Structurally Specific Z-DNA Proteolysis Targeting Chimera Enables Targeted Degradation of Adenosine Deaminase Acting on RNA 1

Abstract

Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.

Graphical Abstract

INTRODUCTION

ADAR1 (Adenosine Deaminase Acting on RNA 1) constitutes a pivotal enzyme intricately involved in the process of RNA editing, particularly the conversion of adenosine to inosine within double-stranded RNA molecules.^1–5 Its implications span a spectrum of ailments, notably marked by its frequent overexpression in cancer^1–3 and its intricate interplay with autoimmune disorders (Figure 1A).^6–10 Notably, recent advancements have led to the development of ADAR catalytic inhibitors as anticancer therapeutic agents, such as 8-Azaadenosine (8-Aza).^11–14 However, the inhibitor has not been fully demonstrated to selectively inhibit ADAR1’s biological functions, in part due to the presence of various isoforms containing the same A to I deaminase catalytic domain.¹³ Given ADAR’s multifaceted domain structure, the reported inhibitor only targets the catalytic editing-dependent domain, leaving the other editing-independent functionalities untouched within the cellular milieu that compromises its potential therapeutic capacity.¹ Thus, a relatively more compelling strategy has emerged in the form of targeting ADAR1 for degradation, potentially eradicating the presence of the protein rather than merely inhibiting it. Considering ADAR1’s distinctive and inherent affinity for Z-DNA, characterized by its Zig-Zag conformation of the sugar–phosphate backbone under previously elucidated physiological conditions (Figure 1B),^15–17 the incorporation of Z-DNA as a foundational element in the design of PROTAC emerges as the optimal and viable choice.

Figure 1. The Z-PROTAC hijacks the VHL E3 ligase for targeted degradation of ADAR1 in cells.

(A) The reported functions of ADAR1 in cancer and immunology. (B) The structure analysis of Z-form versus B-form DNA, with antiparallel orientation of ribose rings marked with red and black arrows in Z-DNA (PDB code: 3P4J), and with parallel orientation in B-DNA (PDB code: 1EN3). (C) Schematic illustration of the Z-DNA-based PROTAC (Z-PROTAC) strategy.

A profusion of DNA-based PROTAC strategies has been innovated by diverse research groups, encompassing approaches ranging from targeting transcription factors, such as TRAFTAC,¹⁸ TF-PROTAC,¹⁹ O’PROTAC,²⁰ and G4-PROTAC,²¹ to targeting the nontranscriptional factor telomere with TeloTAC,²² further expanding to more sophisticated methodologies, such as Myc targeting PROTAC based on a TNA-DNA bivalent binder.²³ In spite of these strides, the imperative persists to expand methodologies of DNA-based modalities to heighten targeting precision and ameliorate off-target effects within cells.^19,24 Z-DNA-based PROTACs (Z-PROTACs) epitomize a distinctive category of PROTACs predicated on the deployment of Z-DNA as a selective targeting entity. Z-DNA, an atypical left-handed helical DNA conformation, ensues under specific circumstances encompassing elevated salt concentrations or well-defined DNA sequences.^25–36 By integrating Z-DNA binding ligands into the PROTAC design, a novel avenue surfaces, enabling the selective targeting of proteins intricately associated with Z-DNA regions within the genome, such as ADAR1.^15,16 Z-PROTAC could be devised by linking a Z-DNA binding moiety meticulously tailored to the ADAR1 oncoprotein with an E3 ligase ligand. Upon interaction, PROTAC orchestrates the intimate proximity of ADAR1 with an E3 ubiquitin ligase, triggering the process of ADAR1 ubiquitination and ensuing subsequent degradation by the 26S proteasome.

Here, we propose the innovative development of a proof-of-concept methodology termed the Z-DNA-based Proteolysis Targeting Chimera (Z-PROTAC) (Figure 1C). To achieve this objective, we synthesized a Z-form oligodeoxynucleotide (Z-DNA) that functions as the ligand to intricately recruit ADAR1, conjugated with a VHL ligand for the selective recruitment of the VHL E3 ligase. This strategic design hijacked the ubiquitination process, ultimately culminating in the precise degradation of ADAR1. The construction of the Z-PROTAC was obtained through a copper-free strain-promoted azide–alkyne cycloaddition (SPAAC) reaction, a method previously elucidated by our group.^19,22,37

RESULTS AND DISCUSSION

Confirmation of Z-DNA Formation by the Z-22 Antibody Binding Using the Electrophoretic Mobility Shift Assay (EMSA).

