Comparison of antiparallel A.AT and T.AT triplets within an alternate strand DNA triple helix.
Comparison of antiparallel A.AT and T.AT triplets within an alternate strand DNA triple helix.
复制标题
交替链 DNA 三螺旋内反平行 A.AT 和 T.AT 三联体的比较。
作者：
E. Washbrook;K. Fox
我们研究了目标序列A11(TC)6上交替链三螺旋的形成。（GA）6T11利用寡核苷酸T11(AG)6和T11(TG)6，通过DNase I足迹。这第三条链被设计成与反平行的g.c gc和A.AT或T.AT三联体形成平行的T.AT三联体。我们发现，尽管这两种寡核苷酸在锰存在的相似浓度（0.3微米）下产生清晰的足迹，但只有T11(TG)6在含镁缓冲液中形成稳定的复合物，尽管浓度更高（10-30微米）。对（AG）6和（TG）6与一半靶位点相互作用的检查证实，含有A.AT三联体的配合物只有在锰存在时才稳定。相反，在两种金属离子存在的情况下，没有检测到（TG）6的结合，这表明反向hoogsteen T.AT三重态比g.c gc更不稳定。我们认为，在g.c gc三元组中，反平行三元组稳定性的秩序为A.AT (Mn2+) > T.AT (Mn2+) > T.AT (Mg2+) > A.AT （Mg2+）。在平行或反平行部分的中心含有单一碱基取代的第三条链在含锰缓冲液中显示出（10倍）弱的相互作用，并且在镁存在时没有相互作用。
We have examined the formation of alternate strand triple-helices at the target sequence A11(TC)6.(GA)6T11 using the oligonucleotides T11(AG)6 and T11(TG)6, by DNase I footprinting. These third strands were designed so as to form parallel T.AT triplets together with antiparallel G.GC and A.AT or T.AT triplets. We find that, although both oligonucleotides yield clear footprints at similar concentrations (0.3 microM) in the presence of manganese, only T11(TG)6 forms a stable complex in magnesium-containing buffers, albeit at a higher concentration (10-30 microM). Examination of the interaction of (AG)6 and (TG)6 with half the target site confirmed that the complex containing A.AT triplets was only stable in the presence of manganese. In contrast no binding of (TG)6 was detected in the presence of either metal ion, suggesting that the reverse-Hoogsteen T.AT triplet is less stable that G.GC. We suggest that, within the context of G.GC triplets, the rank order of antiparallel triplet stability is A.AT (Mn2+) > T.AT (Mn2+) > T.AT (Mg2+) > A.AT (Mg2+). Third strands containing a single base substitution in the centre of either the parallel or antiparallel portion showed a (10-fold) weaker interaction in manganese-containing buffers, and no interaction in the presence of magnesium.