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. 2025 Dec 16;6(1):144–153. doi: 10.1021/jacsau.5c00978

Aptamer-Mediated Covalent Dual Lysosome-Targeting Chimeras Enhance Targeted Degradation of Cell Surface Proteins

Tao Peng †,‡, Min Su ‡, Zuying Zhang †,‡, Ya Wang ‡, Yingdi Zhu ‡, Juan Li †,‡,*
PMCID: PMC12848693  PMID: 41614177

Abstract

Aptamer-based lysosome-targeting chimeras (Apt-LYTACs) have emerged as a promising strategy for the selective degradation of cell surface proteins by linking a target-specific aptamer to a lysosome-trafficking receptor ligand. However, their degradation efficiency is often limited by weak noncovalent interactions, heterogeneous receptor distribution, and the constraints of a 1:1 complex stoichiometry. To address these challenges, we developed aptamer-mediated covalent dual lysosome-targeting chimeras (Apt-cdLYTACs), which enable specific covalent anchoring to the protein of interest with spatiotemporal control by combining the specificity of aptamer recognition with proximity-induced photoreactive cross-linking. These chimeras incorporate two lysosomal receptor ligands to enhance the local avidity and promote multivalent complex formation. Compared to conventional noncovalent or single-ligand covalent Apt-LYTAC, Apt-cdLYTAC forms more stable degradation complexes, exhibits prolonged intracellular retention, and reduces efflux, thereby significantly improving degradation efficiency. Apt-cdLYTAC provides a modular, efficient, and user-friendly platform for the selective degradation of membrane proteins, with broad potential for applications in biochemical research and therapeutic development.

Keywords: aptamer, covalent, dual lysosome-targeting ligand, LYTAC, targeted protein degradation

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Introduction

Cell surface proteins are central to key biological processes, including intercellular communication, immune recognition, receptor–ligand interactions, and antigen presentation. Owing to their pivotal roles and accessible membrane localization, they have emerged as a valuable class of targets for immune and targeted therapies. , Notably, they account for over 60% of current drug targets and serve as key biomarkers for early disease detection, diagnosis, and prognosis. Traditional therapeutic strategies largely rely on occupancy-driven mechanisms to block extracellular receptor–ligand interactions. − However, many surface proteins lack well-defined binding sites or possess multifunctional roles, limiting their druggability. In addition, challenges such as mutation-driven resistance and the inability to modulate undruggable targets further constrain their effectiveness.

Targeted protein degradation (TPD) has emerged as a powerful therapeutic strategy and a versatile tool for modulating cell surface proteins. Among TPD platforms, lysosome-targeting chimeras (LYTACs) employ to simultaneously engage a lysosome-trafficking receptor and a protein of interest (POI), thereby directing membrane-associated proteins toward lysosomal degradation. The pioneering work by the Bertozzi group established the first LYTAC platform by conjugating glycopolypeptide ligands for the cation-independent mannose-6-phosphate (CI-M6PR) to antibodies, enabling selective lysosomal degradation of extracellular and membrane-bound proteins. , Since then, additional lysosome-trafficking receptors, including the asialoglycoprotein receptor (ASGPR), cytokine receptor (e.g., CXCR7), integrins, scavenger receptors, and transferrin receptor, have been explored for their potential in engineered degradation systems. To construct selective LYTAC systems, recognition modules such as antibodies, , nanobodies, peptides, and aptamers , have been employed to target specific surface proteins. Among these, aptamers offer distinct advantages, including high binding specificity, low molecular weight, ease of chemical synthesis, and compatibility with site-specific modifications. These features make aptamer-based lysosome-targeting chimeras (Apt-LYTACs) an attractive and versatile platform for membrane protein degradation. However, current systems often suffer from a suboptimal degradation efficiency.