As reported, the Z-DNA emerges under specific conditions, including specific (GC)n repeats, increased supercoiling, and protein binding during transcription, replication, and chromatin remodeling.³⁸ Environmental factors, such as pH and ions, further contribute to Z-form DNA formation.^25–36 In this study, we devised a synthetic DNA construct with a (CG)₆ sequence,³⁹ denoted as FAM-DNA 2, and strategically introduced and synthesized a 5, 6-FAM fluorescent tag at the 5′ terminus of DNA 2 for tracking. Subsequently, a variety of ions, such as NaCl and hexaammine CoCl₃,^35,36 were used to induce the Z-DNA formation (Figure 2A). These constructs were cultivated under disparate ionic conditions to yield Z-form DNA (Figure 2A), followed by electrophoretic mobility shift assay (EMSA) experiments (Figure 2B).

Figure 2.

Ion induction of FAM-Z-DNA formation and the electrophoretic mobility shift assay (EMSA) for detecting binding between FAM-Z-DNA and the Z22 antibody. (A) Schematic illustration of the ion induction of FAM-Z-DNA formation from FAM-B-DNA. (B) Schematic illustration of binding between the Z22 antibody and FAM-Z-DNA. (C) The induction of FAM-Z-DNA formation was performed using 3 M NaCl. The FAM-Z-DNA was shifted when bound to the Z22 antibody. (D) Induction of FAM-Z-DNA formation with 3 M NaCl, proportional to the FAM-DNA 2 at the indicated dosage. (E) Induction of FAM-Z-DNA formation with 3 M NaCl, proportional to the Z22 antibody at the indicated dosage. The samples in C, D, and E were detected by using 4% gel and separated for 1.5 h. Images were captured with Ruby (left) and Alexa 488 (right) channels. (F) CD detection of the Z-DNA formation of DNA 2 induced by NaCl. The peak shift in the spectrum is indicated by a round dot.

The scheme illustrated in Figure 2B delineates the coculture of FAM-B-DNA 2 or, if successfully triggered, FAM-Z-DNA 2 with the Z-22 antibody for a 30 min duration. Consequently, the induced FAM-Z-DNA 2 exhibited affinity toward the Z-22 antibody, while the nonconverted FAM-B-DNA 2 demonstrated no such interaction, a discernment effectively captured by the EMSA assay. As depicted in Figure 2C, an initial quantity of 0.5 μM FAM-TRF DNA, FAM-Random DNA, and FAM-DNA 2 was introduced into the EMSA buffer supplemented with/without 3 M NaCl, prompting Z-DNA formation. Subsequently, the aforementioned premixed buffer containing DNA was combined with an undiluted Z22 antibody (1 μg) and incubated at room temperature for an additional 30 min.³⁵ The resulting mixture was then subjected to 4% polyacrylamide gel electrophoresis to effectuate the separation of bound (FAM-Z-DNA) and unbound FAM-DNA 2 (FAM-B-DNA 2). Given that the presence of Z22-antibody is detectable on a PAGE gel, albeit manifesting as distinct and shifted green bands exclusively upon interacting with FAM DNA, our observations suggest that 3 M NaCl could induce Z-DNA formation in DNA 2, while the control DNA (TRF or Random DNA) does not exhibit a similar response. Subsequent investigations involved systematic exploration of ion conditions coupled with a heightened Z22 antibody and FAM-Z-DNA 2 concentrations (Figure 2D,E). Furthermore, the Circular Dichroism (CD) assay was used to confirm the formation of the Z-DNA configuration induced by NaCl or CoCl₃, as shown in Figures 2F and S11B, respectively. In conclusion, our findings substantiate the claim that Z-DNA exhibits a propensity to bind to the Z22 antibody in a dose-dependent manner. Notably, we demonstrated the efficient induction of Z-DNA 2 through exposure to 3 M NaCl.

Design of Z-DNA as a Ligand for ADAR1.

