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Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases.

Michalowski D, Chitima-Matsiga R, Held DM, Burke DH - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65211, USA.

ABSTRACT
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.

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Loop sequences modulate ionic sensitivity. RT inhibition by aptamers RT6-B, R1T and SN was carried out in normal buffer (potassium as the primary monovalent cation), or in buffers in which potassium was replaced with sodium. Note that in sodium, R1T effected the most potent inhibition.
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Figure 3: Loop sequences modulate ionic sensitivity. RT inhibition by aptamers RT6-B, R1T and SN was carried out in normal buffer (potassium as the primary monovalent cation), or in buffers in which potassium was replaced with sodium. Note that in sodium, R1T effected the most potent inhibition.

Mentions: Potassium ions strongly favor quadruplex formation, while sodium ions are usually less stabilizing and lithium ions are destabilizing (40,41). We observed equivalent DNA polymerization activity by HIV-1 RT in buffers containing either K+ or Na+. RT inhibition by aptamers SN, R1T and RT6-B was therefore monitored in the original buffer containing K+ and also in a buffer in which K+ was replaced with Na+. The same RT was nearly inactive in buffers containing Li+, preventing analysis of inhibition in the presence of Li+. All three aptamers strongly inhibited RT in the presence of K+. Aptamers SN and R1T were also strongly inhibitory in the Na+-containing buffer, while aptamer RT6-B lost all inhibition under these conditions (Figure 3). Because these three aptamers differ only in the loop sequences connecting the individual guanosine triplets of the quadruplex, we conclude that the loop sequences in RT6-B render that aptamer sensitive to ionic destabilization, while the simplified structures of R1T and SN stabilize those aptamers to ionic substitution.Figure 3.


Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases.

Michalowski D, Chitima-Matsiga R, Held DM, Burke DH - Nucleic Acids Res. (2008)

Loop sequences modulate ionic sensitivity. RT inhibition by aptamers RT6-B, R1T and SN was carried out in normal buffer (potassium as the primary monovalent cation), or in buffers in which potassium was replaced with sodium. Note that in sodium, R1T effected the most potent inhibition.
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Related In: Results  -  Collection

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Figure 3: Loop sequences modulate ionic sensitivity. RT inhibition by aptamers RT6-B, R1T and SN was carried out in normal buffer (potassium as the primary monovalent cation), or in buffers in which potassium was replaced with sodium. Note that in sodium, R1T effected the most potent inhibition.
Mentions: Potassium ions strongly favor quadruplex formation, while sodium ions are usually less stabilizing and lithium ions are destabilizing (40,41). We observed equivalent DNA polymerization activity by HIV-1 RT in buffers containing either K+ or Na+. RT inhibition by aptamers SN, R1T and RT6-B was therefore monitored in the original buffer containing K+ and also in a buffer in which K+ was replaced with Na+. The same RT was nearly inactive in buffers containing Li+, preventing analysis of inhibition in the presence of Li+. All three aptamers strongly inhibited RT in the presence of K+. Aptamers SN and R1T were also strongly inhibitory in the Na+-containing buffer, while aptamer RT6-B lost all inhibition under these conditions (Figure 3). Because these three aptamers differ only in the loop sequences connecting the individual guanosine triplets of the quadruplex, we conclude that the loop sequences in RT6-B render that aptamer sensitive to ionic destabilization, while the simplified structures of R1T and SN stabilize those aptamers to ionic substitution.Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65211, USA.

ABSTRACT
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.

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