Single-nucleobase resolution of a surface energy transfer nanoruler for in situ measurement of aptamer binding at the receptor subunit level in living cells

Abstract

In situ identification of aptamer-binding targets on living cell membrane surfaces is of considerable interest, but a major challenge, specifically, when advancing recognition to the level of membrane receptor subunits. Here we propose a novel nanometal surface energy transfer (NSET) based nanoruler with a single-nucleobase resolution (SN-nanoruler), in which FAM-labeled aptamers and single-sized gold nanoparticle (GNP) antibody conjugates act as a donor and an acceptor. A single nucleobase resolution of the SN-nanoruler was experimentally illustrated by molecular size, orientation, quenching nature, and other dye-GNP pairs. The SN-nanoruler provides high reproducibility and precision for measuring molecule distance on living cell membranes at the nanometer level owing to only the use of single-sized antibody-capped GNPs. In situ identification of the aptamer binding site was advanced to the protein subunit level on the living cell membrane for the utilization of this SN-nanoruler. The results suggest that the proposed strategy is a solid step towards the wider application of optical-based rulers to observe the molecular structural configuration and dynamic transitions on the membrane surface of living cells.

Scheme 1. SN-nanoruler working model for measuring distance on the CD71 receptor of PL45 cells. R represents the distance from FAM dye to the center of G₅NPs, r represents the distance from FAM dye to the surface of G₅NPs, and R′ represents the separation distance between the XQ-2d-FAM binding site and antiCD71 binding site. The d and d_CD71 are the diameters of G₅NPs and antiCD71 antibody, respectively.

Fig. 1. Binding feature analysis of the ligands in living PL45 cells. (A and B) Saturated binding concentration analysis of the XQ-2d-FAM aptamer to the CD71 receptor on PL45 cells. The FCM charts indicated 0, 50, 100, 150, 200, 250, 300, 350, and 400 nM of XQ-2d-FAM from bottom to top in A, respectively. (C and D) Best co-incubation time of the XQ-2d-FAM aptamer with the CD71 receptor on PL45 cells. The FCM charts indicated native PL45 cells and cells with saturated XQ-2d-FAM for about 5, 20, 40, and 60 min and 2, 4, 7 and h from bottom to top in C, respectively. (E and F) Competitive binding analysis of saturated XQ-2d-FAM and different amounts of antiCD71, including 1, 2, 4, and 8 μL, respectively.

Fig. 2. Preparation and characterization of a G₅@antiCD71 conjugate. (A) Normalized absorbance of the G₅@antiCD71 conjugate at varied pH values. (B) Normalized absorbance of the G₅@antiCD71 conjugate for various volumes of antiCD71. (C) UV-VIS absorption spectra of native G₅NPs (blue line) and G₅@antiCD71 (red line), respectively. The inset indicates agarose gel electrophoresis assay of native G₅NPs (channel 1) and G₅@antiCD71 (channel 2). (D) The polydispersity index on hydration dynamics diameters of native G₅NPs and G₅@antiCD71 by DLS measurements, respectively. (E–G) TEM images of native G₅NPs, native antiCD71, and G₅@antiCD71, respectively. The green and yellow circles in G indicate G₅NPs and antiCD71, respectively. The scale bar in (E–G) is 5 nm.

Fig. 3. Feasibility verification of the SN-nanoruler by confocal imaging analysis of PL45 cells. (A) Native PL45 cells without the fluorescence background. (B) PL45 cells with the saturated XQ-2d-FAM aptamer. (C) The cell samples in (B) further treated by DAPI staining. (D) The merged image of (B) and (C). (E) PL45 cells treated with excess saturated G₅@antiCD71, after incubation with saturated XQ-2d-FAM. (F) PL45 cells treated with excess saturated G₅@BSA, after incubation with saturated XQ-2d-FAM, as a negative control. (G–I) Grey values of cell FLI in (B), (E), and (F) that were estimated using Image J software. (J). (J) Normalized grey values of (G–I) that were estimated using Image J software.

Fig. 4. Feasibility verification of the SN-nanoruler by FLI and FLT related experiments. (A) FCM analysis on PL45 cell samples. FCM charts indicated native PL45 cells (cell), cells with saturated XQ-2d-FAM (+FAM), and cells with saturated XQ-2d-FAM and G₅@antiCD71 of different amounts, i.e., 10, 70, 100, 150, 200, 400, and 600 μL from bottom to top, respectively (from “++ 10” to “++ 600”). (B) Fluorescence quenching efficiency as a function of the amounts of G₅@antiCD71. (C) Seven repeats of the Φ test that were calculated by FLI experiments. (D) FLT of cells incubated with saturated XQ-2d-FAM (+FAM, red line) and then co-incubated with excess saturated G₅@antiCD71 (++ G₅@antiCD71, black line). (E) Mean FLT of cells with saturated XQ-2d-FAM treatments before (τ₀) and after co-incubation with the saturated G₅@antiCD71 quencher (τ), respectively. (F) Three repeats of the Φ test that were calculated by FLT experiments. (G) Linear relationship of F₀/F and [Q] when added with different amounts of G₅@antiCD71. (H) The ratio of F₀/F calculated using FLI (blue) and τ₀/τ calculated using FLT (red), respectively. (I) Analysis of distance between two binding sites in the presence of saturated XQ-2d-FAM and saturated G₅@antiCD71 according to the estimated antibody sizes from TEM (group 1), AFM (group 2) and the theoretical calculation (group 3), respectively.