ADAR1 exhibits specific binding to Z-DNA in a cellular context.^15,16 However, it remains relatively unclear whether the induced Z-DNA, especially synthesized in buffer conditions could possess functional binding affinity toward ADAR1. To elucidate the critical role of Z-DNA in facilitating ADAR1 interaction, we engineered two biotin-modified ligands, Biotin-(CG)₃ and Biotin-(CG)₆, characterized by 3 and 6 cytosine-guanine repeats, denoted Biotin-DNA 1 and Biotin-DNA 2, respectively. The Biotin-TRF ODN, which adopts the B-form conformation and is known for its specific binding to TRF1 and TRF2 in telomeres (Figure 3A), was employed as a negative control.²²

Figure 3.

ADAR1 protein binds with biotin-Z-DNA. (A) Schematic structures of the B- and Z-form DNA. (B) The scheme of the pull-down assay for biotin-DNA (biotin-B and biotin-Z) toward myc-ADAR1. (C) Binding of biotin-DNA 1 to myc-ADAR1 extracted from HEK293 cells, monitored with immunoblotting using the anti-myc antibody. (D, E) Pull-down assay of biotin-Z-DNA 2 to myc-ADAR1 extracted from HEK293 cells, monitored with immunoblotting using anti-ADAR1 (D) or myc (E) antibodies as indicated.

To probe the interplay between ADAR1 and the biotinylated oligodeoxynucleotides (ODNs), streptavidin–biotin pull-down assays were performed by using the biotinylated ODNs as bait molecules (Figure 3B–E). pmGFP-ADAR1-p150 was overexpressed in 293T cells to obtain Myc-ADAR1. All the ODNs are annealed or not based on previously described methods.¹⁹ Figure 3C reveals that Biotin-DNA 1 (10 μg), under conditions devoid of ions, as well as those involving 0.15 M NaCl and 3 M NaCl, exhibited an inability to pull down the Myc-ADAR1 protein. This observation may be attributed to the insufficient length of the DNA construct to effectively capture the 110 and 150 kDa proteins (Figure 3C). However, Biotin-Z-DNA 2 successfully demonstrated ADAR1 pull-down capability when Z-DNA formation was induced under 3 M NaCl conditions (Figures 3D,E). It is worth noting that both Myc-ADAR1 p110 and p150 derivatives could be detected due to the alternative splicing. Intriguingly, TRF, representing the B-form of DNA, failed to elicit ADAR1 pull-down under these experimental conditions, as confirmed through ADAR1 and Myc immunoblotting. Notably, Biotin-Z-DNA 2 exhibited comparably weak binding to ADAR1 in the absence of ions, as opposed to the notable binding observed when induced by 3 M NaCl (Figure 3D,E). Despite the confirmation of automatic (CG)₆ sequence conversion to Z-DNA, even in the absence of ions, as indicated by Z22 antibody detection, the significance of 3 M NaCl-induced Z-DNA in ADAR1 binding remained evident (Figure 3D,E). This disparity is likely a consequence of the enhanced efficacy of Z-DNA transformation under conditions involving 3 M NaCl, coupled with the distinct specificity exhibited by ADAR1 and Z22 toward Z-DNA. Therefore, in the subsequent study, we largely utilized 3 M NaCl as the preferred condition for generating Z-DNA to target ADAR1.

Z-PROTAC-Induced ADAR1 Degradation in HeLa Cells.

