Nuclease Resistance Research Articles

Synthesis, Biophysical and Biological Evaluation of Splice-Switching Oligonucleotides with Multiple LNA-Phosphothiotriester Backbones.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2'-O-methyl phosphorothioate nucleotides for therapeutic applications. The nature of the triester alkyl group dictates the success of solid-phase synthesis; tertiary alkyl groups are lost during the P(III) oxidation step, whereas primary alkyl groups are partially cleaved during deprotection. In contrast, oligonucleotides containing secondary phosphothiotriester linkages are stable, and large numbers of triesters can be incorporated. The modified oligonucleotides have excellent duplex stability with complementary RNA and exhibit strong nuclease resistance. To expand synthetic flexibility, oligonucleotides containing multiple internal alkynyl phosphothiotriesters can be conjugated to lipids, carbohydrates, or small molecules through CuAAC click chemistry. Oligonucleotides containing LNA-THP phosphothiotriesters exhibit high levels of pre-mRNA splice switching in eukaryotic cells.

6589 Antisense Oligonucleotides for Reducing ACTH Secretion in a Model of Cushing's Disease

Abstract Disclosure: M.S. Varughese: None. H. Eltumi: None. E.H. Kemp: None. J.D. Newell-Price: None. Antisense Oligonucleotides for Reducing ACTH Secretion in a Model of Cushing’s Disease Background: Cushing’s disease (CD) is a multi-system disorder with high morbidity and mortality characterised by pathologically raised serum cortisol due excess expression of proopiomelanocortin (Pomc) and overproduction of ACTH by a pituitary corticotroph adenoma. Trans-sphenoidal adenomectomy is the treatment of choice, but there is a 30% recurrence rate over time. Advances in medical therapy are needed. Antisense oligonucleotides (ASOs) can silence mRNA translation by specific base-pairing. Locked nucleic acid (LNA) and 2’-O-methyl (OMe) ribose terminals make ASOs more effective by improving affinity and resistance to nuclease degradation. Aims: To investigate the effectiveness of LNA- and OMe-modified anti-Pomc ASOs at reducing ACTH secretion from murine adrenotropic tumour (AtT20) cells. To analyse the nuclease resistance of Pomc ASOs and their ability to drive an immune response. Methods: Two ASOs (ASO2 and ASO3) were designed against Pomc with phosphorothioate backbones modified with either LNA or OMe ribose terminals. These were used in lipofectamine-mediated cell transfections of AtT20 cells and ACTH in the culture medium measured by immunoassay. ASO stability was assessed by nuclease degradation assays and immunogenicity by cytokine ELISAs. Results: When compared with untreated AtT20 cells, LNA- and OMe-modified Pomc ASOs at 1 nM significantly suppressed ACTH secretion for up to 120 h and 96 h, respectively (n = 4; Unpaired t tests, all P values < 0.05). In contrast, control treatments, including scrambled and mismatched ASOs, did not cause a significant reduction. For comparison, 1 nM of ASO2-OMe, ASO2-LNA, ASO3-OMe and ASO3-LNA lowered ACTH at 24 h by 62%, 71%, 63% and 79%, respectively. Modified Pomc ASOs showed 14-31% degradation over a 120-min incubation period with 3’- and 5’-exonucleases, whilst equivalent unmodified Pomc ASOs were completely degraded. No secretion of IFN-α, IFN-β, TNF-α, IL-6 or IL-1β was detected following transfection of AtT20 cells with Pomc ASOs. Conclusions:Pomc ASOs caused significant reduction of ACTH secretion from AtT-20 cells. LNA-modified Pomc ASOs were more effective than those with OMe modifications, with neither stimulating a detectable immune response. These ASOs hold promise as a systemic therapy for Cushing’s disease. Presentation: 6/3/2024

Switchback RNA.

