Journal
OLIGONUCLEOTIDES
Volume 18, Issue 2, Pages 133-143Publisher
MARY ANN LIEBERT, INC
DOI: 10.1089/oli.2008.0103
Keywords
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Funding
- NIGMS NIH HHS [GM051140, R01 GM051140] Funding Source: Medline
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Primer-template-based double-stranded nucleic acids capable of binding human immunodeficiency virus reverse transcriptase (HIV-RT) with high affinity were used as starting material to develop small singlestranded loop-back DNA aptamers. The original primer-templates were selected using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach and consisted of 46- and 50-nt primer and template strands, respectively. The major determinant of the similar to 10-fold tighter binding in selected sequences relative to control primer-templates was a run of 6-8 G residues at the 3' primer end. Sixty, thirty-seven, twenty-seven, and twenty-two nucleotide loop-back single-stranded versions that retained the base pairs near the 3' primer terminus were constructed. Both the 60- and 37-nt versions retained high affinity for RT with K-d values of similar to 0.44 nM and 0.66 nM, respectively. Random sequence primer-templates of the same length had KdS of similar to 20 nM and similar to 161 nM. The shorter 27- and 22-nt aptamers bound with reduced affinity. Several modifications of the 37-nt aptamer were also tested including changes to the terminal 3' G nucleotide and internal bases in the G run, replacement of specific nucleotides with phosphothioates, and alterations to the 5' overhang. Optimal binding required a 4- to 5-nt overhang, and internal changes within the G run had a pronounced negative effect on binding. Phosphothioate nucleotides or the presence of a 3' dideoxy G residue did not alter affinity. The 37-nt was a potent inhibitor of HIV-RT in vitro and functioned by blocking binding of other primer-templates.
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