To assess the degradative potential of Z-PROTAC on ADAR1 protein, we used the HeLa cell line as a representative cellular model. We synthesized a collection of 17 PROTACs, collectively designated as Z-PROTACs, through the conjugation of azide-modified DNA—serving as an ADAR1 ligand—with bicyclooctyne-containing compounds 1b–17b, acting as VHL E3 ligands, via the SPAAC reaction (Figure 4A). Subsequently, the click reaction was executed at room temperature over 16 h to generate the Z-PROTAC degraders 1c–17c, with the reaction efficiency meticulously monitored using PAGE analysis (Figure 4B). Optimal efficiency and robust yields, approximately 80%, were achieved for compounds 7c to 9c (Figure 4B). All of the conjugated ODNs were purified using a column system, followed by the addition of sterile 3 M NaCl to the purified ODNs to induce Z-DNA formation. The acquired Z-PROTACs 1c–17c were introduced into HeLa cells at 5 μg/mL via lipofectamine-mediated transfection, and cell lysates were collected for Western blot analysis. As demonstrated in Figure 4C, ADAR proteins exist in various isoforms; therefore, ADAR1, ADAR2, and ADAR3 containing the A to I deaminase and another Z-DNA binding protein, ZBP1, with a Z_α domain, need to be detected in this experimental condition as well.^40–42 Given the reported importance of Z_α and Z_β domains for Z-DNA binding, and the existence of distinct ADAR1 isoforms (Figure 4C), we conducted a specificity analysis of the Z-PROTACs to ascertain their capacity to degrade different isoforms. Encouragingly, Figure 4D reveals that Z-PROTACs 7c, 8c, and 9c exclusively induced the degradation of ADAR1 p110 and p150 isoforms, displaying pronounced specificity toward these ADAR1 protein variants over other isoforms and the ZBP1 protein, likely due to the differential presence of the Z-DNA-binding motif in different isoforms. Furthermore, HeLa cells expressing GFP were visualized post-transfection with 5 μg/mL vehicle, Z-DNA 2 (ODN), and 9c, with the latter showing evident anticancer efficacy relative to the control groups (Figure 4E). Additionally, we conducted comprehensive investigations of the time and dosage kinetics of ADAR1 p110 degradation, revealing a clear time- and dose-dependent response to 9c (Figure 4F). Importantly, rescue experiments employing VH032 (2.5 μM), MG132 (2.5 μM), and ODN (Z-DNA, 5 μg/mL) as competitive agents alongside 9c indicated that 9c-induced degradation likely operates in a VHL- and proteosome-dependent manner (Figure 4G).

Figure 4. Z-PROTAC targets ADAR1 for degradation.

(A) Schematic illustration of the SPAAC reaction between the VHL ligand and azide-modified Z-DNA for the synthesis of Z-PROTAC 1c - 17c. (B) Click efficiency of obtained 1c–17c detected by PAGE gel analysis. The click efficiency was determined and is indicated below the corresponding bands. (C) The gene constructs of different isoforms of ADAR include ADAR1 (p110 and p150), ADAR2, ADAR3, and ZBP1. ARG: arginine-rich domain; dsRBD: dsRNA-binding domain. (D) Screening of Z-PROTAC 1c–17c for targeted degradation of ADAR protein isoforms in HeLa cells. HeLa cells were treated with 5 μg/mL Z-PROTAC (1c–17c) for 24 h and harvested for Western blot analysis to assess the protein degradation of various ADAR isoforms and ZBP1. Relative protein levels were labeled with numbers below the indicated bands. (E) Induction of cell death by Z-PROTAC 9c in HeLa cells. HeLa cells expressing GFP were treated with ODN (Z-DNA) and 9c at 5 μg/mL for 24 h, and then, cell imaging was acquired to analyze the cell number. Scale bar: 100 μm. (F) Time- and dose-dependent analysis of Z-PROTAC 9c in HeLa cells at different time points and concentrations. (G) Competition experiments with VH032 (2.5 μM), MG132 (2.5 μM), and free ODN (Z-DNA, 5 μg/mL) with 9c for ADAR1 degradation.

Z-PROTAC Exhibits Anticancer Activity by Inducing ADAR1 Degradation.

We showed the specific degradation of ADAR1 by compounds 8c and 9c (structures in Figure 5A) in HeLa cells (Figure 4D). Our exploration was further extended to various cancer cell lines, including MDA-MB-436, MDA-MB-468, and MCF7 (Figure 5B). To comprehensively assess the antiproliferative effects of Z-PROTAC across diverse cancer cell lines, we conducted a series of assays, including cell growth curves, CCK-8 assays, and colony formation assays, as shown in Figure 5C–F. Upon transfection of different cell lines with 5 μg/mL Z-DNA ODN and 9c, our findings revealed a potent inhibitory effect of 9c on cancer cells in comparison to the reported ADAR1 inhibitors 8-Aza^11–14,43 and Z-DNA ODN, as evident from the cell growth curve depicted in Figure 5C. Notably, while normal cells (MRC5 and LF1) exhibited some degree of inhibition, this effect was relatively weaker than that observed in cancer cells (Figure S13). However, it is worth noting that 9c displayed cytotoxicity comparable to that of 8-Aza in the CCK-8 assay (Figure 5D). Further investigation is required to elucidate the activity of 9c. Subsequently, we performed a colony formation assay to gain deeper insights into the effect of Z-PROTAC on cancer cells (Figure 5E). Quantitative assessment of colony numbers, presented in Figure 5F, affirmed the substantial inhibition of the colony formation potential across various cancer cell lines by Z-PROTAC 9c. In contrast, 8-Aza and Z-DNA ODN exhibited a comparatively lower efficiency in this regard (Figure 5F). These findings collectively indicate that Z-PROTAC exhibits enhanced potency compared to inhibitor and Z-DNA ODN in relation to ADAR1.

