PMC

Sc5-c3 binding to HPV-16 L1 VLPs in murine cervical washes. (A) Murine cervical washes do not interact with Sc5-c3. Binding assays were performed incubating labeled Sc5-c3 (30 pM) with increasing concentrations (0, 50, 100, 500 and 1,000 ng) of HPV-16 L1 VLPs (VLPs) or cervical wash protein (CP). The plot represent the mean and standard deviation of bound RNA relative to the input (n=3). (B) Specific Sc5-c3 binding to HPV-16 L1 VLPs in murine cervical washes. Five hundred nanograms of cervical wash protein were mixed with increasing concentrations of HPV-16 L1 VLPs (triangles) and 500 ng of HPV-16 L1 VLPs were added to increasing concentrations of cervical wash protein (squares). The plot represent the mean and standard deviation of bound RNA relative to the input (n=3).

Mutational analysis of Sc5-c3 binding to HPV-16 L1 VLPs. (A) Several Sc5-c3 mutants (M1 to M8) were produced by site-directed mutagenesis. Underlined positions correspond to constant nucleotides presented in all co-isolated aptamers against HPV-16 L1 VLPs and mutations on specific Sc5-c3 nucleotide positions are indicated by squares. Nucleotide numbering is indicated. (B) Sc5-c3 mutants binding to HPV-16 L1 VLPs. Binding assays were performed using end labeled aptamer RNA incubated with 100 ng of BSA protein (open bars), HPV-16 L1 VLPs (closed bars), or binding buffer alone (slotted bars). The plots represent the mean and standard deviation of relative binding from three independent experiments.

Deletion analysis of Sc5-c3 binding to HPV-16 L1 VLPs. Binding assays were performed using α-[³²P]-UTP labeled RNA from Sc-5c3 and 5′ or 3′-deleted derivatives (30 pM) incubated with BSA protein (open bars), HPV-16 L1 VLPs (closed bars), and binding buffer alone (slotted bars). The deleted Sc5-c3 schematic maps and name are included as reference (B1, bubble 1; ML, main loop; S1, stem 1; S2, stem2; UR, unstructured region). Initial and end nucleotide positions are indicated for each deleted derivative. The bars show the mean fractional saturation (n=3) and standard deviation.

Dissociation constant (K_d) determination of Sc5-c3 binding to HPV-16 L1 VLPs. (A) K_d determination was performed on “sandwich” slot blot binding assays using α-[³²P]-UTP labeled aptamer RNA (30 pM) incubated with increasing amounts of the HPV-16 L1 VLPs (left panel) and BSA (right panel). Protein–aptamer complexes were retained on the nitrocellulose membrane (NC), whereas the free aptamer is bound to the positively charged nylon membrane (NY). (B) The K_d for Sc5-c3 binding with HPV-16 L1 VLP were obtained using fractional saturation [defined as the bound RNA relative to the total RNA in a binding reaction vs. VLP protein concentration (pM)] (left panel) and Scatchard [fractional saturation vs. fractional saturation/VLP protein concentration (pM)] (right panel) plots. The plots represent the mean and standard deviation from three experiments.

Sc5-c3 binding sequence requirements. (A) Multiple alignment of co-isolated aptamers against HPV-16 L1 VLPs (Sc5-c1, Sc5-c3, and Sc-5-c8). Differences in nucleotide positions are indicated. Nucleotide numbering is shown. (B) Binding analysis of co-isolated aptamers against HPV-16 L1 VLPs. Labeled RNA (30 pM) from Sc5-c1(open bars), Sc5-c3 (closed bars), and Sc5-c8 (slashed bars) aptamers were incubated with 500 ng of HPV-16 L1 VLPs (VLP), bacteria-produced HPV-16 L1 (L1), bovine serum albumin (BSA), or green fluorescent protein (GFP) using a sandwich slot-blot binding assay. A negative control of the background binding to the nitrocellulose membrane was included (Ctrl). The plot displays the mean and standard deviation of three independent experiments.

