Nuclease Resistance Research Articles

Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: A potential new tool for improving RNA-based plant protection

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either are not currently controlled by artificial means or have developed resistance against chemical pesticides. Long dsRNAs are capable of inducing RNAi in insects and are emerging as novel, highly selective alternatives for sustainable insect management strategies. However, there are significant challenges associated with RNAi efficacy in insects. In this study, we synthesized a range of chemically modified long dsRNAs in an approach to improve nuclease resistance and RNAi efficacy in insects. Our results showed that dsRNAs containing phosphorothioate modifications demonstrated increased resistance to southern green stink bug saliva nucleases. Phosphorothioate-modified and 2′-fluoro-modified dsRNA also demonstrated increased resistance to degradation by soil nucleases and increased RNAi efficacy in Drosophila melanogaster cell cultures. In live insects, we found chemically modified long dsRNAs successfully resulted in mortality in both stink bug and corn rootworm. These results provide further mechanistic insight into the dependence of RNAi efficacy on nucleotide modifications in the sense or antisense strand of the dsRNA in insects and demonstrate for the first time that RNAi can successfully be triggered by chemically modified long dsRNAs in insect cells or live insects.

Relative Nuclease Resistance of a DNA Aptamer Covalently Conjugated to a Target Protein.

A major obstacle to the therapeutic application of an aptamer is its susceptibility to nuclease digestion. Here, we confirmed the acquisition of relative nuclease resistance of a DNA-type thrombin binding aptamer with a warhead (TBA3) by covalent binding to a target protein in the presence of serum/various nucleases. When the thrombin-inhibitory activity of TBA3 on thrombin was reversed by the addition of the complementary strand, the aptamer was instantly degraded by the nucleases, showing that the properly folded/bound aptamer conferred the resistance. Covalently binding aptamers possessing both a prolonged drug effect and relative nuclease resistance would be beneficial for in vivo translational applications.

Hairpin-inserted cross-shaped DNA nanoprobe for ultrasensitive microRNA detection based on built-in target analogue cycle amplification

It remains technically challenging to develop a sensitive assay system to isothermally amplify the signal for miRNA detection because of its low abundance in tested sample, sequence similarities and existence in complex biological environments. In this study, using miRNA-21 as target model, a hairpin-inserted cross-shaped DNA nanoprobe (CP) with four functional arms is constructed for the ultrasensitive detection of miRNA via one-step built-in target analogue (BTA) cycle-mediated signal amplification. BTA is pre-locked in one arm of CP probe and inactive. In the presence of target miRNA, BTA can be unlocked and initiate an isothermal amplification process. Utilizing as-designed CP probe, miRNA-21 can be detected to down to 500 fM, and the linear response range spans over five orders of magnitude. The nonspecific signal is less than 1% upon nontarget miRNAs. CP probe exhibits ∼six times enhancement in resistance to nuclease degradation and no obvious degradation-induced fluorescence change is detected during the assay period. The recovery yield ranges from 98.2～105.5% in FBS solution. Because of the high sensitivity, desirable specificity, strong anti-interference ability and substantial increase in nuclease resistance, CP probe is a promising tool for the detection of miRNAs in a complex biological milieu.

Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides.

We previously reported that the formation of guanine-quadruplex (G4) structures provides phosphodiester oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG ODNs) with higher nuclease resistance and cellular uptake, thereby increasing their immunostimulation efficiency through TLR9 activation. CpG ODNs forming G4 structures (G4 CpG ODNs) are thus potential vaccine adjuvants against infectious diseases. However, the G4 structure changes topology depending on the surrounding environment. Recently, G4 ligands, which are small molecules that bind to G4 ODNs with high affinity, were reported to improve the stability of G4. In this study, we propose to increase the stability and function of G4 CpG ODNs using G4 ligands. We show the effects of two G4 ligands, named L2H2-6OTD (L2H2) and L2G2-2M2EG-6OTD (L2G2), on the topology, stability, and immunostimulatory properties of a monomeric hybrid-type G4 CpG ODN containing CpG motifs in the central loop, named GD3. We found that L2H2 helps maintain the hybrid G4 topology of GD3, whereas L2G2 induces parallel G4 formation. Both G4 ligands increase the thermodynamic and nuclease stability of GD3. However, only GD3 associated with L2H2 binds efficiently to TLR9 and evokes a higher immune response from mouse macrophage-like RAW264 cells. GD3 associated with L2G2 does not bind efficiently to TLR9 and elicits lower cytokine production. Our results demonstrate that the potential to enhance immunostimulatory properties depends on the ability of G4 ligands to maintain and stabilize the hybrid G4 of GD3. We anticipate that our findings will facilitate the development of more effective G4 CpG ODN-based vaccine adjuvants against infectious diseases.