Intricately designed DNA and RNA motifs guide the assembly of robust and functional nucleic acid nanostructures. In this work, we present a globally left-handed RNA motif with two parallel strands called switchback RNA and report its assembly, biophysical, and biochemical characterization. Switchback RNA can be assembled in buffers without Mg2+, with improved thermal stability in buffers containing Mg2+, Na+, or K+. Differences in the binding of small molecules to switchback RNA and conventional RNA indicate design-based approaches for small molecule loading on RNA nanostructures. Further, the differential affinity of the two component strands in switchback or conventional duplex conformations allows for toehold-less strand displacement. Enzyme studies showed that the switchback and conventional RNA structures have similar levels of nuclease resistance. These results provide insights for employing switchback RNA as a structural motif in RNA nanotechnology. Our observation that RNA strands with switchback complementarity can form stable complexes at low magnesium concentrations encourages studies into the potential occurrence of switchback RNA in nature.

Next generation APOBEC3 inhibitors: Optimally designed for potency and nuclease stability.

APOBEC3 (or A3) enzymes have emerged as potential therapeutic targets due to their role in introducing heterogeneity in viruses and cancer, often leading to drug resistance. Inhibiting these enzymes has remained elusive as initial phosphodiester (PO) linked DNA based inhibitors lack stability and potency. We have enhanced both potency and nuclease stability, of 2'-deoxy-zebularine (dZ), substrate-based oligonucleotide inhibitors for two critical A3's: A3A and A3G. While replacing the phosphate backbone with phosphorothioate (PS) linkages increased nuclease stability, fully PS-modified inhibitors lost potency (1.4-3.7 fold) due to the structural constraints of the active site. For both enzymes, mixed PO/PS backbones enhanced potency (2.3-9.2 fold), while also vastly improving nuclease resistance. We also strategically introduced 2'-fluoro sugar modifications, creating the first nanomolar inhibitor of A3G-CTD2. With hairpin-structured inhibitors containing optimized PS patterns and LNA sugar modifications, we characterize the first single-digit nanomolar inhibitor targeting A3A. These extremely potent A3A inhibitors, were highly resistant to nuclease degradation in serum stability assays. Overall, our optimally designed A3 oligonucleotide inhibitors show improved potency and stability, compared to previous attempts to inhibit these critical enzymes, opening the door to realize the therapeutic potential of A3 inhibition.

Tuning assembled DNA nanoarchitecture into disassembled state to enhance accurate and ultrasensitive detection of KRAS G12D mutation

Cancer remains a formidable global health challenge, necessitating precise diagnostic tools. The KRAS gene, particularly the G12D mutation, holds pivotal clinical significance across various cancer subtypes. Existing DNA sensing techniques using single-stranded and double-stranded oligonucleotide probes are susceptible for enzymatic degradation, especially in complex biological samples like blood or serum. To address these challenges, we present an innovative approach that involves tuning assembled DNA nanoarchitecture into a disassembled state to accurate and ultrasensitive detection of the KRAS G12D mutation. Our method utilized a pair of multifunctional assembly hairpin probes to construct a DNA wire with both of a fluorophore labeled probe and a quencher labeled probe attached. Upon binding with the target DNA, the DNA wire undergoes gradual disassembly, separating all fluorophores from quenchers to significantly amplify fluorescence signals. This approach offers exceptional sensitivity and reaction kinetics, capable of detecting the KRAS G12D at femtomolar levels, and demonstrates outstanding specificity in discriminating single-base mutations. Furthermore, the DNA nanoarchitecture with well-demonstrated ability to nuclease resistance makes our method exhibits robustness in detecting KRAS G12D mutation in human serum samples. This innovative approach represents an advancement in cancer diagnostics, providing a promising tool for early disease diagnosis and biomedical study.

In-vivo Polyvalent Aptamer@Protein Based Nanocarrier with Synergistic Charge Effect for High Drug Loading, High Nuclease Resistance, and High Receptor Accessibility

<i>In-vivo</i> Polyvalent Aptamer@Protein Based Nanocarrier with Synergistic Charge Effect for High Drug Loading, High Nuclease Resistance, and High Receptor Accessibility

Two-layer cascaded catalytic hairpin assemblies based on locked nucleic acids for one-step and highly sensitive ctDNA detection.