Apt-LYTAC is typically constructed by linking an aptamer that binds a target membrane protein to a ligand that engages a lysosome-trafficking receptor (Scheme A). This design enables the formation of a 1:1:1 ternary complex involving the target protein, the Apt-LYTAC, and a single lysosomal receptor molecule. However, such stoichiometry may limit degradation efficiency due to local receptor heterogeneity or restricted receptor availability on the cell surface. To overcome this, multivalent ligand strategies targeting lysosomal receptors have been reported to significantly enhance binding avidity by increasing local ligand density, thereby promoting more efficient formation of degradation complexes. , Moreover, most Apt-LYTACs rely on noncovalent interactions, , which can compromise their binding stability and reduce on-target retention, ultimately diminishing degradation efficacy. To address these challenges, covalent Apt-LYTACs have recently been explored to improve the degradation efficiency of targets such as PD-L1. However, the reported method requires a two-step, metabolic labeling approach involving the preincubation of cells with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) for several days to install azide groups on the cell surface, followed by the application of dibenzocyclooctyne (DBCO)-modified aptamers. While effective, this approach is labor-intensive and operationally complex, limiting its broader applicability.

1. Design Principle for Aptamer-Mediated Covalent Dual Lysosome-Targeting Chimeras .

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a (A) Comparison of conventional Apt-LYTAC with Apt-cdLYTAC. (B) Schematic representation of Apt-cdLYTAC for targeted degradation of cell surface protein.

To this end, we developed Aptamer-mediated Covalent Dual Lysosome-Targeting Chimeras (Apt-cdLYTACs), leveraging both the high specificity of aptamer-based recognition and the stability provided by proximity-induced photoreactive chemistry. Photoreactive manipulation has become an attractive tool due to its precise spatiotemporal control, rapid action, and noninvasive nature. This design enables covalent cross-linking of Apt-cdLYTAC with surface proteins on cancer cells, resulting in more stable degradation complexes that prolong the intracellular residence time, reduce molecular efflux, and ultimately enhance membrane protein degradation (Scheme B). To the best of our knowledge, a covalent, dual lysosome-targeting Apt-LYTAC designed to enhance membrane protein degradation has not yet been reported. Compared to conventional noncovalent or single-ligand covalent systems, Apt-cdLYTAC exhibited markedly improved degradation efficiency across a broad range of membrane protein targets. We anticipate that this modular and generalizable strategy will serve as a valuable platform for the selective degradation of cell surface proteins.

Results and Discussion

Fabrication and Optimization of Aptamer-Mediated Covalent Dual Lysosome-Targeting Chimeras (Apt-cdLYTACs)

Trivalent N-acetylgalactosamine (GalNAc) serves as a high-affinity ligand for the asialoglycoprotein receptor, enabling mediation of cell surface protein internalization and lysosome-targeted degradation through LYTACs. This targeting strategy is applicable to hepatocyte-specific studies and has been demonstrated in hepatocellular carcinoma models to effectively degrade oncogenic membrane-associated proteins, thereby suppressing tumor cell proliferation. , The Apt-cdLYTAC structure is formed by three functional modules through DNA self-assembly: a protein recognition probe (abc) and two photoreactive lysosome-trafficking receptor recruitment probes (NB-b*-GalNAc and NB-c*-GalNAc). Region a contains the aptamer sequence, which selectively binds the target membrane protein. Regions b, c and b*, c* are designed with complementary bases to form a stable duplex. Regions b* and c* serve as functionalized with photoreactive moieties (o-nitrobenzyl alcohol, o-NB) and GalNAc to form lysosome-trafficking receptor recruitment probes (NB-b*-GalNAc and NB-c*-GalNAc) for binding to the ASGPR (Figure A). Upon light activation, proximity-induced covalent cross-linking occurs between Apt-cdLYTAC and the surface protein, anchoring the complex to facilitate efficient lysosomal trafficking via ASGPR.