Construction of Fast-Walking Tetrahedral DNA Walker with Four Arms for Sensitive Detection and Intracellular Imaging of Apurinic/Apyrimidinic Endonuclease 1.

Herein, a novel tetrahedral DNA walker with four arms was engineered to travel efficiently on the 3D-tracks via catalyzed hairpin assembly autonomously, realizing the sensitive detection and activity assessment as well as intracellular imaging of apurinic/apyrimidinic endonuclease 1 (APE1). In contrast to traditional DNA walkers, the tetrahedral DNA walker with the rigid 3D framework structure and nonplanar multi-sites walking arms endowed with high collision efficiency, showing a fast walking rate and high nuclease resistance. Impressively, the initial rate of the tetrahedral DNA walker with four arms was 4.54 times faster than that of the free bipedal DNA walker and produced a significant fluorescence recovery in about 40 min, achieving a sensitive detection of APE1 with a low detection limit of 5.54× 10-6 U/μL as well as ultrasensitive intracellular APE1 fluorescence activation imaging. This strategy provides a novel DNA walker for accurate identification of low-abundance cancer biomarker and potential medical diagnosis.

A versatile strategy for convenient circular bivalent functional nucleic acids construction.

Functional nucleic acids (FNAs), such as aptamers, nucleic acid enzymes and riboswitches play essential roles in various fields of life sciences. Tailoring of ingenious chemical moieties toward FNAs can enhance their biomedical properties and/or confer them with exogenic biological functions that, in turn, can considerably expand their biomedical applications, or even improve their clinical translations. Herein, we report the first example of a general chemical tailoring strategy that enables the divergent ligation of DNA sequences. By applying this technology, different types of aptamers and single-stranded nucleic acids of various lengths could be efficiently tailored to deliver the designed circular bivalent aptamers (CBApts) and cyclized DNA sequences with high yields. It is worth noting that CBApts exhibited significantly enhanced nuclease resistance, as well as considerably improved binding, targeting and tumor tissue enrichment abilities, which may pave the way for different investigations for biomedical purposes.

Rapid Determination of RNA Modifications in Consensus Motifs by Nuclease Protection with Ion-Tagged Oligonucleotide Probes and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry.

The reversible and substoichiometric modification of RNA has recently emerged as an additional layer of translational regulation in normal biological function and disease. Modifications are often enzymatically deposited in and removed from short (~5 nt) consensus motif sequences to carefully control the translational output of the cell. Although characterization of modification occupancy at consensus motifs can be accomplished using RNA sequencing methods, these approaches are generally time-consuming and do not directly detect post-transcriptional modifications. Here, we present a nuclease protection assay coupled with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to rapidly characterize modifications in consensus motifs, such as GGACU, which frequently harbor N6-methyladenosine (m6A). While conventional nuclease protection methods rely on long (~30 nt) oligonucleotide probes that preclude the global assessment of consensus motif modification stoichiometry, we investigated a series of ion-tagged oligonucleotide (ITO) probes and found that a benzylimidazolium-functionalized ITO (ABzIM-ITO) conferred significantly improved nuclease resistance for GGACU targets. After optimizing the conditions of the nuclease protection assay, we applied the ITO and MALDI-MS-based method for determining the stoichiometry of GG(m6A)CU and GGACU in RNA mixtures. Overall, the ITO-based nuclease protection and MALDI-MS method constitutes a rapid and promising approach for determining modification stoichiometries of consensus motifs.

Optimization of Automated Synthesis of Amide-Linked RNA

The recent FDA approval of several antisense and siRNA drugs illustrates the utility of nucleic acid chemical modifications, but numerous challenges remain for generalized nucleic acid therapeutics, urging the exploration of new modification strategies. Replacing backbone phosphates with amides has shown promise for enhancing siRNA activity, specificity, and nuclease resistance; however, amide-linked RNA has not been fully explored due to lengthy and low yielding manual amide coupling procedures. We have addressed this by automating the assembly of amide-linked RNA using an Expedite 8909 nucleic acid synthesizer and optimizing solid-phase synthesis conditions to achieve 91–95% yields in just 5 min of coupling time. The optimized protocol allowed synthesis of a 21-nucleotide-long siRNA guide strand having six consecutive amide linkages at the 3′-end with an overall yield of ∼1%. Our results show that the steric hindrance caused by bulky 2′-O protecting groups and steric hindrance of the solid support are the key optimization variables for improving the amide couplings.

Quantitative Analysis of In Situ Locked Nucleic Acid and DNA Competitive Displacement Events on Microspheres.

