Gamper H B, Cimino G D, Hearst J E
HRI Research Incorporated, Berkeley, CA 94710.
J Mol Biol. 1987 Sep 20;197(2):349-62. doi: 10.1016/0022-2836(87)90128-8.
Several DNA oligonucleotides have been photochemically modified with the furocoumarin 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) such that each contained a single HMT furan side monoadduct to thymidine at a unique 5' TpA 3' sequence. When these oligonucleotides were hybridized to their respective complements, the HMT adduct could be driven to form an interstrand crosslink by irradiation of the hybrid with 360 nm light. The ability to crosslink probe-target complexes has allowed us to determine the kinetics and the extent of hybridization in solution between these oligonucleotides and their complementary sequences in single-stranded bacteriophage M13 DNA. Our data indicate that these parameters are strongly influenced by the existence of local as well as global secondary structure in the viral DNA. During hybridization, rearrangement of this secondary structure so as to expose the target sequence can be rate-limiting. Upon attainment of equilibrium, only a portion of the target sequence may be hybridized to the probe with the remainder involved in intrastrand base-pairing. Using crosslinkable oligonucleotide probes hybridized and irradiated near the melting temperature of the respective probe-target complex one can partially overcome these secondary structure effects.
几种DNA寡核苷酸已用光化学方法与呋喃香豆素4'-羟甲基-4,5',8-三甲基补骨脂素（HMT）进行修饰，使得每个寡核苷酸在独特的5'TpA3'序列处都含有一个与胸腺嘧啶的单个HMT呋喃侧单加合物。当这些寡核苷酸与它们各自的互补链杂交时，通过用360nm光照射杂交体，可促使HMT加合物形成链间交联。交联探针-靶标复合物的能力使我们能够确定这些寡核苷酸与单链噬菌体M13 DNA中的互补序列在溶液中的杂交动力学和程度。我们的数据表明，这些参数受到病毒DNA中局部和整体二级结构的强烈影响。在杂交过程中，这种二级结构的重排以暴露靶序列可能是限速步骤。达到平衡后，只有一部分靶序列可能与探针杂交，其余部分参与链内碱基配对。使用在各自探针-靶标复合物的解链温度附近杂交并照射的可交联寡核苷酸探针，可以部分克服这些二级结构效应。
