DNA aptamers to human immunodeficiency virus reverse transcriptase selected by a primer-free SELEX method: characterization and comparison with other aptamers

Abstract

A 30-nucleotide DNA aptamer (5'-AGGAAGGCTTTAGGTCTGAGATCTCGGAAT-3', denoted PF1) selected for high affinity to human immunodeficiency virus reverse transcriptase (HIV RT) using a primer-free SELEX (systematic evolution of ligands by exponential enrichment) method was characterized to determine features promoting tight binding. PF1's equilibrium dissociation constant for RT was ∼80 nM, over 10-fold lower than a random 30-mer. Changing the 2 terminal diguanosine repeats (underlined above) to diadenosine or dithymidine modestly decreased binding. Any changes to the 2 central diguanosines dramatically decreased binding. Binding was highly sensitive to length, with any truncations that deleted part of the 4 diguanosine motifs resulting in a 6-fold or more decrease in affinity. Even a construct with all the diguanosine motifs but lacking the 5' terminal A and 3 nucleotides at the 3' end showed ∼3-fold binding decrease. Changes to the nucleotides between the diguanosines, even those that did not alter PF1's low secondary structure (free energy of folding ΔG=-0.61 kcal/mol), dramatically decreased binding, suggesting sequence specificity. Despite the diguanosine motifs, circular dichroism (CD) spectra indicated that PF1 did not form a G-quartet. PF1 inhibited HIV RT synthesis with a half-maximal inhibitory value (IC(50)) of ∼60 nM. Larger, more structured RT DNA aptamers based on the HIV polypurine tract and those that formed G-quartets (denoted S4 and R1T) were more potent inhibitors, with IC(50) values of ∼4 and ∼1 nM, respectively. An RNA pseudoknot aptamer (denoted 1.1) showed an IC(50) near 4 nM. Competition binding assays with PF1 and several previously characterized RT aptamers indicated that they all bound at or near the primer-template pocket. These other more structured and typically larger aptamers bound more tightly than PF1 to RT based on filter binding assays. Results indicate that PF1 represents a new class of RT aptamers that are relatively small and have very low secondary structure, attributes that could be advantageous for further development as HIV inhibitors.

FIG. 1.

Representative gel-shift analysis of the PF1 aptamer and various derivatives. Increasing amounts of HIV RT (as indicated above wells; note different amounts with different constructs) were mixed with 5′ ³²P end-labeled PF1 or other modified constructs (see Table 1). Samples were run on a native 6% polyacrylamide gel. Positions of shifted and unshifted material are indicated. For PF1-4G, G-quartets (G-quart, boxed lanes) were observed migrating above the unshifted DNA. A small portion of the material remained in the gel wells with some aptamers, but this occurred even when no RT was added, indicating it was not dependent on the added protein. In general, PF1 and the other derivatives tested did not form a single discrete band in gel-shift assays. A concentrated shifted band was observed just below the wells and a diffuse area of shifted material appeared underneath the band. This probably resulted from some complexes dissociating during the electrophoresis process.

FIG. 2.

Predicted structures of some aptamers and derivatives. Structures and free energy of folding values (ΔG, in kcal/mol) were obtained using mfold as described in Materials and Methods.

FIG. 3.

Competition filter binding experiment. Unlabeled (cold) aptamers (5′-N₃₀-3′, PF1, PF1-C6, 38 NT SELEX, S4, R1T, or 1.1 RNA pseudoknot, as indicated) were used as competitors with radiolabeled (hot) 38 NT SELEX (10 nM). Increasing amounts of cold aptamer (x-axis) were added to incubations with the hot aptamer and 3 nM human immunodeficiency virus reverse transcriptase (HIV RT) and incubated at room temperature for 1.5 hours. Samples were then filtered over nitrocellulose and counted by liquid scintillation. The y-axis shows the amount of hot 38 NT SELEX bound to the filters at a given aptamer concentration, relative to the amount in the absence of added cold aptamer. Plotted points are average values from 2–3 experiments, with error bars representing standard deviations.

FIG. 4.

A representative autoradiogram using various concentrations of PF1 in the RT inhibition assay. (A) Schematic diagram of the primer (black)-template (gray) used to conduct the reverse transcriptase (RT) inhibition assay. The primer strand was radiolabeled with ³²P at the 5′ end. (B) The assay was conducted by incubating the prehybridized primer–template (50 nM) with 100 μM dNTPs and various concentrations of aptamers as indicated. Reactions were initiated with HIV RT (0.25 nM), and aliquots were removed at 2, 5, 10, 15, and 20 minutes. The samples were run on a denaturing 10% polyacrylamide gel and quantified as described in Materials and Methods.

FIG. 5.

Inhibition of HIV RT primer extension in the presence of various amounts of PF1 and starting material (5′-N₃₀-3′) (5A), or R1T and 38 NT SELEX ddG (5B). *Graphs of counts (photo-stimulated luminescence radiation units from phosphoimager) versus time derived from experiment of the type shown in Fig. 4. The difference in magnitude of the y-axis in the experiment in A and B is not meaningful, as the counts are dependent on the specific activity of the primer and the time of exposure. The assay was conducted by incubating the primer-template (5′ γ-³²P end-labeled primer, 50 nM) with various concentrations of aptamers. Reactions were initiated with HIV RT (0.25 nM), and aliquots were removed at the indicated times. The samples were run on a denaturing 10% polyacrylamide gel and quantified as described in Materials and Methods. In Fig. 5B, the 38 NT SELEX aptamer contained a 3′ terminal dideoxy G residue in place of the normal dG to prevent extension of the aptamer by RT. Refer to Tables 1 and 2 for information on the specific oligonucleotides used as inhibitors. Color images available online at www.liebertonline.com/nat

FIG. 6.

Stability of various aptamers in cell culture media (RPMI+10% fetal bovine serum), shown in an autoradiogram of a gel with various 5′ end-labeled aptamers (as indicated above lanes) incubated in culture medium for increasing times. Each set has a control (C) time 0 sample, followed by a 30-minute incubation in cell media that included nuclease (N). Either DNase I (5 units) for the DNA aptamers or RNase (0.25 μgs) for the 1.1 RNA aptamer was used. This lane is followed by samples incubated for 15, 30, 60, and 120 minutes. *For the 1.1 RNA pseudoknot aptamer, an extra lane where 20 units of RNasin RNase inhibitor was added to the media and incubated for 15 minutes is shown. Note that 38 NT SELEX [38 nucleotides (nts)] runs faster on the gel than either PF1 (30 nts) or S4 (35 nts), despite being longer. This is likely do to it being highly structured.

FIG. 7.

CD spectra of various aptamers and derivatives. Spectra were acquired as described in Materials and Methods. The various aptamers and derivatives used are shown in Tables 1 and 2. (A) Spectra acquired in potassium buffer: 50 mM Tris-HCl (pH 8), 80 mM KCl, 6 mM MgCl₂, and 1 mM DTT; (B) Spectra acquired in sodium buffer: 50 mM Tris-HCl (pH 8), 80 mM NaCl, 6 mM MgCl₂, and 1 mM DTT. Color images available online at www.liebertonline.com/nat