Ribonuclease footprinting assay. (A) Footprinting assay of the Sc5-c3-VLP complex using increasing amounts (0.3 to 4.8 μg) of BSA (left panel) or HPV-16 L1 VLP protein (right panel), in the presence of RNaseV1. Cleavage products were resolved in 20% polyacrylamide/7 M urea denaturing gels. Brackets indicate the protected sequences. MWM, molecular weight marker. Lane 1 contains the undigested Sc5-c3 RNA and lane 2 includes the Sc5-c3 transcript incubated with the ribonuclease reaction buffer but no ribonuclease. Arrows indicate G residues (G*) within in the ML region. (B) Footprinting of the Sc5-c3-VLP complex using RNaseT1 (left panel) and RNaseA (right panel) showing a higher resolution view of the protected nucleotides. Arrows indicate G residues (G*) within in the ML region. (C) Schematic representation of Sc5-c3 contact region with HPV-16 L1 VLPs. Positions protected from ribonuclease cleavage by VLP binding are indicated by boxes. Sc5-c3 structural features are shown as reference (B1, bubble 1; ML, main loop; S1, stem 1; S2, stem2; UR, unstructured region).

Ribonuclease mapping of Sc5-c3 secondary structure. (A) 5′-end labeled Sc5-c3 RNA was partially digested with ribonuclease A (RNaseA), RNaseV1, and RNaseT1. Cleavage products were resolved in 20% polyacrylamide/7M urea denaturing gels. Molecular weight markers (MWM) were obtained from diverse radioactively labeled transcripts. IVT, in vitro transcribed Sc5-c3 RNA. IVT 37, Sc5-c3 transcript incubated with RNase reaction buffer at 37°C in the absence of ribonuclease. Brackets indicate the boundaries of the structural domains stem 1 (S1), bubble (B1), stem 2 (S2), main loop (ML) and an unstructured region (UR). (B) Schematic representation of Sc5-c3 secondary structure. In silico secondary structure analysis of Sc5-c3 ERNA showed five well-defined structural domains: stem 1 (S1), bubble (B1), stem 2 (S2), main loop (ML), and an unstructured region (UR) which were adjusted according to ribonuclease mapping data. Ribonuclease cleavage sites are indicated as follows: RNaseA (squares), RNaseV1 (triangles), and RNaseT1 (circles). ΔG, Gibbs' free energy.

Binding of RNA aptamers to human papillomavirus (HPV)-16 L1 virus-like particles (VLPs). (A) Interaction assays. Radioactively labeled aptamers obtained by systematic evolution of ligands by exponential enrichment (SELEX) (Sc5-c2, Sc5-c3 and Sc5-c7) were incubated with 500 ng of polyvinylidene fluoride (PVDF)-immobilized bovine serum albumin (BSA), 500 ng HPV-16 L1 VLP (VLPs) or PVDF membranes alone (PVDF) in binding reactions. Membranes were thoroughly washed, and bound aptamer RNA was recovered and resolved by denaturing electrophoresis in 8% polyacrylamide/7 M urea gels. Arrows indicate migration of bound aptamer RNA. (B) Interaction assay digital analysis. The plot shows the average percentage of bound RNA relative to the input expressed as the mean and standard deviation of three independent experiments. (C) Sc5-c3 specificity control. A “sandwich” slot-blot binding assay was made to confirm specific Sc5-c3-VLP complex formation after SELEX. Binding assays were performed incubating the 5′ end-labeled unselected RNA pool (Aptlib) and the Sc5-c3 aptamer with 200 ng HPV-16 L1 VLPs (closed bars), 200 ng BSA (open bars), or binding buffer only (slotted bars). Aptamer–protein interactions were represented as relative to Sc5-c3 binding to VLPs. The bars represent the mean and standard deviation of three independent experiments.