Enzyme-free signal amplification of catalytic hairpin assembly (CHA) has enabled sensitive detection of circulating tumor DNA (ctDNA) in early clinical diagnosis. Conventional CHA strategies are restrained by the limited amplification efficiency of the single-stage system, and signal leakage from "breathing" influence and nuclease degradation. Here, we introduced two-layer cascaded locked nucleic acid (LNA)-assisted CHA circuits with the intelligent incorporation of LNA in the hairpins and reporter for the highly sensitive one-step detection of scarce ctDNA. The target-triggered upstream CHA reaction continuously generates hybrid products to catalyze the downstream CHA reaction for transducing the primary sensing event, and the released target and the produced hybrid product trigger the next catalytic reaction round at the same time and finally cascade to amplify the target ctDNA fluorescence output signal. Meanwhile, the stronger binding affinity of the LNA-DNA duplex endows the two-layer LNA-assisted CHA system with thermodynamic stability and nuclease resistance, and thus our designed system exhibits an excellent detection performance for target ctDNA in the range from 2 pM to 5 nM with a low detection limit of 0.6 pM. Significantly, the two-layer LNA-assisted CHA circuits have been successfully implemented for the feasible analysis of clinical samples. This two-layer cascaded LNA-assisted CHA strategy provides a promising high sensitivity tool for one-step detection of scarce ctDNA from complex clinical samples and would facilitate the reconfiguration of DNA circuit-based DNA nanotechnology for the precise analysis of other biomarkers in clinical research fields.

Improvement of the Nuclease Resistance and Immunostimulatory Activity of CpG Oligodeoxynucleotides by Conjugation to Sugar-Immobilized Gold Nanoparticles.

Adjuvants are essential substances for vaccines and immunotherapies that enhance antigen-specific immune responses. Single-stranded oligodeoxynucleotides containing an unmethylated CpG motif (CpG ODNs) are agonistic ligands for toll-like receptor 9 that initiate an innate immune response. They represent promising adjuvants for antiviral and antitumor immunotherapies; however, CpG ODNs have some limitations, such as poor nuclease resistance and low cell membrane permeability. Therefore, an effective formulation is needed to improve the nuclease resistance and immunostimulatory effects of CpG ODNs. Previously, we demonstrated the selective delivery of a small molecule toll-like receptor 7 ligand to immune cells through sugar-binding receptors using sugar-immobilized gold nanoparticles (SGNPs), which significantly enhanced the potency of the ligand. In this study, we examined SGNPs as carriers for partially phosphorothioated A-type CpG ODN (D35) and an entirely phosphorothioated B-type CpG ODN (K3) and evaluated the functionality of the sugar moiety on SGNPs immobilized with CpG ODN. SGNPs immobilized with D35 (D35-SGNPs) exhibited improved nuclease resistance and the in vitro and in vivo potency was significantly higher compared with that of unconjugated D35. Furthermore, the sugar structure on the GNPs was a significant factor in enhancing the cell internalization ability, and enhanced intracellular delivery of D35 resulted in improving the potencies of the A-type CpG ODN, D35. SGNPs immobilized with K3 (K3-SGNPs) exhibited significantly higher induction activities for both humoral and cellular immunity compared with unconjugated K3 and D35-SGNPs. On the other hand, sugar structure on K3-SGNPs did not affect the immunostimulatory effects. These results indicate that the sugar moiety on K3-SGNPs primarily functions as a hydrophilic dispersant for GNPs and the formulation of K3 to SGNPs contributes to improving the immunostimulatory activity of K3. Because our CpG ODN-SGNPs have superior induction activities for antigen-specific T-cell mediated immune responses, they may be effective adjuvants for vaccines and immunotherapies.

Synthesis and Properties of Oligonucleotides Containing LNA-Sulfamate and Sulfamide Backbone Linkages.

Oligonucleotides hold great promise as therapeutic agents but poor bioavailability limits their utility. Hence, new analogues with improved cell uptake are urgently needed. Here, we report the synthesis and physical study of reduced-charge oligonucleotides containing artificial LNA-sulfamate and sulfamide linkages combined with 2'-O-methyl sugars and phosphorothioate backbones. These oligonucleotides have high affinity for RNA and excellent nuclease resistance.

