Nucleic Acids Research Advance Access originally published online on November 7, 2008
Nucleic Acids Research 2008 36(22):7124-7135; doi:10.1093/nar/gkn891
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Nucleic Acids Research, 2008, Vol. 36, No. 22 7124-7135
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases
1Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine and 2Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA
*To whom correspondence should be addressed. Tel: +1 537 884 1316; Fax: +1 573 884 9676; Email: burkedh{at}missouri.edu
Correspondence may also be addressed to Daniel Michalowski. Tel: +1 301 668 4991; Fax: +1 301 668 4991; Email: michalowskid{at}missouri.edu
Received June 20, 2008. Revised October 17, 2008. Accepted October 21, 2008.
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 IC50 values. These data provide insight into the requirements for broad-spectrum RT inhibition by nucleic acid aptamers.