Fig. 5. SN-nanoruler with a single-nucleobase resolution identified by FLI and FLT experiments. (A) Single-nucleobase resolution model by lengthening the “n TA” spacer, i.e., one TA base pair at a time, between the 5′ end of XQ-2d and FAM, where n is the number of TA base pairs. (B) FCM analysis of native PL45 cells (cell), cells incubated with saturated XQ-2d-FAM (0 TA), XQ-2d-1TA-FAM (1 TA), XQ-2d-2TA-FAM (2 TA), and XQ-2d-3TA-FAM (3 TA), respectively. (C) FCM analysis of cells, cells incubated with saturated 0 TA, and cells incubated with saturated n TA and then saturated G₅@antiCD71, respectively. (D) Linear fitting relationship between the number of TA and Φ_FLI (red line) and Φ_FLT (black line), respectively, calculated according to the change in FAM FLI and FLT. (E) FLT analysis of cells incubated with saturated 0 TA and cells incubated with saturated n TA and then co-incubated with excess saturated G₅@antiCD71, respectively. (F) Comparisons of our experimental data with the theoretical simulation curves. Theoretical curves show the r-dependent Φ of GNPs with diameters of 4.3 nm (blue line), 5.3 nm (red line), and 6.6 nm (black line), respectively, adopted from the literature. The color triangles indicate experimental Φ values calculated from FLI.

Fig. 6. Binding feature analysis of DML7-FAM and two antibodies on the integrin α3β1 receptor of DU145 cells. (A) Illustration on the SN-nanoruler for measuring α3β1 subunit-level distance. DML7 and each antibody bring FAM dye and G₅NP to their binding sites, respectively. and represent the separation distance between the corresponding binding sites, and d_α3 and d_β1 represent the size of each antibody, respectively. (B and C) Saturated binding concentration analysis of DML7-FAM. The numbers in the FCM chart indicated each final concentration (nM) of DML7-FAM incubated with native DU145 cells (cell). (D and E) Best co-incubation time analysis of DML7-FAM. The numbers in the FCM chart indicated each incubation time of saturated DML7-FAM with native DU145 cells (cell). (F and G) Competitive binding analysis of saturated DML7-FAM and different amounts of antiα3 (P1B5) (left) and antiβ1 (K-20) (right) including 1, 2, 4, and 8 μL, respectively. And the order of incubation with cells is DML7-FAM and antibody.

Fig. 7. Synthesis and characterization of the G₅@antibody conjugate. (A and B) Normalized absorbance of antiα3 (P1B5) and G₅@antiβ1 (K-20) conjugates at varied pH values. (C and D) Normalized UV-VIS absorption spectra of native G₅NP (gray line), G₅@antiα3 (P1B5) (blue line), and G₅@antiβ1 (K-20) (red line). The insets of C and D indicate agarose gel electrophoresis assay of G₅@antiα3 (P1B5) and G₅@antiβ1 (K-20) (channels 1 and 3) and native G₅NPs (channels 2 and 4). (E and F) The polydispersity on hydration dynamics diameters of native G₅NPs (gray line), G₅@antiα3 (P1B5) (blue line) and G₅@antiβ1 (K-20) (red line), respectively. (G–J) TEM images of native antiα3 (P1B5), G₅@antiα3 (P1B5), native antiβ1 (K-20), and G₅@antiβ1 (K-20), respectively. The green and yellow circles in (G–J) indicate G₅NPs and antibodies, respectively. The scale bar in (G–J) is 5 nm.

Fig. 8. Measurement of the DML7-FAM binding integrin α3β1 receptor protein subunit domain in living cell systems. (A–E) Confocal imaging analysis of DU145 cells including native cells (A), incubation with the saturated DML7-FAM aptamer (B), and subsequent co-incubation with excess saturated G₅@BSA as a negative control (C), G₅@antiα3 (P1B5) (D), and G₅@antiβ1 (K-20) (E), respectively. (F) FLI analysis of grey values that were estimated using Image J software. Group 1, 2, 3, and 4 indicated the cells in (B), (C), (D), and (E). (G and H) FCM assay on FLI quenching of DML7-FAM induced by different amounts of G₅@antiα3 (P1B5) and G₅@antiβ1 (K-20), respectively. FCM charts included native DU145 cells (cell), incubation with saturated DML7-FAM (+DML7-FAM), and the subsequent co-incubation with G₅@antibody of different microliters as indicated by the numbers in each panel (++ number). (I) The Φ_FLI curves plotted as a function of the amounts of G₅@antiα3 (P1B5) (blue data) and G₅@antiβ1 (K-20) (red data), respectively. (J and K) Linear relationship of F₀/F and [Q] before the saturation binding of G₅@antiα3 (P1B5) (J) and G₅@antiβ1 (K-20) (K), respectively. (L) The calculated R′ values between the binding sites of DML7-FAM and each antibody according to the antibody size parameters obtained from TEM (group 1), AFM (group 2) and the theoretical calculation (group 3), respectively.