Lima W F, Monia B P, Ecker D J, Freier S M
Department of Molecular and Cellular Biology, Isis Pharmaceuticals, Carlsbad, California 92008.
Biochemistry. 1992 Dec 8;31(48):12055-61. doi: 10.1021/bi00163a013.
A 47-nucleotide transcript of the activated Ha-ras gene was prepared and determined, by enzymatic structure mapping, to form a stable hairpin structure. Six antisense decaribonucleotides were designed, and association constants (Ka) for the hairpin- and length-matched complements were measured. Two of the antisense oligonucleotides targeted to the loop had nearly equal affinity for the transcript compared to the complement. The others, including one oligonucleotide complementary to the 3' side of the single-stranded loop, bound 10(5)-10(6)-fold less tightly to the transcript than to the short complement. We propose the difference in affinity is due to the target structure, both the secondary structure of the stem and the structure in the loop. Measurement of the bimolecular association rate constant, k1, and the dissociation rate constant, k-1, for these oligonucleotides indicates the observed relationship between affinity and structure is primarily due to k1.
制备了活化的Ha-ras基因的47个核苷酸的转录本，并通过酶促结构图谱确定其形成稳定的发夹结构。设计了六种反义十碳寡核苷酸，并测量了与发夹和长度匹配的互补序列的缔合常数（Ka）。与互补序列相比，靶向环的两种反义寡核苷酸对转录本的亲和力几乎相等。其他的，包括一种与单链环3'侧互补的寡核苷酸，与转录本的结合比与短互补序列的结合紧密程度低10^5 - 10^6倍。我们认为亲和力的差异是由于靶标结构，即茎的二级结构和环中的结构。对这些寡核苷酸的双分子缔合速率常数k1和解离速率常数k-1的测量表明，观察到的亲和力与结构之间的关系主要归因于k1。