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

High-resolution crystal structure of RNA kinase ArK1 from G. acetivorans

U47 phosphorylation (Up47) is a novel tRNA modification discovered recently; it can confer thermal stability and nuclease resistance to tRNAs. U47 phosphorylation is catalyzed by Archaeal RNA kinase (Ark1) in an ATP-dependent manner. However, the structural basis for tRNA and/or ATP binding by Ark1 is unclear. Here, we report the expression, purification, and crystallization studies of Ark1 from G. acetivorans (GaArk1). In addition to the Apo-form structure, one GaArk1-ATP complex was also determined in atomic resolution and revealed the detailed basis for ATP binding by GaArk1. The GaArk1-ATP complex represents the only ATP-bound structure of the Ark1 protein. The majority of the ATP-binding residues are conserved, suggesting that GaArk1 and the homologous proteins share similar mechanism in ATP binding. Sequence and structural analysis further indicated that endogenous guanosine will only inhibit the activities of certain Ark1 proteins, such as Ark1 from T. kodakarensis.

Separation of the diastereomers of phosphorothioated siRNAs by anion-exchange chromatography under non-denaturing conditions

In recent years, several small interfering RNA (siRNA) therapeutics have been approved, and most of them are phosphorothioate (PS)-modified for improving nuclease resistance. This chemical modification induces chirality in the phosphorus atom, leading to the formation of diastereomers. Recent studies have revealed that Sp and Rp configurations of PS modifications of siRNAs have different biological properties, such as nuclease resistance and RNA-induced silencing complex (RISC) loading. These results highlight the importance of determining diastereomeric distribution in quality control. Although various analytical approaches have been used to separate diastereomers (mainly single-stranded oligonucleotides), it becomes more difficult to separate all of them as the number of PS modifications increases. Despite siRNA exhibits efficacy in the double-stranded form, few reports have examined the separation of diastereomers in the double-stranded form. In this study, we investigated the applicability of non-denaturing anion-exchange chromatography (AEX) for the separation of PS-modified siRNA diastereomers. Separation of the four isomers of the two PS bonds tended to improve in the double-stranded form compared to the single-stranded form. In addition, the effects of the analytical conditions and PS-modified position on the separation were evaluated. Moreover, the elution order of the Sp and Rp configurations was confirmed, and the steric difference between them, i.e., the direction of the anionic sulfur atom, appeared to be important for the separation mechanism in non-denaturing AEX. Consequently, all 16 peak tops of the four PS modifications were detected in one sequence, and approximately 30 peak tops were detected out of 64 isomers of six PS bonds, indicating that non-denaturing AEX is a useful technique for the quality control of PS-modified siRNA therapeutics.

Rational design of prodrug-type apoB-targeted siRNA for nuclease resistance improvement without compromising gene silencing potency

Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2'-O-MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2'-O-MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2'-O-methyl (2'-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2'-OMe and 2'-O-MDTM groups should be incorporated. siRNA bearing the 2'-O-MDTM and 2'-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum.

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.

4'-C-Acetamidomethyl-2'-O-methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs.

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

Exceptional Nuclease Resistance of DNA and RNA with the Addition of Small-Molecule Nucleobase Mimics.

Nucleases present a formidable barrier to the application of nucleic acids in biology, significantly reducing the lifetime of nucleic acid-based drugs. Here, we develop a novel methodology to protect DNA and RNA from nucleases by reconfiguring their supramolecular structure through the addition of a nucleobase mimic, cyanuric acid. In the presence of cyanuric acid, polyadenine strands assemble into triple helical fibers known as the polyA/CA motif. We report that this motif is exceptionally resistant to nucleases, with the constituent strands surviving for up to 1 month in the presence of serum. The conferred stability extends to adjacent non-polyA sequences, albeit with diminishing returns relative to their polyA sections due to hypothesized steric clashes. We introduce a strategy to regenerate stability through the introduction of free polyA strands or positively charged amino side chains, enhancing the stability of sequences of varied lengths. The proposed protection mechanism involves enzyme failure to recognize the unnatural polyA/CA motif, coupled with the motif's propensity to form long, bundling supramolecular fibers. The methodology provides a fundamentally new mechanism to protect nucleic acids from degradation using a supramolecular approach and increases lifetime in serum to days, weeks, or months.

