Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation

Abstract

As a crucial element of proteolysis targeting chimeras (PROTACs), the choice of E3 ubiquitin ligase significantly influences degradation efficacy and selectivity. However, the available arsenal of E3 ligases for PROTAC development remains underexplored, severely limiting the scope of targeted protein degradation. In this study, we identify a non-inhibitory aptamer targeting ZYG11B, a substrate receptor of the Cullin 2-RING ligase complex, as an E3 warhead for targeted protein degradation. This aptamer-based PROTAC platform, termed ZATAC, is facilely produced through bioorthogonal chemistry or self-assembly and shows promise in eliminating several undruggable target proteins, including nucleolin (NCL), SRY-box transcription factor 2 (SOX2), and mutant p53-R175H, underscoring its universality and versatility. To specifically deliver ZATACs into cancer cells, we further develop DNA three-way junction-based ZATACs (3WJ-ZATACs) by integrating an additional aptamer that selectively recognizes the protein overexpressed on the surface of cancer cells. The 3WJ-ZATACs demonstrate in vivo tumor-specific distribution and achieve dual-target degradation, thereby suppressing tumor growth without causing noticeable toxicity. In summary, ZATACs represent a general, modular, and straightforward platform for targeted protein degradation, offering insights into the potential of other untapped E3 ligases.

Fig. 1. A non-inhibitory DNA aptamer targeting ZYG11B was identified.

a Schematic of the SELEX procedure. Created with BioRender.com. b Positive and negative retention rates of the enriched DNA pool using RT-qPCR across rounds 1 to 7. c, d ITC was used to characterize the binding affinity of the aptamer candidates (c), with detailed ITC titration and fitting curves for Apt#Z6 shown (d). e Biotin-labeled Apt#Z6 was used to pull GFP-tagged ZYG11B down in HEK293T cells via a streptavidin pull-down assay, and the pellets were subjected to immunoblotting. The averages of n = 2 (B) and n = 3 (c, d) biologically independent samples are shown. Data are shown as the mean ± SD. The data presented in panel (e) are representative of three independent experiments.

Fig. 2. Construction of Halo-ZATACs to degrade GFP-HaloTag protein.

a Structure of the Halo-ZATACs. “Tn”, the number of thymidine nucleotides added to the 5’ end of Apt#Z6, serves as linkers. b Schematic of Halo-ZATAC-mediated degradation of GFP-HaloTag fusion protein through the ubiquitin-proteasome system. c, d GFP-HaloTag-expressing HEK293T cells were transfected with Halo-ZATAC#Tn for 24 h, and cell lysates were subjected to immunoblotting (c). The remaining GFP-HaloTag was quantified (d). #Tn (n = 1, 3, 5) represents Halo-ZATACs with one, three, or five thymine linkers. e, f Fluorescence imaging (e) and quantification (f) of GFP-HaloTag-expressing HEK293T cells treated with Halo-ZATAC#T3 in the presence or absence of MG132 (20 µM). Scale bar, 100 µm. g, h GFP-HaloTag-expressing HEK293T cells were transfected with escalating doses of Halo-ZATAC#T3 for 24 h and cell lysates were subjected to immunoblotting (g). The remaining GFP-HaloTag was quantified (h). i, j GFP-HaloTag-expressing HEK293T cells were transfected with Halo-ZATAC#T3 (500 nM) for the specified time points, and cell lysates were subjected to immunoblotting (i). The remaining GFP-HaloTag was quantified (j). k, l GFP-HaloTag-expressing HEK293T cells were transfected with Halo-ZATAC#T3 (500 nM) in the presence or absence of MG132 (20 µM) for 24 h, and cell lysates were subjected to immunoblotting (k). The remaining GFP-HaloTag was quantified (l). m, n GFP-HaloTag-expressing HEK293T cells were transfected with siRNAs targeting ZYG11B, followed by treating with Halo-ZATAC#T3 (500 nM) for 24 h. Cell lysates were subjected to immunoblotting (m), and the remaining GFP-HaloTag was quantified (n). o, p GFP-HaloTag-expressing HEK293T cells were transfected with Halo-ZATAC#T3 (500 nM) and Apt#Z6 for 24 h. Cell lysates were subjected to immunoblotting (o), and the remaining GFP-HaloTag was quantified (p). Figure 2a & b were created with BioRender.com. The averages of n = 3 (d, h, f, j, l, n, p) biological replicates are presented as mean ± SD. Statistical significance was determined using one-way ANOVA with multiple comparisons (d, f, j, n, p) and the t test (and nonparametric tests) in (l). The data presented in (c, e, g, i, k, m, o) are representative of three independent experiments.

