Wilds C J, Damha M J
Department of Chemistry, Otto Maass Chemistry Building, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 2K6, Canada.
Nucleic Acids Res. 2000 Sep 15;28(18):3625-35. doi: 10.1093/nar/28.18.3625.
Recently, hybrids of RNA and D-arabinonucleic acids (ANA) as well as the 2'-deoxy-2'-fluoro-D-arabinonucleic acid analog (2'F-ANA) were shown to be substrates of RNase H. This enzyme is believed to be involved in the primary mechanism by which antisense oligonucleotides cause a reduction in target RNA levels in vivo. To gain a better understanding of the properties of arabinose based oligonucleotides, we have prepared a series of 2'F-ANA sequences of homopolymeric (A and T) and mixed base composition (A, T, G and C). UV thermal melting and circular dichroic (CD) studies were used to ascertain the thermodynamic stability and helical conformation of 2'F-ANA/RNA and 2'F-ANA/DNA hybrids. It is shown that 2'F-ANA has enhanced RNA affinity relative to that of DNA and phosphorothioate DNA. The 2'-fluoroarabino modification showed favorable pairing to single-stranded DNA also. This is in sharp contrast to ANA, which forms weak ANA/DNA hybrids at best. According to the measured thermodynamic parameters for duplex formation, the increased stability of hybrids formed by 2'F-ANA (e.g., 2'F-ANA/RNA) appears to originate from conformational pre-organization of the fluorinated sugars and a favorable enthalpy of hybridization. In addition, NMR spectroscopy revealed a five-bond coupling between the 2'F and the base protons (H6/H8) of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. This observation is suggestive of a through-space interaction between 2'F and H6/H8 atoms. CD experiments indicate that 2'F-ANA/RNA hybrids adopt an 'A-like' structure and show more resemblance to DNA/RNA hybrids than to the pure RNA/RNA duplex. This feature is believed to be an important factor in the mechanism that allows RNase H to discriminate between 2'F-ANA/RNA (or DNA/RNA) and RNA/RNA duplexes.
最近，RNA与D-阿拉伯核酸（ANA）的杂交体以及2'-脱氧-2'-氟-D-阿拉伯核酸类似物（2'F-ANA）被证明是核糖核酸酶H的底物。据信，这种酶参与了反义寡核苷酸在体内导致靶RNA水平降低的主要机制。为了更好地了解基于阿拉伯糖的寡核苷酸的特性，我们制备了一系列具有同聚体（A和T）以及混合碱基组成（A、T、G和C）的2'F-ANA序列。利用紫外热变性和圆二色性（CD）研究来确定2'F-ANA/RNA和2'F-ANA/DNA杂交体的热力学稳定性和螺旋构象。结果表明，相对于DNA和硫代磷酸酯DNA，2'F-ANA对RNA具有更高的亲和力。2'-氟阿拉伯糖修饰对单链DNA也表现出良好的配对。这与ANA形成了鲜明对比，ANA充其量只能形成弱的ANA/DNA杂交体。根据双链形成的测量热力学参数，由2'F-ANA形成的杂交体（如2'F-ANA/RNA）稳定性增加似乎源于氟化糖的构象预组织和有利的杂交焓。此外，核磁共振光谱显示2'-脱氧-2'-氟-β-D-阿拉伯核苷的2'F与碱基质子（H6/H8）之间存在五键耦合。这一观察结果表明2'F与H6/H8原子之间存在空间相互作用。CD实验表明，2'F-ANA/RNA杂交体采用“A样”结构，与DNA/RNA杂交体的相似性高于纯RNA/RNA双链体。这一特征被认为是核糖核酸酶H区分2'F-ANA/RNA（或DNA/RNA）与RNA/RNA双链体机制中的一个重要因素。