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Design, synthesis, and analysis of Apt-cdLYTAC. (A) Synthetic strategy of Apt-cdLYTAC, tri-GalNAc (red), photoreactive group (gray), complementary bases (blue or green). (B) MS analysis confirmed the modification of b* and c* with DBCO-GalNAc, as well as the subsequent modification of the resulting b*-GalNAc and c*-GalNAc with NB-(PEG)3-N3 and NB-(PEG)10-N3. (C) Schematic representation of Apt-dLYTAC with different spacer lengths (dashed circle). (D) Native PAGE analysis of the assembly of recognition probe abc with b*-GalNAc and c*-GalNAc bearing different spacer lengths. DNA strands from all groups (500 nM) in 20 μL of PBS (pH = 7.4) were denatured at 95 °C for 5 min and then rapidly cooled on ice for 30 min to facilitate preassembly. Lane 1: abc alone; Lane 2: abc + b*-GalNAc (0 nt) + c*-GalNAc (0 nt); Lane 3: abc + b*-GalNAc (0 nt) + c*-GalNAc (9 nt); Lane 4: abc + b*-GalNAc (9 nt) + c*-GalNAc (0 nt); Lane 5: abc + b*-GalNAc (9 nt) + c*-GalNAc (9 nt). M: DNA Marker. (E) Western blot analysis of Met levels in HepG2 cells treated with 500 nM of four types of Apt-dLYTAC for 24 h. Lane 1: cell (control); Lanes 2–5: Apt-dLYTAC with different spacer lengths corresponding to those in lanes 2–5 of Figure 1D were used. Uncropped full-size gel/blot images for Figure 1D and E are shown in Supporting Information Figures S16 and S17.

We initiated the synthesis of DBCO-GalNAc and the photoreactive moieties (NB-(PEG)3/5/10-N3). The major structures of the newly synthesized compounds were validated by mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (1H NMR, 13C NMR) (Figures S29–S36). Subsequently, two-step click reactions were employed to covalently conjugate DBCO-GalNAc and the photoreactive moieties (NB-(PEG)3-N3, NB-(PEG)10-N3) to DNA probes (b* and c*). MS analysis confirmed the successful synthesis of the DNA probes b*-GalNAc, c*-GalNAc, NB-b*-GalNAc, and NB-c*-GalNAc (Figure B). Given the critical influence of spacer length on the degradation efficiency of Apt-LYTACs, we first screened a series of Cy3-labeled RS-dLYTACs bearing GalNAc modifications with varying spacer lengths (0–18 nt) in both the b*-GalNAc and c*-GalNAc probes, assessing their ASGPR-targeting capability using flow cytometry. The results indicated that the 9 nt spacer conferred the strongest targeting ability (Figure S1). Therefore, we selected the 0 and 9 nt spacers to construct dual-terminal spacer variants, placed between the complementary duplex and the GalNAc moieties (as indicated by dashed boxes in the oligonucleotide chains) for subsequent comparative analysis (Figure C). The successful formation of four types of aptamer-mediated dual lysosome-targeting chimera constructs (Apt-dLYTAC) incorporating two distinct spacer lengths at both termini was confirmed via native polyacrylamide gel electrophoresis (PAGE) analysis (Figure D). These four Apt-dLYTAC constructs were subsequently applied to evaluate their Met protein degradation capabilities. Western blot analysis revealed that the construct incorporating 9 nt spacers on both the b*-GalNAc and c*-GalNAc probes exhibited the highest degradation efficiency and was identified as the optimal design for subsequent degradation of Met membrane proteins (Figure E).