Fig. 3. Bispecific aptamer-based ZATACs induce NCL degradation in a ZYG11B- and ubiquitin proteasome-dependent manner.

a Schematic of NCL-ZATACs-mediated degradation of NCL. b, c Microscale thermophoresis (MST) to determine the binding affinity of GFP-NCL to NCL-ZATAC#20 (b) and to the NCL-ZATAC#20/ZYG11B complex (c). d Streptavidin pulldown assays to determine the interaction between NCL and ZYG11B proteins mediated by NCL-ZATAC#20 in GFP-NCL overexpressing HEK293T cells. For Biotin-Scramble ctrl, only NCL-targeted aptamer (AS1411) is scrambled, and the Apt#Z6 remains unchanged. e Co-immunoprecipitation assays to assess the interaction between endogenous NCL and ZYG11B in the presence or absence of NCL-ZATAC#20 (500 nM) in MG132-treated (20 µM) MCF-7 cells. f–h Immunoblotting analysis of MCF-7 cells treated with the specified concentrations of Scr-ZATAC#20 (f) or NCL-ZATAC#20 for 24 h (g). The remaining NCL was quantified (h). i Immunoblotting analysis of NCL levels in MCF-7 cells treated with NCL-ZATAC#20 (500 nM) for the specified time points. j, k Immunoblotting analysis of NCL levels in MCF-7 cells treated with NCL-ZATAC#20 and cycloheximide (CHX) for specified time points (j). The remaining NCL was quantified (k). l Fluorescence images of GFP-NCL-expressing HEK293T cells treated with NCL-ZATAC#20 in the presence or absence of MG132 (20 µM). Scale bar: 100 µm. m MCF-7 cells were transfected with His-Myc-Ub plasmids, followed by treating with NCL-ZATAC#20 and MG132 (20 µM) for 5 h. Cell lysates were subjected to immunoprecipitations. n Immunoblotting analysis of MCF-7 cells treated with NCL-ZATAC#20 (500 nM) in the presence or absence of MG132 (20 µM) for 24 h. o Immunoblotting analysis of MCF-7 cells transfected with ZYG11B-targeted siRNAs, followed by treating with NCL-ZATAC#20 (500 nM) for 24 h. p Immunoblotting analysis of MCF-7 cells treated with NCL-ZATAC#20 (500 nM) and varying concentrations of Apt#Z6. Figure 3a–c were created with BioRender.com. The averages of n = 3 (b, c, h, k) biologically independent samples are shown. Data are shown as the mean ± SD. Statistical significance in (k) was assessed using the t-tests (and nonparametric tests). The data shown in (d–g, i, j, l–o) are representative of three independent experiments.

Fig. 4. SOX2-ZATAC#20 efficiently degrades SOX2 and inhibits cancer cell proliferation.

a Schematic of SOX2 degradation mediated by SOX2-ZATACs. Created with BioRender.com. b Immunoblotting analysis of A549 cells treated with SOX2-ZATACs with different lengths of double-stranded flexible linkers for 24 h. c Immunoblotting analysis of A549 cells treated with scramble control (Scr-ZATAC-2#20) or SOX2-ZATAC#20 for 24 h. d Immunoblotting analysis of A549 cells treated with SOX2-ZATAC#20 (500 nM) for the specified time points. e Co-immunoprecipitation assays to evaluate the interaction between endogenous SOX2 and ZYG11B in the presence or absence of SOX2-ZATAC#20 (500 nM) in MG132-treated (20 µM) A549 cells. f, g Immunoblotting analysis of A549 cells treated with SOX2-ZATAC#20 (500 nM), together with or without CQ (10 µM) or MG132 (20 µM) for 24 h. h Immunoblotting analysis of A549 cells treated with SOX2-ZATAC#20 (500 nM) and varying concentrations of Apt#Z6. i Immunoblotting analysis of A549 cells transfected with ZYG11B-targeted siRNAs, followed by treating with SOX2-ZATAC#20 (500 nM) for 24 h. j, k A549 cells were treated with PBS, Apt#SOX2 (500 nM), or SOX2-ZATAC#20 (500 nM) for 24 h, and were subjected to EdU incorporation assays (j). The percentage of EdU-positive cells was quantified (k). Scale bar: 100 µm. l A549 cells were treated with PBS, Apt#SOX2 (500 nM), or SOX2-ZATAC#20 (500 nM) for specified time points, and viable cells were evaluated using CCK-8 assays. m, n A549 cells were treated with PBS, Apt#SOX2 (500 nM), or SOX2-ZATAC#20 (500 nM) for two weeks and then subjected to crystal violet staining. The colonies were imaged (m) and quantified (n). o, p Representative 3D tumor spheroidic image (o) and quantification (p) of A549 cells treated with PBS, Apt#SOX2 (500 nM), or SOX2-ZATAC#20 (500 nM). Scale bar, 65 µm. The averages of n = 5 (k), n = 4 (l), n =  3 (n, p) biological replicates are shown. Data are shown as the mean ± SD. Statistical significance in (k, l, n, p) was assessed using one-way ANOVA with multiple comparisons. The data shown in (b–j, m, o) are representative of three independent experiments.

