations to be corrected. TFO-induced recombination in mammalian cells betweena plasmid vector and a donor oligonucleotide was detected at distances ranging from 24 to 750 bp. Together,these results indicate that TFO-induced recombination requires high-affinity binding but can affect siteshundreds of base pairs away from the position of triplex formation.