Apt-cdLYTAC Variants Facilitate Covalent Membrane Protein Labeling and Specific Recognition of ASGPR

Aptamers possess superior tissue penetration capabilities, making them advantageous for targeted therapy in solid tumors, such as hepatocellular carcinoma. However, their inherent conformational instability can lead to off-target effects during internalization and degradation. To overcome this limitation, we introduce a strategy based on site-specific covalent labeling of target proteins. Photoaffinity labeling offers a proximity-driven approach, in which the o-NB group functions as a widely used photoreactive moiety for site-specific covalent modification of proteins (Figure A). We selected the Met receptor in HepG2 cells as the target protein and accordingly designed specific recognition and photoreactive recruitment probes. The efficiency of covalent labeling by the recruitment probes was evaluated using a three-step strategy: (i) aptamer-mediated target recognition, (ii) photoactivation-induced covalent labeling, and (iii) displacement of the aptamer by complementary DNA (cDNA) to remove noncovalently bound components. Before labeling, potential UV phototoxicity was assessed (375 nm, 20 mW/cm2, 0–10 min). No effect on cell viability was observed under these conditions (Figure S2), consistent with prior reports.

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Covalent labeling of membrane protein and ASGPR recognition by Apt-cdLYTAC variants. (A) Schematic diagram of the dual site-specific covalent labeling process of membrane proteins in living cells, Cy3 (green), Cy5 (red), inactive photosensitive group (gray), and activated photosensitive group (green). (B) Detection of the dual site-specific covalent labeling of membrane proteins in HepG2 cells by flow cytometry. HepG2 cells were incubated with the indicated constructs (500 nM) after preassembly, followed by light activation (375 nm, 2 min). A displacement group was subsequently treated with a 10-fold excess of cDNA. All incubations were carried out at 37 °C. (C) Schematic diagram of ASGPR-mediated specific recognition and subsequent lysosome-mediated internalization in HepG2 cells. (D) Flow cytometry analysis of GalNAc-dependent binding. HepG2 cells were incubated for 4 h with 500 nM Cy3-labeled RS-dLYTAC, with or without GalNAc modification, followed by analysis. (E) Median fluorescence intensity (MFI) relative to the control for HepG2 and HeLa cells as shown in Figure 2D and Figure S5. Data are presented as the mean ± SD. Statistics were performed using one-Way ANOVA (n = 3, ****P < 0.0001, ns: not significant). (F) Confocal microscopy images of HepG2 cells incubated at 37 °C for 4 h with 500 nM Cy3-labeled RS-dLYTAC with or without GalNAc modification. Nuclei stained by Hoechst 33342 (blue); lysosomes stained by Lysotracker (red); internalized Cy3-labeled RS-dLYTAC with or without GalNAc (green) (scale bar: 10 μm). Fluorescence colocalization curves in the right panel were derived from Image J analysis.

To optimize the covalent labeling efficiency, we synthesized a series of photoreactive recruitment probes NB-(PEG)3/5/10-b*-Cy3 and NB-(PEG)3/5/10-c*-Cy5 (Figure S3). Among them, NB-(PEG)3-b*-Cy3 and NB-(PEG)10-c*-Cy5 were selected as the labeling pairs for targeting the Met protein in HepG2 cells. Flow cytometry analysis demonstrated that, following photoirradiation and cDNA displacement, 86.9% of the cells localized within the Q2 region, indicating successful dual labeling. This signal was significantly higher than that of the control group (1.94%) (Figure B). To validate the specificity of Apt-cdLYTAC, we replaced the recognition region (region a) with a randomized sequence to generate RS-cdLYTAC. Flow cytometry analysis revealed that cells treated with RS-cdLYTAC predominantly localized in the Q4 region (76.3%), indicating minimal labeling. Similarly, cells treated with Apt-cdLYTAC but without photoirradiation also showed predominant localization in the Q4 region (87.9%). These results confirm that the covalent labeling strategy is both sequence-specific and photoactivation-dependent (Figure B, Figure S4).

