All oligonucleotides used in this study were purchased from a commercial supplier (IBA, Göttingen, Germany). Their concentration was routinely determined by the ultraviolet (UV) absorption spectroscopy and the individual extinction coefficients. The integrity was controlled applying denaturing PAGE [20% (w/v) acrylamide, 7 M urea] followed by staining with Stains-All (Sigma–Aldrich, Deisenhofen, Germany). Oligonucleotides were 5′ end-labeled with T4 polynucleotide kinase (MBI Fermentas, St Leon-Rot, Germany). Briefly, 10 pmol of oligonucleotide were incubated with polynucleotide kinase and 30 μCi of [γ-32P]ATP (PerkinElmer, Boston, MA) for 30 min at 37°C. Reactions were stopped by heating the samples for 5 min at 95°C. Labeled oligonucleotides were analyzed by using a PhosphorImager after denaturing PAGE [20% (w/v) acrylamide, 7 M urea].
Recombinant heterodimeric wild-type HIV-1, HIV-2, equine infectious anemia virus (EIAV) RT and the p51 subunit of HIV-1 RT were expressed in Escherichia coli
and purified as described before (9
). Enzyme concentrations were routinely determined using an extinction coefficient at 280 nm of 260450 (HIV-1 RT), 238150 (HIV-2 RT), 223180 (EIAV RT) and 124180 M−1
(HIV-1 p51). The purified RTs were free of nuclease contamination. T7 RNA polymerase (12
) was expressed in E.coli
and purified as described (13
). Enzyme concentration was routinely determined using an extinction coefficient at 280 nm of 140000 M−1
. BSA was purchased from Promega (Mannheim, Germany).
Combinatorial screening of a random pool of hexadeoxyribonucleotides for binding to HIV-1 RT
A random library of hexanucleotides (10 nM) was mixed with HIV-1 RT (10 μM) and incubated at 37°C for 10 min in buffer containing 50 mM Tris–HCl (pH 8.0), 50 mM KCl, 5 mM MgCl2 in a final volume of 20 μl. The mixture was soaked through a nitro-cellulose filter BA85 (Schleicher & Schuell, Dassel, Germany) and rinsed with 3 ml of binding buffer. The hexamers retained on the filter were extracted from the nitro-cellulose by heating for 10 min at 95°C in 1 ml of water. The eluted hexanucleotides were subjected to poly(C) extension of the 3′ termini for 10 min at 37°C using terminal deoxynucleotidyl transferase (MBI Fermentas, St Leon-Rot, Germany). Tailed products were then precipitated in ethanol and ligated at their 5′ termini with a primer PA (TGCAGGCTCGAGTTAATTAACTGA) by T4-RNA ligase (MBI Fermentas, St Leon-Rot, Germany). Reactions were stopped by heating at 95°C for 5 min. Ligated products were precipitated in ethanol and amplified by PCR in the presence of Taq polymerase (New England Biolabs, Ipswich, MA) and primers PA, PB (TGTATTGCGGCCGCTGATCTAGA) and adaptor-PB [TGTATTGCGGCC GCTGATCTAGA(G)14]. PCR products were subsequently cloned in the pCRII-TOPO vector (Invitrogen, Karlsruhez, Germany) and sequenced.
It should be noted that our attempts to use the RNA analogs, i.e. hexaribonucleotides as substrates for terminal deoxynucleotidyl transferase, poly(A)polymerase, and T4-RNA ligase showed extremely little product or failed. We suspect that very short RNAs are poor substrates for those enzymes.
Filter binding assay
Typically, radiolabeled oligonucleotides (1 nM) and target proteins (5–25 μM) were incubated in 30 μl of binding buffer [50 mM Tris–HCl (pH 8.0), 5 mM KCl, 5 mM MgCl2 and 1 mM DTT] for 2 min at 25°C. An aliquot (10 μl) of this mixture was filtered under suction through a prewet nitro-cellulose filter BA85 (Schleicher & Schuell, Dassel, Germany) and rinsed with 3 ml of binding buffer. Radioactivity retained on the filters was measured by scintillation counting (PerkinElmer, Boston, MA).
An amount of 250 pmol of HIV-1 RT and of 100 fmol 32P-labeled 4-thio U-modified Hex-S3R (position 4) were pre-incubated in binding buffer (see above) for 2 min at 25°C. Samples were then transferred on a piece of Parafilm, placed on ice and irradiated for 20 min at 366 nm using a hand-held fluotest lamp (Heraeus, Hanau, Germany), followed by heating for 5 min at 95°C in 1× Laemmli loading buffer and resolved by 12% SDS–PAGE. Gels were stained with Coomassie blue, dried and exposed to a PhosphorImager screen.
CNBr cleavage of HIV-1 RT tagged with 32P-labeled 4-thio U-modified Hex-S3R
HIV-1 RT cross-linked to 32P-labeled 4-thio U-modified Hex-S3R was precipitated by 20% (w/v) trichloroacetic acid (TCA), followed by a washing with acetone. The pellet was then air-dried and dissolved in 100 μl formic acid (70%) containing 3.5 g/l cyanbromide (CNBr). Cleavage reaction was performed at room temperature for 24 h. Formic acid and CNBr were removed by centrifugal-lyophilization in a speed-vac. The pellet was dissolved in 50 mM Tris–HCl (pH 8.0) buffer, heated 5 min at 95°C in 1× Laemmli loading buffer and resolved by SDS–PAGE [10–20% (w/v) linear gradient]. Gels were stained with Coomassie blue, dried and exposed to a PhosphorImager screen. All reagents were purchased from Sigma–Aldrich (Deisenhofen, Germany).
Modeling and docking studies
Rigid-body docking studies of HIV-1 RT (Brookhaven Protein Data Bank accession nos: 1HMV, 1RTH, 1RTD and 1HVU) and Hex-S3 were performed with the program Hex 4.2 (15
). Molecular graphics images were produced using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (16
Virus replication assay
Hexamers were co-transfected by calciumphosphate co-precipitation with pGJ3-Luci and pczVSV-G in 106
293T cells as described in detail recently (17
). In brief, 60 min thereafter medium was exchanged and the cells were incubated for 40 h followed by filtration (0.45 μm). The cleared supernatant (0.2–0.3 ng of p24 core antigen per ml) was used to infect a number of 2 × 104
293 cells in a 96-well format for 24 h in the presence of polybrene (8 μg/ml). Cell viability was monitored by the conversion of fluorescein diacetate (FDA) (18
) and the expression of luciferase was measured in a Fluoroskan Ascent® FL (Thermo Labsystems, Dreieich, Germany) after addition of 50 μl of the following buffer: 28 mM Tricine (pH 7.8), 500 μM ATP, 250 μM coenzyme A, 250 μM luciferin, 33 mM DTT, 200 μM EDTA, 15 mM MgSO4
, 1.5% (v/v) Triton X-100 and 5% (v/v) glycerol. All reagents were purchased from Sigma–Aldrich (Deisenhofen, Germany).