Figure 5. ADAR1 degradation by Z-PROTAC efficiently induces cell death in tumor cells.

(A) The structures of compounds used for further detection included ODN (Z-DNA), ADAR1 inhibitor 8-Aza, Z-PROTAC 8c, and 9c. (B) 8c and 9c were used for targeted degradation of ADAR1 in MDA-MB-436, MDA-MB-468, and MCF7 cells. Cells were treated with 5 μg/mL Z-PROTAC for 24 h and harvested for Western blot analysis to assess ADAR1 protein degradation. (C) Effect of Z-PROTAC 9c on the proliferation of cancerous and noncancerous normal cells (green box) after 24 h. Cells were treated with 5 μg/mL of 9c for 3 days, and the cell number was counted daily. (D) IC₅₀ values of Z-PROTAC 9c in cancer cells. Cells were treated with the indicated concentrations of 9c for 72 h, and the cell viability was measured. (E) Z-PROTAC 9c inhibits colony formation in cancer cells. Cells were treated with 2.5 μg/mL Z-PROTAC 9c and 2.5 μM 8-Aza, and the colony number was calculated 2 weeks after treatment. (F) The quantification of the number of colonies of various cancer cell lines. Data were analyzed by one-way or two-way ANOVA with Tukey’s correction using GraphPad Prism software and are presented as mean ± SD. Differences were considered statistically significant at p < 0.05. NC: negative control was added with the same solvents as 9c. *, **, ****: p < 0.05, p < 0.01, p < 0.0001.

Z-PROTAC Modulates Both Apoptosis and Necroptosis Signaling in Cancer Cells.

First, as ADAR1 is also known as an oncoprotein in the cancer setting,^9,44–47 the expression levels of target proteins (ADAR1 p110, p150, and ZBP1 as controls) were assessed using Western blot analysis in both cancer and normal cells. Figure 6A illustrates the over-expression of ADAR1 protein isoforms in various cancer cell lines compared to normal cells. In addition to Western blotting, the mRNA levels of ADAR1 and ZBP1 were examined in various cell lines (Figure 6B). It has also been previously reported that ADAR1 inhibits the necroptosis pathway by inhibiting ZBP1 to downregulate RIPK3-mediated necroptosis in cancer.^9,42,48–50 Consequently, other marker proteins in necroptosis, such as RIPK1 and RIPK3, were monitored. While RIPK1 exhibited similar expression levels in both normal and cancer cells, in keeping with a previous report,⁵¹ RIPK3 expression was found to be absent in most of the cell lines derived from solid tumors, such as HeLa cells. However, we detected RIPK3 expression in the A375 cancer cell line and in normal cell lines, such as MRC5. Given that ADAR1 is implicated in both apoptosis and necroptosis pathways,^42,52 experimental assays were conducted in cancer and normal cell lines to verify whether Z-PROTAC induces downstream pathway alterations. Following treatment of various cancer and normal cells with 5 μg/mL vehicle, ODN, 8c, and 9c for 24 h, Western blot analysis was employed to assess the apoptosis marker proteins. Figure 6C depicts a significant decrease in total caspase 3 and a noticeable increase in cleaved PARP1 upon Z-PROTAC 8c or 9c treatment in cancer cell lines, with a slight increase in cleaved PARP1 observed in normal cells. Notably, necroptosis marker proteins, such as p-MLKL, exhibited a significant increase in cancer cells A375 following treatment with 8c or 9c, while such an effect was largely absent in normal cells, such as MRC5 (Figure 6D). The differential response of tumor versus normal cells to Z-PROTAC treatment in activating downstream MLKL pathway is possibly in part due to the differential expression pattern of ADAR1 isoforms in these cell lines. To this end, the overexpression of ADAR1 in tumor cells might confer an ‘oncogene addiction feature^53,54 for tumor cells to the ADAR1 oncoprotein, thus rendering them more sensitive to Z-PROTAC-induced ADAR1 degradation to trigger the downstream MLKL pathway. Based on these observations, we postulate that Z-PROTAC activates both apoptosis and necroptosis pathways in cancer cells, while only slightly triggering apoptosis in normal cells. The apoptosis in noncancerous normal cells also accounted for the induction of cell death by Z-PROTAC (Figures 6C, S14, and S15). Consequently, we conclude that Z-PROTAC-induced rewriting of the signaling pathway likely involves both apoptosis and necroptosis pathways in cancer cells, as illustrated in Figure 6E.