Fig. 5. R175H-ZATAC#25 suppresses the growth of p53-R175H harboring cancer cells through degrading p53-R175H mutant.

a Schematic of the degradation of p53-R175H induced by R175H-ZATACs. Created with BioRender.com. b, c Immunoblots (b) and quantification (c) of H1299-R175H cells treated with R175H-ZATACs (500 nM) for 24 h. d Immunoblotting analysis of H1299-R175H cells treated with R175H-ZATAC#25 for 24 h. e, f Immunoblotting analysis of H1299-R175H cells treated with R175H-ZATAC#25 (500 nM) for the indicated time points (e). The remaining p53-R175H was quantified (f). g Immunoblotting analysis of H1299-R175H cells treated with R175H-ZATAC#25 (500 nM) in the presence or absence of MG132 (20 µM) for 24 h. h Immunoblotting analysis of H1299-R175H cells treated with R175H-ZATAC#25 (500 nM) and Apt#Z6. i Cell proliferation assays to determine the effects of R175H-ZATAC#25 (500 nM) on H1299-R175H cells. j, k Fluorescence images (j) and quantification (k) of H1299-R175H cells treated with PBS, p53-mDA (500 nM), or R175H-ZATAC#25 (500 nM), followed by EdU staining. Scale bar, 100 µm. l, m H1299-R175H cells were treated with PBS, p53-mDA (500 nM), or R175H-ZATAC#25 (500 nM) for two weeks and then subjected to crystal violet staining. The colonies were imaged (l) and quantified (m). n, o Representative spheroidic images (n) and quantification (o) of H1299-R175H cells treated with PBS, p53-mDA (500 nM), or R175H-ZATAC#25 (500 nM). Scale bar, 65 µm. The averages of n =  3 (c, f, m), n = 4 (i, o), n = 5 (k) biological replicates are shown. Data are shown as the mean ± SD. Statistical significance in (c, f, i, k, m, o) was assessed using one-way ANOVA with multiple comparisons. The data shown in (d, e, g, h, j, l, n) are representative of three independent experiments.

Fig. 6. 3WJ-ZATACs enable targeted delivery and dual-target degradation.

a Schematic depicting membrane protein-mediated 3WJ-ZATAC uptake and dual-target degradation. Created with BioRender.com. b Native PAGE showing the successful assembly of 3WJ-ZATAC. c Fluorescence images of A549 cells incubated with Cy3-3WJ-Scr^AS1411 (1000 nM), Cy3-3WJ-Scr^Apt#Z6 (1000 nM), or 3WJ-ZATAC (1000 nM) for 6 h. Scale bar, 20 µm. d Immunoblotting analysis of A549 cells treated with Cy3-3WJ-Scr^Apt#Z6 or 3WJ-ZATAC#T10 for 24 h. e Immunoblotting analysis of A549 cells treated with 3WJ-ZATAC#T10 (1000 nM) for the specified time points. f Immunoblotting analysis of A549 cells treated with 3WJ-ZATAC#T10 (1000 nM) in the presence of MG132 (20 µM) for 24 h. g, h A549 cells were treated with PBS, 3WJ-Scr^Apt#6 (1000 nM), or 3WJ-ZATAC#T10 (1000 nM) for two weeks and then subjected to crystal violet staining (g). The colonies were quantified (h). i, j Representative spheroidic images (i) and quantification (j) of A549 cells treated with PBS, 3WJ-Scr^Apt#6 (1000 nM), or 3WJ-ZATAC#T10 (1000 nM). Scale bar, 65 µm. k Immunoblotting of A549 cells treated with 3WJ-ZATAC#PTK7 for 24 h. l Immunoblotting analysis of H1299-R175H cells treated with 3WJ-ZATAC#p53 for 24 h. The averages of n =  3 (h), n = 4 (j) biological replicates are shown. Data are shown as the mean ± SD. Statistical significance in (h, j) was assessed using one-way ANOVA with multiple comparisons. The data shown in (b–g, i, k, l) are representative of three independent experiments.

Fig. 7. 3WJ-ZATAC#T10 inhibits tumor growth in vivo.

a Images of xenograft mice injected with PBS, Cy5, or Cy5-3WJ-ZATAC#T10 via tail vein. b Schematic of the administration of tumor-bearing mice. Created with BioRender.com. c, d Images (c) and volumes (d) of tumors isolated from xenograft mice intravenously inoculated with PBS, 3WJ-Scr^Apt#Z6 (5 mg/kg), or 3WJ-ZATAC (5 mg/kg). e Measurement of tumor volumes at specified times. f Measurement of body weight at the indicated intervals. g Histopathological examination of major organs post-treatment. Scale bar, 100 μm. h, i Immunohistochemistry images (h) and quantification (i) of tumor sections stained with the indicated antibodies. Scale bar, 100 μm. The averages of n = 4 (d–f) and n = 3 (i) biological replicates are shown. Data are shown as the mean ± SD. Statistical significance in (d–f, i) was assessed using one-way ANOVA with multiple comparisons. The data shown in (g, h) are representative of three independent experiments.