DNA mechanocapsules for programmable piconewton responsive drug delivery

The mechanical dysregulation of cells is associated with a number of disease states, that spans from fibrosis to tumorigenesis. Hence, it is highly desirable to develop strategies to deliver drugs based on the “mechanical phenotype” of a cell. To achieve this goal, we report the development of DNA mechanocapsules (DMC) comprised of DNA tetrahedrons that are force responsive. Modeling shows the trajectory of force-induced DMC rupture and predicts how applied force spatial position and orientation tunes the force-response threshold. DMCs functionalized with adhesion ligands mechanically denature in vitro as a result of cell receptor forces. DMCs are designed to encapsulate macromolecular cargos such as dextran and oligonucleotide drugs with minimal cargo leakage and high nuclease resistance. Force-induced release and uptake of DMC cargo is validated using flow cytometry. Finally, we demonstrate force-induced mRNA knockdown of HIF-1α in a manner that is dependent on the magnitude of cellular traction forces. These results show that DMCs can be effectively used to target biophysical phenotypes which may find useful applications in immunology and cancer biology.

Improved synthesis and polymerase recognition of 7-deaza-7-modified α-l-threofuranosyl guanosine analogs.

Threofuranosyl nucleic acid (TNA), an artificial genetic polymer known for its nuclease resistance and acid stability, has grown in popularity as a genetically-encoded material for applications in synthetic biology and biomedicine. TNA oligonucleotide synthesis requires enzymatic or solid phase synthesis pathways that rely on monomer building blocks that are not commercially available and can only be obtained by chemical synthesis. Here we present a synthetic route to 7-deaza-7-modified tGTP and phosphoramidite analogs that is operationally simpler than our previously described strategy. The new methodology offers an HPLC-free route to tGTP analogs that are recognized by engineered TNA polymerases and can be incorporated with continued TNA synthesis.

Self-assembly of DNA parallel double-crossover motifs.

DNA double-crossover motifs, including parallel and antiparallel crossovers, serve as the structural foundation for the creation of diverse nanostructures and dynamic devices in DNA nanotechnology. Parallel crossover motifs have unique advantages over the widely used antiparallel crossover design but have not developed as substantially due to the difficulties in assembly. Here we created 29 designs of parallel double-crossover motifs varying in hybridization pathways, central domain lengths, and crossover locations to investigate their assembly mechanism. Arrays were successfully formed in four distinct designs, and large tubular structures were obtained in seven designs with predefined pathways and central domains appoximately 16 nucleotides in length. The nanotubes obtained from parallel crossover design showed improved nuclease resistance than the ones from the antiparallel counterpart design. Overall, our study provides a basis for the development of generalized assembly rules of DNA parallel crossover systems and opens new opportunities for their potential use in biological systems.

A Tumor-Specific Cascade-Activating Smart Prodrug System for Enhanced Targeted Therapy.

Developing intelligently targeted drugs with low side effects is urgent for cancer treatment. Toward this goal, a tumor-specific cascade-activating smart prodrug system consisting of a G-quadruplex(G4)-modulated tumor-targeted DNA vehicle and a well-designed cellular stimuli-responsive ligand-drug conjugates (LDCs) is proposed. An original "donor-acceptor" binary fluorescent ligand, with ultrahigh affinity, brightness, and photostability, is engineered to tightly bind G4 structures and significantly improve the nuclease resistance of the DNA vehicle, which serves as a bridge contributing to the construction of the prodrug system, named ApG4/LDCs. Sodium nitroprusside and doxorubicin are loaded into ApG4/LDCs in one pot and generate nitric oxide and superoxide anion in response to cancer cellular environments, which in cascade generates peroxynitrite to cause DNA damage while promoting the self-monitored drug release to achieve enhanced targeted therapy. Such a cascade activation and self-reinforcement process is executed only when the prodrug system targets the tumor tissue followed by cell uptake, showing significant antitumor efficacy and greatly weakening the damage to normal tissues. Given the unique features, the innovative strategy for prodrug design may open a new door to precision disease treatment.