Next, to validate the ASGPR-mediated specificity of RS-dLYTAC, we used HepG2 cells (ASGPR+) as the positive model and HeLa cells (ASGPR–) as the negative control. RS-dLYTAC with or without GalNAc modification, was incubated with both cell types (Figure C). Flow cytometry revealed that in HepG2 cells, RS-dLYTAC with GalNAc showed significantly higher fluorescence intensity compared to its unmodified counterpart (Figure D and E). In contrast, no significant difference was observed in HeLa cells, confirming the ASGPR-dependent internalization (Figure E and Figure S5). Moreover, RS-dLYTAC with GalNAc exhibited time-dependent internalization kinetics (Figure S6), whereas the unmodified counterpart showed only baseline nonspecific uptake. Confocal laser scanning microscopy (CLSM) further confirmed the efficient lysosomal trafficking of the Cy3-labeled, GalNAc-bearing RS-dLYTAC in HepG2 cells, as demonstrated by its strong colocalization with LysoTracker (Figure F). This pronounced colocalization highlights the essential role of the GalNAc moiety in promoting receptor-mediated endocytosis, likely via ASGPR, which is highly expressed on hepatocytes. In contrast, the GalNAc-free counterpart exhibited weak and diffuse fluorescence, reflecting its lack of a targeting ligand and resulting in minimal cellular internalization. Taken together, Apt-cdLYTAC Variants enable target-specific and photoactivation-dependent proximity covalent labeling of membrane proteins, followed by ASGPR-mediated lysosomal trafficking, offering a promising strategy for targeted protein degradation.

High-Efficiency Degradation of Membrane Protein Met Using Apt-cdLYTAC

Building upon the validated performance of Apt-cdLYTAC in covalent labeling and ASGPR-mediated internalization, we engineered Region a with a Met-targeted recognition sequence. The resultant conjugate enables the lysosomal delivery and degradation of target proteins through ASGPR-mediated trafficking. To delineate the covalent dual-ligand advantage of Apt-cdLYTAC in membrane protein degradation, we engineered two covalent monovalent variants (Apt-cmLYTAC-b and Apt-cmLYTAC-c, they structurally correspond to the 3′ and 5′ termini of the DNA strand, respectively) alongside a noncovalent control Apt-dLYTAC (no-hv) (Figure A). CLSM demonstrated predominant intracellular localization of the Apt-cdLYTAC in HepG2 cells, with significant colocalization observed with the LysoTracker, confirming efficient lysosomal delivery (Figure B). Similar spatial distribution patterns were observed for Apt-cmLYTAC-b and Apt-cmLYTAC-c (Figure S7).

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Degradation of the membrane protein Met by Apt-cdLYTAC. (A) Schematic diagram of protein degradation of Met in a HepG2 cell by Apt-cdLYTAC, three variants of Apt-cmLYTAC-b, Apt-cmLYTAC-c, and Apt-cdLYTAC (no-hv). (B) Validation of the lysosomal targeting transport of Apt-cdLYTAC in HepG2 Cells. Nuclei stained by Hoechst33342 (blue); lysosome visualized by lysotracker (red); Cy3-labeled Apt-cdLYTAC was used to visualize the internalization pathway of the degradation complex (green) (scale bar: 10 μm). (C) The Western blot was used to analyze the effect of the degradation time on the degradation of Met by Apt-cdLYTAC (from 3 to 24 h) in HepG2 Cells. The degradation assay was carried out with 500 nM Apt-cdLYTAC in DMEM at 37 °C. (D) The Western blot was used to analyze the effect of degradation concentration on the degradation of Met by Apt-cdLYTAC in HepG2 Cells. The degradation assay with Apt-cdLYTAC was conducted over 24 h in DMEM at 37 °C. (E) Western blot analysis was performed to assess the effects of Apt-cdLYTAC and its variants on the total Met protein levels in HepG2 cells. In all experimental groups, except the untreated control, cells were incubated with different Apt-cdLYTAC variants at a concentration of 500 nM for 24 h. Lane 1: cell (no-hv) (control); Lane 2: Apt-cmLYTAC-b; Lane 3: Apt-cmLYTAC-c; Lane 4: Apt-cdLYTAC; Lane 5: Apt-dLYTAC (no-hv); Lane 6: cell (hv). (F) Quantitative analysis of protein degradation efficiency was performed for Figure 3E. Data are presented as the mean ± SD. Statistics were performed using one-Way ANOVA (n = 3, ***P < 0.001, ****P < 0.0001). (G) Western blot analysis was performed to evaluate the effects of Apt-cdLYTAC and variants on total Met protein levels in HeLa cells, with all experimental groups consistent with those depicted in Figure 3E. (H) Quantitative analysis of protein degradation efficiency was performed for Figure G. Uncropped blot images for Figure 3C,D,E and G are shown in Supporting Information Figures S18, S19, S21 and S23.

