Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

Abstract

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance.

Figure 1.

Drug resistance mutations in M3 HIV-1 RT. (a) Sequence substitutions of M3 (right) relative to WT (left). Numbers indicate position in the amino acid sequence. (b) Sequence substitutions in M3 HIV-1 RT (pink) mapped onto the known 3D structure. The chain in blue is the WT HIV-1 RT p66 subunit and the chain in magenta is the p51 subunit. A 33 residue, pseudoknot RNA aptamer that interacts with the RT template/primer-binding site is shown in green. This image was generated through Protein Explorer using the 1hvu PDB file as a template.

Figure 2.

Sequences of aptamers binding to M3 HIV-1 RT. (a) Sequences from 29 clones isolated from the selected Round 12 pool that bound to M3 HIV-1 RT. The constant regions are not shown. The number of repeats of each sequence is shown to the right. (b) Sequences from 30 clones isolated from the selected Round 8 doped Aptamer 12.01 pool that bound to M3 HIV-1 RT. Repeats are again indicated to the right. Sequence M302 from the previous selection was found again in this selection, likely indicating a small amount of cross-contamination.

Figure 3.

Secondary structural analysis of aptamers. (a) Predicted secondary structure of Aptamer 12.01. Numbers are nucleotide positions relative to the 5′-end of the RNA (+1). The dots are nucleotides whose 2′-hydroxyl groups are available for NMIA-modification as revealed by SHAPE. (b) Predicted secondary structure of Aptamer M302. Numbers and dots are the same as in (a). (c) Probing the secondary structure of Aptamer 12.01 using SHAPE. Lanes ‘U’, ‘C’, ‘G’ and ‘A’ are dideoxy-sequencing ladders. The lane marked as ‘+’ is transcription-mediated primer extension of NIMA-modified Aptamer 12.01. Bands are offset by 1 nt compared to the sequencing ladder. Nucleotide positions corresponding 2′-O-adduct formation are numbered according to NMIA lanes and were marked in the predicted secondary structure for Aptamer 12.01, shown in (a). The lane marked as ‘–’ is primer extension of unmodified Aptamer 12.01. (d) Probing the secondary structure of Aptamer M302 using SHAPE. Modified positions were marked in the predicted secondary structure for Aptamer M302, shown in (b). Conventions are as in (c).

Figure 4.

Engineering minimal aptamers. (a) Functional boundary determination for Aptamer M302. 3′-boundaries were determined by binding partially hydrolyzed, 5′-end-labeled Aptamer M302 to M3 HIV-1 RT. Recovered RNA fragments were analyzed by PAGE (lane 2), along with unreacted Aptamer M302 (lane 1), a partially hydrolyzed Aptamer M302 ladder (lane 3) and a G-ladder generated by nuclease T1 hydrolysis (lane 4). G75 is the functional 3′-boundary of Aptamer. (b) Predicted secondary structure of minimal Aptamer M302-S. (c) Predicted secondary structure of minimal Aptamer 12.01-S.

Figure 5.

Dissociation constants of anti-HIV-1 RT aptamers. (a) Binding curve of Aptamer 12.01 to M3 (squares) and WT (dots) HIV-1 RTs. (b) Binding curve of Aptamer M302 to M3 (squares) and WT (dots) HIV-1 RTs. (c) Binding curve of unselected pool RNA to M3 (squares) and WT (dots) HIV-1 RTs. (d) Summary of aptamers’ binding constants (at 25°C) and inhibition constants (at 37°C). All values are in nanomolar.

Figure 6.

Binding specificities of anti-RT aptamers. Different reverse transcriptases (0.5 μM) were incubated with Aptamer M302 for 30 min in Selection Buffer at 25°C. Protein-bound RNA was retained on the nitrocellulose filter and free RNA flowed through to the nylon filter during vacuum filtration.

Figure 7.

Inhibitory activities of anti-RT aptamers. (a) Polymerization activity of RTs. A (5′-³²P)-labeled primer was extended by M3 HIV-1 RT or by WT HIV-1 RT. Either 0, 15, 50 or 500 nM HIV-1 RT was used. Labeled primer and extended cDNA are denoted by arrows to the left. (b) RNase H activity of RTs. A (5′-³²P)-labeled template was digested by M3 HIV-1 RT or WT HIV-1 RT. M3 HIV-1 RT does not show obvious RNase H activity. Labeled template and degradation products are denoted by arrows to the left. (c) Aptamer inhibition of polymerase activity. Aptamer 12.01 and Aptamer M302 at varying concentrations (0, 50, 200, 1000 nM) were incubated with radiolabeled primer and an RNA template. Labeled primer and extended cDNA are denoted by arrows to the left. (d) Aptamer inhibition of RNase H activity. Aptamer 12.01 and Aptamer M302 at varying concentrations (0, 50, 200, 1000 nM) were incubated with radiolabeled primer and an RNA template. Labeled template and degradation products are denoted by arrows to the left.

Figure 8.

Competitive binding of aptamers to M3 and WT HIV-1 RTs. Aptamers M302, 12.01, TPK1.1 and the unselected pool RNA (N50) were end-labeled (all at 0.1 nM, indicated on x-axis) competed with different cold versions of the same RNAs (all at 1000 nM, indicated in inset) in the presence of either M3 or WT RT (indicated on x-axis). Bound, radiolabeled RNA was collected by filtration and quantitated.

Figure 9.

Specific detection of M3 versus WT RT by selected aptamers. Biotin-labeled aptamers were immobilized onto neutravidin-coated slides. Immobilized aptamers were sequentially incubated with M3 or WT HIV-1 RT, a polyclonal anti-HIV-1 RT antibody from rabbit and Cy3-labeled anti-rabbit IgG. The slide was scanned for fluorescence on a microarray scanner—GenePix 4000 B.