Rumney Squire, Kool Eric T
Department of Chemistry, University of Rochester, Rochester, New York 14627.
J Am Chem Soc. 1995;117:5635-5646. doi: 10.1021/ja00126a004.
Described are studies systematically exploring structural effects in he use of ethylene glycol (EG) oligomers as non-nucleotide replacements for nucleotide loops in duplex and triplex DNAs. The new structurally optimized loop replacements are more stabilizing in duplexes and triplexes than previously described EG-based linkers. A series of compounds ranging in length from tris(ethylene glycol) to octakis(ethylene glycol) are derivatized as monodimethoxytrityl ethers on one end and phosphoramidites on the other, to enable their incorporation into DNA strands by automated methods. These linker molecules span lengths ranging from 13 to 31 Å in extended conformation. They are incorporated into a series of duplex-forming and triplex-forming sequences, and the stabilities of the corresponding helixes are measured by thermal denaturation. In the duplex series, results show that the optimum linker is the one derived from heptakis(ethylene glycol), which is longer than most previous loop replacements studied. This affords a helix with greater thermal stability than one with a natural T(4) loop. In the triplex series, the loop replacements were examined in four separate situations, in which the loop lies in the 5' or 3' orientation and the central purine target strand is short or extends beyond the loop. Results show that in all cases the loop derived from octakis(ethylene glycol) (EG(8)) gives the greatest stability. In the cases where the target strand is short, the EG(8)-linked probe strands bind with affinities in some cases greater than those with a natural pentanucleotide (T(5)) loop. For the cases where the target strand extends beyond the linker, the EG(8)-linked strands are much lower in the 5' loop orientation than in the 3' loop orientation. It is found that extension by one additional nucleotide in one of the bonding domains in the EG-linked series can result in considerably greater stabilities with long target strands. Overall, the data show that optimum loop replacements are longer than would be expected from simple distance analysis. The results are discussed in relation to expected lengths and geometries for double and triple helixes. The findings will be usefull in the design of synthetically modified nucleic acids for use as diagnostic probes, as biochemical tools, and as potential therapeutic agents.
本文描述了一系列系统性研究，旨在探索乙二醇（EG）低聚物作为双链和三链DNA中核苷酸环的非核苷酸替代物时的结构效应。新的结构优化的环替代物在双链和三链结构中比先前描述的基于EG的连接子更具稳定性。一系列长度从三乙二醇到八乙二醇的化合物，一端衍生为单二甲氧基三苯甲基醚，另一端衍生为亚磷酰胺，以便通过自动化方法将它们掺入DNA链中。这些连接子分子在伸展构象下的长度范围为13至31 Å。它们被掺入一系列双链形成和三链形成序列中，并通过热变性测量相应螺旋的稳定性。在双链系列中，结果表明最佳连接子是源自七乙二醇的连接子，其长度比大多数先前研究的环替代物更长。这提供了一个比具有天然T(4)环的螺旋具有更高热稳定性的螺旋。在三链系列中，在四种不同情况下检查环替代物，其中环位于5'或3'方向，并且中央嘌呤靶链短或延伸超过环。结果表明，在所有情况下，源自八乙二醇（EG(8)）的环具有最大的稳定性。在靶链短的情况下，EG(8)连接的探针链在某些情况下的结合亲和力大于具有天然五核苷酸（T(5)）环的链。对于靶链延伸超过连接子的情况，EG(8)连接的链在5'环方向上比在3'环方向上低得多。研究发现，在EG连接系列的一个结合域中额外延伸一个核苷酸可以导致与长靶链相比具有显著更高的稳定性。总体而言，数据表明最佳环替代物比简单距离分析预期的要长。结合双螺旋和三螺旋的预期长度和几何形状对结果进行了讨论。这些发现将有助于设计用作诊断探针、生化工具和潜在治疗剂的合成修饰核酸。