Then, to evaluate Met degradation efficiency, HepG2 cells were treated with Apt-cdLYTAC over a time course ranging from 3 to 24 h (Figure C). Apt-cdLYTAC concentration also played a crucial role in the degradation efficiency. A concentration-dependent effect was observed (Figure D). Maximum degradation was achieved at 500 nM, followed by a slight decrease at higher concentrations, likely due to the common hook effect in LYTAC systems. Similar dose- and time-dependent degradation was observed in the Apt-cdLYTAC, whereas the RS-cdLYTAC exhibited no significant effect on total Met protein levels (Figure S8). Using the optimal concentration (500 nM, 24 h), Apt-dLYTAC­(no-hv) (Lane 5) exhibited minimal degradation, likely due to the short half-life of the aptamer and compensation by cellular protein renewal. Notably, the Apt-cdLYTAC (Lane 4) demonstrated significantly higher degradation efficiency (∼54%) compared to the Apt-cmLYTAC-b (Lane 2) and Apt-cmLYTAC-c (Lane 3) (∼14% and ∼27%) (Figure E and F).

To assess the potential for off-target degradation, we first performed total protein staining. Because this gel-based assay showed no appreciable differences due to its limited resolution (Figure S9), we next employed quantitative mass-spectrometry-based proteomics to obtain a global view of protein-level changes following Apt-cdLYTAC or RS-cdLYTAC treatment. The proteomic analysis confirmed a pronounced reduction in Met expression after Apt-cdLYTAC treatment, whereas RS-cdLYTAC produced no detectable change (Figure S10A–C). To identify specific proteomic alterations, we analyzed proteins that were significantly downregulated (P < 0.05, Log2 (FC) < −2) by each agent versus untreated controls. Apt-cdLYTAC triggered coordinated downregulation of ten proteins functionally linked to the Met network, including receptor tyrosine kinase signaling components , (TGFBR2, ARRB1), membrane ion channels (TRPC4/5), nucleocytoplasmic transport receptors (KPNA5), and RNA splicing and translation regulators − (SF1, GPATCH3, UBR2) (Figure S10D). This suggests that Met degradation disrupts its signaling axis, leading to synchronous destabilization of these core interactors and demonstrating a systemic effect beyond the target clearance. In contrast, the few proteins downregulated by RS-cdLYTAC were unrelated to Met signaling and mostly did not meet the threshold for statistical significance (Figure S10E), further underscoring the essential role of aptamer-mediated targeting.

Western blot analysis further confirmed that Met protein was not degraded in ASGPR-negative HeLa cells after treatment with the same set of degrader variants for 24 h (Figure G and H), indicating that Met degradation requires ASGPR receptor-mediated internalization. This result is consistent with the design principle that, without receptor recognition and subsequent internalization, the degraders cannot enter the endolysosomal pathway and, therefore, fail to induce Met degradation. Taken together, our Apt-cdLYTAC efficiently degrades Met protein through the lysosomal pathway, demonstrating superior performance compared with both monovalent variants and noncovalent configurations.