Figure 6.

Analysis of the functional impact of ODN (Z-DNA), 8c, and 9c on cell signaling in cancer cells compared to noncancerous normal cells. (A) The Western blot analysis of whole-cell lysates was obtained from the indicated cell lines for ADAR1 (p110 and p150), RIPK3, ZBP1, and RIPK1. (B) Kaplan–Meier analysis of ADAR1 and ZBP1 mRNA expression. (C) Assessment of the apoptotic pathway in cancer cells (HeLa, A375, and MDA-MB-231) and normal cells (LF1 and MRC5) upon treatment by Z-PROTAC. (D) Assessment of the necroptosis pathway in cancer cells (A375) and normal cells (MRC5) after treatment by Z-PROTAC. (E) Schematic representation of the mechanisms underlying apoptosis and necroptosis induced by Z-PROTAC in cancer cells.

CONCLUSION

In light of the prevailing advancements in DNA- or RNA-based proteolysis-targeting chimeras (PROTACs), there remains a substantial avenue for the exploration and expansion of DNA-based tools, especially those that exhibit heightened specificity. In contrast to the traditional A- or B-forms of DNA, our investigation focused on the utilization of Z-form DNA, a distinctive conformation that arises exclusively within precise nucleotide sequences and under critical conditions. This innovative pursuit has markedly enhanced the precision and targeting efficacy of DNA-based strategies. Further setting our approach apart, Z-DNA demonstrates a distinctive affinity for select cellular proteins containing the Z domain, including ADAR1 and ZBP1, both of which have pivotal implications in cancer biology and immunology. Our investigation has unearthed intriguing facets of this interaction. Notably, the interaction between Z-DNA and ADAR1 has been found to impede the function of ZBP1, resulting in the suppression of apoptotic cascades via the caspase pathway and the inhibition of necroptotic processes through the MLKL pathway within the context of cancer biology.^9,42,48–50 Concomitantly, ADAR1 binding exerts a regulatory influence on immune responses, acting as a negative modulator of intracellular innate immunity, chiefly by interfering with the MDA5/MAV5 axis, thereby fostering the progression of cancer cells.^49,55 Using the Z-PROTAC approach, we achieved the targeted degradation of ADAR1, while leaving ZBP1 unaffected, specifically within cancer cells. Moreover, Z-PROTAC manipulation has been shown to induce PANoptotic responses, encompassing both apoptosis and necroptosis, exclusively within cancerous cellular contexts as opposed to normal cells. However, the translation of Z-PROTAC into the clinical realm is underpinned by several formidable challenges. Notable among these are issues pertaining to cellular permeability, molecular stability, and the limited spectrum of viable delivery methods. Moreover, modifications of the DNA backbone are indispensable for future in vivo animal evaluation, thereby exacerbating the intricacies associated with clinical translation. While concerted efforts have been directed toward unraveling the mechanism of ADAR1-mediated signaling pathways, the immunological mechanisms that ensue subsequent to ADAR1 degradation warrant a deeper and more comprehensive exploration. Finally, the nondegradation of ZBP1 can be attributed to a triad of key factors: a) the protein abundance of ZBP1 is much lower than that of ADAR1 in HeLa cells, as shown in Figure 4D; b) the affinities of ADAR1 and ZBP1 toward Z-DNA are different, which leads to the less influence on ZBP1 by Z-PROTAC; and c) meticulous linker screening led to the identification of a specialized degrader, akin to the precedent set by CDK4/6 targeting agents.^56,57 Further structural analyses are imperative to substantiate these findings. Nonetheless, Z-PROTAC is a pioneering DNA-based PROTAC strategy that capitalizes on the distinctive structural attributes of Z-DNA to enhance the specificity of PROTAC-mediated protein targeting. The present study revealed the untapped potential of ADAR1, a well-documented oncoprotein, as a promising candidate for anticancer drug discovery via Z-PROTAC, both in cancer and immunology, signifying a promising avenue for further advancement.