Michalowski Daniel, Chitima-Matsiga Rebecca, Held Daniel M, Burke Donald H
Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65211, USA.
Nucleic Acids Res. 2008 Dec;36(22):7124-35. doi: 10.1093/nar/gkn891. Epub 2008 Nov 7.
DNA aptamers RT5, RT6 and RT47 form a group of related sequences that inhibit HIV-1 reverse transcriptase (RT). The essential inhibitory structure is identified here as bimodular, with a 5' stem-loop module physically connected to a 3'-guanosine quadruplex module. The stem-loop tolerates considerable sequence plasticity. Connections between the guanosine triplets in the quadruplex could be simplified to a single nucleotide or a nonnucleic acid linker, such as hexaethylene glycol. All 12 quadruplex guanosines are required in an aptamer retaining most of the original loop sequence from RT6; only 11 are required for aptamer R1T (single T residue in intra-quadruplex loops). Circular dichroism (CD) spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. The simplified aptamers displayed increased overall stability. An aptamer carrying the original intra-quadruplex loops from RT6 inhibited RT in K(+) buffers but not in Na(+) buffers and displayed significant CD spectral broadening in Na(+) buffers, while R1T inhibited RT in both buffers and displayed less broadening in Na(+) buffers. The bimodular ssDNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC(50) values. These data provide insight into the requirements for broad-spectrum RT inhibition by nucleic acid aptamers.
DNA适配体RT5、RT6和RT47构成了一组相关序列，可抑制HIV-1逆转录酶（RT）。在此确定其基本抑制结构为双模块结构，5'端的茎环模块与3'端的鸟嘌呤四链体模块物理相连。茎环结构能够耐受相当程度的序列可塑性。四链体中鸟嘌呤三联体之间的连接可以简化为单个核苷酸或非核酸连接子，如六甘醇。保留RT6大部分原始环序列的适配体需要全部12个四链体鸟嘌呤；适配体R1T（四链体内环中有单个T残基）仅需要11个。圆二色（CD）光谱在240 nm和264 nm处出现椭圆率最小值和最大值，表明四链体链呈平行排列。简化后的适配体整体稳定性增强。携带RT6原始四链体内环的适配体在K⁺缓冲液中可抑制RT，但在Na⁺缓冲液中则不能，并且在Na⁺缓冲液中CD光谱显著展宽，而R1T在两种缓冲液中均能抑制RT，且在Na⁺缓冲液中的展宽程度较小。这种双模块单链DNA适配体以低纳摩尔IC₅₀值抑制多种灵长类慢病毒的RT。这些数据为核酸适配体实现广谱RT抑制的要求提供了见解。
