A highly stable RNA aptamer probe for the retinoblastoma protein in live cells

Abstract

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (K _d = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.

Fig. 1. Characterisation of 18E16 's G-quadruplex structure by (A) molecular recognition using an anti G-quadruplex antibody BG4 (inset: a schematic view of the 18E16 -sandwiched binding). The assay was performed in the binding buffer (5 mM MgCl₂, 5 mM KCl, 1 mM CaCl₂, 150 mM NaCl, 20 mM Hepes, pH 7.4). Changing Hepes to Tris in the binding buffer does not affect binding of the aptamer to RB. The binding curve was obtained by fitting the binding data to Langmuir isotherm model using Igor (WaveMetrics Inc.). (B) CD spectrum of 18E16 showing the characteristic peak for the parallel RNA G-quadruplex structures at 262 nm. The CD measurements were performed using 10 μM 18E16 in the binding buffer using a Chirascan (Applied Photo-Physics). (C) Staining of 18E16 using SYBR® Gold and NMM (a parallel G-quadruplex specific dye) on native (TBE) and denaturing (TBE-urea) gels.

Fig. 2. (A) Gel electrophoresis analysis. Lane 1: reference (HEK293 cell lysate with streptavidin magnetic beads in the absence of the aptamer). Lane 2: sample (HEK293 cell lysate captured by the aptamer and separated by streptavidin magnetic beads). A biotin labelled at the 3′-end of the aptamer allowed the complex to be separated from the lysate using streptavidin magnetic beads. The bound molecules were then eluted from beads for gel electrophoresis analysis. The top band of lane 2 showed a molecular weight corresponding to that of RB, where it was not shown from lane 1 of a parallel experiment in the absence of biotinylated 18E16. (B) Direct detection of RB in HEK293 cell lysate by 2-site ELISA using 18E16 as the capture molecule. 3′-end biotin labelled 18E16 was immobilized on a streptavidin-coated microplate to capture RB from the cell lysate. An anti-RB antibody labelled with HRP was used to detect the protein. The assays were run in the binding buffer.

Fig. 3. (A) Single molecule FRET (smFRET) histogram of the RNA aptamer in the binding buffer. The aptamer was doubly labelled at the 5′-end with a donor and the 3′-end with an acceptor. It showed high FRET efficiency of the pair when at the 2 ends of the aptamer. (B) Confocal images of Cy3/Cy5 FRET shown by acceptor photobleaching in live cells more than a day after aptamer transfection. U87MG, MCF7, HEK293 cells were incubated with the doubly labelled RNA aptamer (5′-end Cy3 and 3′-end Cy5). For each cell type Cy3 (donor) and Cy5 (acceptor) images are shown before and after photobleaching of the acceptor. Pre-bleach images show very low Cy3 (donor) fluorescence, post-bleach images (bleached region is indicated) demonstrate FRET by Cy3 fluorescence due to the absence of Cy5, quantification as described in methods, scale bar 10 μm.

Fig. 4. Co-localisation of a Cy3-labelled aptamer and an Alexa Fluor 647-labelled anti-RB antibody IgG1 kappa light chain (sc-73598, Santa Cruz Biotechnology) transfected into live cells, U87MG, MCF7 and HEK293. Quantification of co-localisation in the cytoplasm and nucleus using Pearson's coefficient. The Pearson's coefficients of 0.47 ± 0.03 (SEM, n = 15), 0.67 ± 0.03 (SEM, n = 15) and 0.53 ± 0.01 (SEM, n = 16) were measured for U87MG, MCF7 and HEK293 cells respectively. Data from three independent co-localisation experiments were used for the calculation. Scale bar 10 μm.