Synthetic DNA was purchased from Integrated DNA Technologies (Coralville, IA). Radiolabeled nucleotides for 5′ labeling ([γ-32
P]ATP) were purchased from Perkin-Elmer (Waltham, MA). The p51 and p66 subunits of RT from HIV-1 subtype B (HXB2 strain GenBank accession number K03455) were cloned into the protein expression construct pRT-Dual, kindly provided by Dr Stefanos G. Sarafianos, expressed in Escherichia coli
strain BL21(DE3) and purified essentially as described.28
Aptamer RNA was transcribed in vitro
from synthetic DNA oligonucleotides or from polymerase chain reaction products amplified from plasmids4
using phage T7 RNA polymerase. Transcripts were gel-purified as described7
and resuspended in deionized water.
RT enzymatic inhibition assays.
Primer extension was carried out essentially as described.7
Briefly, a Cy3-labeled, 18-mer DNA oligonucleotide corresponding to the 3′ end of tRNALys, 3
was mixed with a 31-mer template in a 1:3 ratio to ensure that all primer was pre-bound to template. This mixture was heated to 90
°C in a heat block for 2 minutes and then annealed by cooling to room temperature. A reaction mster mix was assembled to contain (final concentrations) 30 µmol/l dNTPs, 0.5 mmol/l ethylenediaminetetraacetic acid (EDTA), 50 mmol/l Tris–HCl pH 7.8, 50 mmol/l NaCl2
, 10 mmol/l dithiothreitol and 20 nmol/l RT, with the RT and dithiothreitol added last. The reaction master mix of 14 µl was aliquoted to each tube, along with either 2 µl of aptamer solution (final concentration 100 nmol/l unless otherwise noted) or water. Reactions were initiated by adding 4 µl of a solution containing annealed primer/template and MgCl2
((final concentration 20 nmol/l and 6 mmol/l, respectively). After incubating at 37
°C for 10
min, reactions were stopped by adding 20 µl of 90% formamide, 50 mmol/l EDTA, and a trace amount of bromophenol blue. Samples were heated to 90
°C for 2
min immediately before loading onto a 15% polyacrylamide, 8 mol/l urea denaturing gel. Gels were scanned for Cy3 fluorescence with a FLA9000 phosphorimager (Fujifilm, Valhalla, NY). The fraction of primer converted to full-length product was determined by quantifying band intensities using ImageQuant software (Pharmacia, Piscataway, NJ) and normalized by setting the fraction converted to full-length product in the absence of aptamer to 100%. Aptamer concentrations required for half-maximal inhibition (IC50
values) were calculated as described7
by fitting the data with GraphPad Prism 6 software to a standard two-state sigmoidal dose response curve: Y = 1/(1 + 10^[x − log(IC50
)]), where Y is the normalized fraction full-length product at a given aptamer concentration (x). Enzyme inhibition assays were performed in triplicate for all reactions from which IC50
values were calculated.
HTS and analysis pipeline.
HTS data for the 80HRT14
population were obtained and analyzed as described previously for 70HRT14
and other populations.13
Flanking sequences required for Illumina sequencing and indexing were appended during two sequential polymerase chain reaction amplification steps using plaque-forming unit DNA polymerase. Final amplified products from 80HRT14
were pooled with other populations, loaded onto a single lane, bridge amplified and then run through 100 sequencing cycles. Illumina's analysis software was used to generate fastq files with sequence calls and associated quality scores for >3 million raw sequence reads per population. 613,313 quality-filtered reads for the 80HRT14
populations were aligned, clustered, and used to identify converged structures.
Secondary structures in solution were assessed by enzymatic digestion as described.29
For each reaction, 50,000–200,000 cpm of 5′ radiolabeled RNA was digested under native conditions at 37
°C with ribonuclease T1 (0.005
U/µl for 2
min; Ambion; Life Technologies, Grand Island, NY), or S1 nuclease (4.75
U/µl for 10
min; New England Biolabs, Ipswich, MA), or ribonuclease V1 (5 × 10–5
U/µl for 8
min; Ambion). All reactions were quenched with equal volumes of colorless gel loading buffer (10 mol/l urea, 15 mmol/l EDTA) and quickly cooled in a dry ice/ethanol bath. Products of digestions were separated on 8 mol/l urea denaturing 15% polyacrylamide gels and analyzed as above.
Cell lines, plasmids and viral assays.
Plasmids for directing aptamer expression were constructed as previously described17
and utilize a human cytomegalovirus (CMV) immediate early promoter. Proviral plasmid (pNL4-3-Δenv-CMV-EGFP) was kindly provided by Vineet KewalRamani (National Cancer Institute [NCI], Fredrick, MD) and carries the genome of HIV-1 strain NL4-3, in which the genes encoding vif
, and env
have been deleted, and a CMV-driven enhanced green fluorescent protein (EGFP) reporter gene replaces nef
. Cell culture, virus production and evaluation of single-cycle viral infectivity were carried out as described.17,30
The human cell line, 293FT (Invitrogen, Carlsbad, CA), was transfected with polyethyleneimine in 6-well cell culture dishes. Aptamer-expressing plasmids were transfected first (1 µg), followed four hours later by transfection with a mixture of pNL4-3-Δenv-CMV-EGFP (250
ng) and pMD-G (125
ng; Invitrogen) to produce pseudotyped HIV-1 in the presence of aptamer. Medium was changed between transfections and again four hours after the second transfection. Virus was harvested 48 hours posttransfection by filtering the medium through 0.45
µm filters, and was quantified by p24 enzyme-linked immunosorbent assay. Fresh 293FT cells were infected with 25 µl of filtrate so that 5–10% of the target cells would become infected in the no-aptamer control; this level of infection provides sensitive readout of aptamer-mediated viral suppression.17
Cells were collected 48 hours post-infection, fixed with 4% paraformaldehyde, and analyzed for EGFP fluorescence on an Accuri Flow Cytometer (BD Biosciences, San Jose, CA). The percentage of infected (EGFP positive) cells was normalized to p24 levels in each sample, and the average of the control samples was set to 1. One-way analysis of variance and Student's t
test were used to determine statistical significance between samples.
Complete sequences of isolates from aptamer populations 70HRT14
) and 80HRT14
), as determined by low-throughput sequencing (LTS) of plasmids that were shotgun cloned from the library.
Initial screen and prioritization of 99 aptamers for inhibition of RT from HIV-1 strain HXB2.
Results from screen of proposed F1Pk and F2Pk cores.
Aligned sequences of the UCAA family members.
Enzymatic probing of aptamer 80.111(9–73).
Effects of internal mutations of 80.103 and 80.111 on RT inhibition.
Effects of helical swaps among subfamilies of UCAA aptamers.
Aptamer concentration dependence of RT inhibition used in calculating IC50