Modulation of Cellular Behavior via Apt-cdLYTAC-Induced Targeted Protein Degradation

Encouraged by the observed Met receptor degradation, we next investigated whether this degradation elicited functional consequences in the cells. This investigation was motivated by reports that the HGF/Met signaling pathway influences cell proliferation and invasion and is implicated in cancer metastasis. We found that cells have lower cellular viability after treatment with the Apt-cdLYTAC as assayed with the CCK-8 test, indicating that lower levels of Met on cell membranes may have functional impacts on the cells (Figure A). Additionally, wound healing assays quantitatively evaluated the migratory capacity of HepG2 cells following 24-h treatment with distinct protein degraders. The Apt-cdLYTAC treatment significantly impaired migratory capacity compared to the control (Figure B and C). Moreover, treatment with Apt-cmLYTAC-b or Apt-cmLYTAC-c also moderately reduced the level of wound closure. Furthermore, actin network organization in lamellipodia governs the mechanical flexibility of migrating cell fronts, playing a key role in cell motility. , Compared to control groups (HGF alone, Apt-dLYTAC­(no-hv), or Apt-cmLYTAC-b/c), the Apt-cdLYTAC significantly reduced F-actin-enriched lamellipodia at cell edges, thereby impairing cell motility (Figure D). Collectively, these results demonstrated that Apt-cdLYTAC could effectively restrain cell migration through weakened Met membrane localization.

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Regulation of cellular behavior by Apt-cdLYTAC. (A) Cell viability by CCK-8 assay of HepG2 cells post-treatment with 500 nM Apt-cmLYTAC-b, Apt-cmLYTAC-c, Apt-cdLYTAC, or Apt-dLYTAC (no-hv) for 24 h. Data are presented as the mean ± SD. Statistics were performed using one-Way ANOVA (n = 3, *P < 0.1, **P < 0.01, ****P < 0.0001). (B) Cell mobility regulated by Apt-cmLYTAC-b, Apt-cmLYTAC-c, Apt-cdLYTAC, or Apt-dLYTAC (no-hv) degrader in HepG2 cells was determined by a wound healing assay. Following an initial 6-h coincubation with 500 nM degraders (excluding the control group), all groups were treated with 30 ng/mL HGF. Cell migration was recorded at 0 and 24 h after HGF addition. (C) Quantification of the wound area in the wound healing assay in Figure 4B. Data are presented as the mean ± SD. Statistics were performed using one-Way ANOVA (n = 3, ****P < 0.0001). (D) CLSM images of F-actin-rich lamellipodia outside the cell membrane. Following a 12-h HGF treatment under the same conditions as the wound healing assay, the cells were fixed, permeabilized, incubated with FITC-phalloidin for 1 h, and finally imaged by confocal microscopy.

A Universal Aptamer-Mediated Platform for Selective Degradation of Membrane Proteins via Apt-cdLYTAC

To expand the therapeutic applicability of the aptamer-mediated covalent dual lysosome-targeting chimera (Apt-cdLYTAC) platform, we selected protein tyrosine kinase 7 (PTK7), a key transmembrane receptor recognized by the sgc8 aptamer, as an additional target. The corresponding Apt-cdLYTAC was engineered through rational design by replacing the aptamer recognition region a with the sgc8 sequence. Four types of Apt-dLYTAC constructs were applied to assess targeted PTK7 degradation. Initially, spacer optimization was performed, and based on degradation efficiency, constructs with 9 nt spacers on both b*-GalNAc and c*-GalNAc were selected for subsequent studies (Figure S11). We then used NB-(PEG)3-b*-Cy3 and NB-(PEG)5-c*-Cy5 as the labeling pair to target PTK7 in HepG2 cells. Flow cytometry following photoirradiation and cDNA displacement showed that 70.9% of cells resided in the Q2 region with dual fluorescence signals (Cy3 and Cy5), confirming the successful covalent labeling of PTK7 using this strategy (Figure S12).

