Enhanced Live-Cell Delivery of Synthetic Proteins Assisted by Cell-Penetrating Peptides Fused to DABCYL

Abstract

Live-cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell-penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca-arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and a Ub variant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.

Keywords: activity-based probes; cell-penetrating peptides; fluorescence; small ubiquitin like modifier (SUMO); ubiquitin.

Figure 1

Chemical synthesis of model peptides 1 and 2. a) Synthetic scheme for P1. b) Synthetic scheme for cR10D. c) Conjugation of activated cR10 to P1 and HPLC‐MS analysis of purified 1. d) Conjugation of activated cR10D to P1 and HPLC‐MS analysis of purified 2.

Figure 2

Delivery of model peptides 1 and 2 to live U2OS cells. a) FITC signal from 1 (green). b) FITC signal from 2 (green) (scale bars 10 μm).

Figure 3

Synthesis of Ub‐conjugates: a) Synthetic scheme of 4 and 5 starting from 3. b) HPLC‐MS analysis of purified conjugate 4 (observed mass=11 473.2±0.2 Da, calcd mass=11 473 Da). c) HPLC‐MS analysis of purified conjugate 5 (observed mass=11 852.2±0.2 Da, calcd mass=11 852 Da).

Figure 4

Representative images of delivery of Ub analogues 4 (a–d) and 5 (d–g) to live U2OS cells. a,d) 4 and 5 (TAMRA, red). b,e) Hoechst (blue). c,f) TAMRA and Hoechst channels combined. d,g) Bright field channel. (Scale bars 10 μm). The experiment was repeated twice. i) Quantification of nuclear TAMRA intensity relative to the untreated cells from Figure 4 a & e. Results are the average of two independent experiments (over 150 cells per experiment). Error bars are the standard deviation of the averages. j) Fluorescent gel assay in U2OS cells after cell delivery.

Figure 5

TAMRA‐Cys‐Ub‐CONH₂ construct (6) and the four fluorescently labelled Ub‐CPP conjugates (7–10) for live‐cell delivery.

Figure 6

Representative images of delivery of 7, 8, 9, 10 to live HeLa cells. a,f,k,p) TAMRA signal (red) from 7, 8, 9, and 10, respectively. b,g,l,q) Hoechst (blue) from 7, 8, 9, and 10, respectively. c,h,m,r) CT‐DR (pink) from 7, 8, 9, and 10, respectively. d) 7 (TAMRA) and Hoechst channel from (a) and (b) combined. i) 8 (TAMRA) and Hoechst channel from (f) and (g) combined. n) 9 (TAMRA) and Hoechst channel from (k) and (l) combined. s) 10 (TAMRA) and Hoechst channel from (p) and (q) combined. e,j,o,t) Bright field images from 7, 8, 9, and 10, respectively. (Scale bars 10 μm). The experiment was repeated three times.

Figure 7

Quantification of cellular and nuclear TAMRA intensity in a) HeLa cells after treatment with 7–10, relative to untreated cells (based on the average of 450 cells from three repetitions). b) Quantification of nuclear TAMRA intensity after treatment of U2OS cells with 7 and 8, as determined by flow imaging cytometry. Unpaired t‐test, p<0.0001. Results are the average of two independent sets of experiments. Error bars are the standard deviation of averages.

Figure 8

Synthesis of cell‐permeable DUB ABP. a) Ub sequence with S20C mutation and synthetic scheme for Ub‐based DUB ABP (11) for live‐cell delivery. b) Ubv M6 sequence with S20C mutation and synthetic scheme for Ubv‐based DUB ABP (12) for live‐cell delivery. c) HPLC‐MS analysis of purified conjugate 11 (observed mass=11 600.4±2.4 Da, calcd mass=11 602 Da). d) HPLC‐MS analysis of purified conjugate 12 (observed mass=11 833.1±2.1 Da, calcd mass=11 835 Da).

Figure 9

Representative images of delivery of 11 and 12 to live U2OS cells. a,e) TAMRA signal (red) from 11 and 12, respectively. b,f) Hoechst (blue) from 11 and 12, respectively. c) 11 (TAMRA) and Hoechst channel from (a) and (b) combined. g) 12 (TAMRA) and Hoechst channels from (e) and (f) combined. d,h) Bright field from 11 and 12, respectively. (Scale bars 20 μm). The experiment was repeated thrice (imaging of at least 150 cells per experiment for each construct).

Figure 10

Synthesis of SUMO2‐CPP conjugates: a) Synthetic scheme of 14 and 15 starting from 13. b) HPLC‐MS analysis of purified conjugate 14 (observed mass=13 214.6±0.6 Da, calcd mass=13 214 Da). c) HPLC‐MS analysis of purified conjugate 15 (observed mass=13 594.2±0.2 Da, calcd mass=13 594 Da).

Figure 11

Representative images of delivery of 14 and 15 to live U2OS cells. a,e) TAMRA signal (red) from 14 and 15, respectively. b,f) Hoechst (blue) from 14 and 15, respectively. c) 14 (TAMRA) and Hoechst channel from (a) and (b) combined. g) 15 (TAMRA) and Hoechst channels from (e) and (f) combined. d,h) Bright field from 14 and 15, respectively. (Scale bars 20 μm). The experiment was repeated thrice (imaging of at least 150 cells per experiment for each construct). i) Representative zoomed image of 14 from TAMRA channel and j) TAMRA and Hoechst channels combined. k) Representative zoomed image of 15 from TAMRA channel and l) TAMRA and Hoechst channels combined. (Scale bars 5 μm). m) Quantification of nuclear TAMRA intensity of 14 and 15 relative to the untreated cells. The result is the average of the three independent sets of experiments. Error bars are the standard deviation of averages.