The concentration-dependent degradation kinetics of PTK7 demonstrated that Apt-cdLYTAC achieved peak degradation efficacy at 500 nM (Figure A). With increasing incubation time of Apt-cdLYTAC, the PTK7 levels of HepG2 cells displayed a significant decrease (Figure B). PTK7 degradation was dose- and time-dependent with Apt-cdLYTAC but was unaffected by RS-cdLYTAC (Figure S13). At the established optimal dose and treatment time (500 nM, 24 h), a 41% reduction in PTK7 levels was observed following Apt-cdLYTAC treatment (Lane 4), significantly exceeding Apt-dLYTAC (∼9%, Lane 5), Apt-cmLYTAC-b (∼4%, Lane 2), and Apt-cmLYTAC-c (∼11%, Lane 3) (Figure C).

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Degradation of the membrane protein PTK7 and regulation of cellular behavior by Apt-cdLYTAC. (A) The Western blot was used to analyze the effect of degradation concentration on the degradation of PTK7 by Apt-cdLYTAC. (B) The Western blot was used to analyze the effect of the degradation time on the degradation of PTK7 by Apt-cdLYTAC. (C) Western blot analysis was performed to assess the effects of Apt-cdLYTAC and variants on the total PTK7 protein levels in HepG2 cells. Lane 1: cell (no-hv) (control); Lane 2: Apt-cmLYTAC-b; Lane 3: Apt-cmLYTAC-c; Lane 4: Apt-cdLYTAC; Lane 5: Apt-dLYTAC (no-hv); Lane 6: cell (hv). Data are presented as the mean ± SD. Statistics were performed using one-Way ANOVA (n = 3, *P < 0.1, **P < 0.01, ***P < 0.001, ns: not significant). Uncropped blot images for Figure 5A, B, and C are shown in Supporting Information Figures S26, S27 and S28. The experimental conditions and grouping for PTK7 degradation in HepG2 cells were identical to those used for Met protein degradation.

Given the established role of PTK7 in regulating cell proliferation and invasion, we assessed the functional consequences of its degradation using Apt-cdLYTAC. Depletion of PTK7 significantly impaired cellular viability (CCK-8 assay) (Figure S14) and reduced migration capacity (wound healing assay) (Figure S15), demonstrating that reduced membrane PTK7 levels exert functional impacts on these cellular processes. Collectively, these findings highlight the efficient membrane protein degradation capability of Apt-cdLYTAC and its promise as a versatile therapeutic platform.

Conclusions

In summary, we present a proof-of-concept for the Apt-cdLYTAC platform, which enables covalent labeling of cell surface proteins and dual recruitment of lysosome-trafficking receptors to enhance the protein degradation efficiency. This universal strategy allows for the targeted degradation of various membrane proteins (Met and PTK7 in this work) by simply substituting the recognition aptamer. Systematic comparisons show that Apt-cdLYTAC achieves significantly higher degradation efficiency than Apt-dLYTAC or Apt-cmLYTAC-b/c. Notably, degradation of target proteins led to marked inhibition of cell proliferation and invasion, implicating potential antimetastatic effects. However, an important limitation is that the short-wavelength photoactivated covalent binding mode suffers from poor tissue penetration, which significantly restricts its applicability in vivo. The development of photoaffinity labeling reagents that operate in the near-infrared or infrared range, which provide superior tissue penetration, may offer promising opportunities for future in vivo therapeutic applications. Collectively, this study establishes Apt-cdLYTAC as a robust and versatile platform for membrane protein degradation with promising therapeutic potential for diseases characterized by aberrant membrane protein expression.

Supplementary Material

au5c00978_si_001.pdf (1.8MB, pdf)

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 22174161, 21991080, 22304179), the Zhejiang Leading Innovation and Entrepreneurship Team (No. 2022R01006), and the Pioneer R&D Program of Zhejiang (No. 2024SDYXS0003). The authors acknowledge the use of instruments from the Shared Instrumentation Core Facility at the Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c00978.

  • Supplementary figures and tables, materials and methods, details of statistical tests, detailed synthetic procedures and compound ESI-MS analyses, confocal microscopy images, flow cytometry analysis. Supplementary Figures S1–S36 (PDF)

The authors declare no competing financial interest.

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