Synthetic analogues of natural oligonucleotides known as locked nucleic acids (LNAs) offer superior nuclease resistance and cytocompatibility for numerous scenarios ranging from in vitro detection to intracellular imaging of nucleic acids. While recognized as stronger hybridization partners than equivalent DNA residues, quantitative analysis of LNA hybridization activity is lacking, especially with respect to competitive displacement of the original hybridization partner by another oligonucleotide. In the current study, we perform in situ measurements of toehold-mediated competitive displacement of soluble, fluorescently labeled primary targets from probe strands immobilized on microspheres using high throughput flow cytometry. Both LNA-DNA hybrid sequences and pure DNA sequences are employed as the immobilized strands, as soluble, fluorescently labeled 9-base-long primary targets, and as unlabeled 15-base-long secondary or competitive targets. In addition to comparing chemically substituted and unsubstituted sequences, we explore the effects of mismatched primary targets and the location of the toehold segment within the primary duplexes on the resulting displacement profiles. The primary duplex or double-stranded probe (dsprobe) systems implemented here exhibited varying responses to unlabeled secondary targets ranging from surprisingly modest primary target displacement activity despite the presence of a six base-long nucleotide toehold segment at the dsprobe free end to distinctive displacement profiles sensitive to LNA substitutions and the placement of the toehold segment closer to the microsphere surface.

The minor groove topology of nucleic acid ligands plays a key role in nuclease resistance and targeting of HMGB1

The architectural DNA‐binding protein High Mobility Group B1 (HMGB1) binds distorted double stranded DNA (e.g. kinked, cruciform) in the nucleus with extremely high affinity to modulate nuclear homeostasis. HMGB1 is a multifunctional protein that also plays a pivotal role outside of the nucleus. It is now clear that HMGB1 can also function as a Damage Molecular Pattern Molecule. In this capacity, HMGB1 can act as a friend or foe to: i)bolster innate immunity to ward off pathogens or ii)exacerbate unwanted immune responses that worsen disease states (e.g. lupus, rheumatoid arthritis). Research shows that cruciform DNA [four‐way junctions (4WJs)] and single strand DNA (ssDNA) can effectively bind/sequester HMGB1. The 4WJs and ssDNA investigated contain natural and phosphorothioate (PS) bonds. PS bonds are introduced to enhance nuclease resistance. Our prior study shows that intramolecular 4WJs composed of natural and PS bonds possess enhanced resistance against the endo‐ and exonucleases. We hypothesize that the increase in nuclease resistance is due to a combination of three factors: i)restricting access to the helical termini (end‐capping), ii)the presence of PS bonds and iii)altered minor groove topology. The objective of this study is to more stringently investigate the potential connection between minor groove topology and nuclease binding. Here, three nucleases are used that cleave double and ssDNA: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). The nucleases bind within the minor groove to initiate hydrolysis via a non‐specific (DNase I) or specific manner (Exo III and T5 Exo). We hypothesize that nucleases do not bind strongly to nucleic acid substrates with altered (likely wider) minor groove dimensions. As a result, nucleic acids with wider minor grooves tend to have enhanced nuclease resistance. Circular dichroism (CD) studies are used to investigate the minor groove topology of intramolecular 4WJs and ssDNA. The minor groove binder 4’,6‐diamidino‐2‐phenylindole (DAPI) is used to estimate the gross minor groove topology of each nucleic acid ligand. The data clearly shows the DNA substrates with altered minor groove dimensions, as reflected by weakened DAPI binding interactions, possess enhanced nuclease resistance. DNA substrates with enhanced nuclease resistance will be investigated further to target the DNA‐binding cytokine, HMGB1.

Amplification of the Fluorescence Signal with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a Based on Au Nanoparticle-DNAzyme Probe and On-Site Detection of Pb2+ Via the Photonic Crystal Chip.

Although great headway has been made in DNAzyme-based detection of Pb2+, its adaptability, sensitivity, and accessibility in complex media still need to be improved. For this, we introduce new ways to surmount these hurdles. First, a spherical nucleic acid (SNA) fluorescence probe (Au nanoparticles-DNAzyme probe) is utilized to specifically identify Pb2+ and its suitability for precise detection of Pb2+ in complex samples due to its excellent nuclease resistance. Second, the sensitivity of Pb2+ detection is greatly enhanced via the use of a clustered regularly interspaced short palindromic repeats-Cas12a with target recognition accuracy to amplify the fluorescent signal upon the trans cleavage of the SNA (signal probe), and the limit of detection reaches as low as 86 fM. Third, we boost the fluorescence on photonic crystal chips with a bionic periodic arrangement by employing a straightforward detection device (smartphone and portable UV lamp) to achieve on-site detection of Pb2+ with the limit of detection as low as 24 pM. Based on the abovementioned efforts, the modified Pb2+ fluorescence sensor has the advantages of higher sensitivity, better specificity, accessibility, less sample consumption, and so forth. Moreover, it can be applied to accurately detect Pb2+ in complex biological or environmental samples, which is of great promise for widespread applications.

Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance

Post-transcriptional modifications have critical roles in tRNA stability and function1–4. In thermophiles, tRNAs are heavily modified to maintain their thermal stability under extreme growth temperatures5,6. Here we identified 2′-phosphouridine (Up) at position 47 of tRNAs from thermophilic archaea. Up47 confers thermal stability and nuclease resistance to tRNAs. Atomic structures of native archaeal tRNA showed a unique metastable core structure stabilized by Up47. The 2′-phosphate of Up47 protrudes from the tRNA core and prevents backbone rotation during thermal denaturation. In addition, we identified the arkI gene, which encodes an archaeal RNA kinase responsible for Up47 formation. Structural studies showed that ArkI has a non-canonical kinase motif surrounded by a positively charged patch for tRNA binding. A knockout strain of arkI grew slowly at high temperatures and exhibited a synthetic growth defect when a second tRNA-modifying enzyme was depleted. We also identified an archaeal homologue of KptA as an eraser that efficiently dephosphorylates Up47 in vitro and in vivo. Taken together, our findings show that Up47 is a reversible RNA modification mediated by ArkI and KptA that fine-tunes the structural rigidity of tRNAs under extreme environmental conditions.

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings.

The internal design of DNA nanostructures defines how they behave in different environmental conditions, such as endonuclease-rich or low-Mg2+ solutions. Notably, the inter-helical crossovers that form the core of such DNA objects have a major impact on their mechanical properties and stability. Importantly, crossover design can be used to optimize DNA nanostructures for target applications, especially when developing them for biomedical environments. To elucidate this, two otherwise identical DNA origami designs are presented that have a different number of staple crossovers between neighboring helices, spaced at 42- and 21-basepair (bp) intervals, respectively. The behavior of these structures is then compared in various buffer conditions, as well as when they are exposed to enzymatic digestion by DNase I. The results show that an increased number of crossovers significantly improves the nuclease resistance of the DNA origami by making it less accessible to digestion enzymes but simultaneously lowers its stability under Mg2+ -free conditions by reducing the malleability of the structures. Therefore, these results represent an important step toward rational, application-specific DNA nanostructure design.

Optimization of Short RNA Aptamers for TNBC Cell Targeting.

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2′Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.

Synthesis of 4′-C-(aminoethyl)thymidine and 4′-C-[(N-methyl)aminoethyl]thymidine by a new synthetic route and evaluation of the properties of the DNAs containing the nucleoside analogs

A gapmer-type antisense oligonucleotide is an oligonucleotide therapeutic that targets pathogenic mRNA directly, and it is expected to be a next-generation therapeutic drug. In this study, we designed and synthesized 4′-C-[(N-methyl)aminoethyl]-thymidine (4′-MAE-T) as a novel nucleoside analog and compared its properties with those of 4′-C-aminoethyl-thymidine (4′-AE-T). Furthermore, we designed a new synthetic route for 4′-C-aminoethyl-modified nucleosides and accomplished the synthesis of 4′-AE-T via a novel pathway with high total yield. DNA containing 4′-MAE-T analogs decreased RNA affinity slightly more than unmodified DNA and DNA containing 4′-AE-T, but significantly improved nuclease resistance compared to unmodified DNA in a solution containing bovine serum. In addition, the impact of 4′-MAE-T on DNA stability was higher than that of 4′-AE-T. Also, DNA containing these analogs can activate Escherichia coli-derived RNase H. Thus, 4′-MAE-T has the potential to be used in gapmer-type antisense nucleic acids as a suitable candidate for the development of therapeutic antisense oligonucleotides.

Altered Biodistribution and Hepatic Safety Profile of a Gapmer Antisense Oligonucleotide Bearing Guanidine-Bridged Nucleic Acids.

Guanidine-bridged nucleic acid (GuNA) is a novel 2',4'-bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA) analog containing cations that exhibit strong affinity for target RNA and superior nuclease resistance. In this study, Malat1 antisense oligonucleotide (ASO) bearing GuNA was evaluated for target knockdown (KD) activity and tolerability. The GuNA ASO did not interfere with RNase H recruitment on the target RNA/ASO heteroduplex and did show potent target KD activity in a skeletal muscle-derived cell line equivalent to that of the LNA ASO under gymnotic conditions, whereas almost no KD activity was observed in a hepatocyte-derived cell line. The GuNA ASO exhibited potent KD activity in various tissues; the KD activity in the skeletal muscle was equivalent with that of the LNA ASO, but the KD activities in the liver and kidney were clearly lower compared with the LNA ASO. In addition, despite the higher accumulation of the GuNA ASO in the liver, levels of aspartate aminotransferase and alanine aminotransferase with the GuNA ASO administration were not elevated compared with those induced by the LNA ASO. Our data indicate that the GuNA ASO is tolerable and exhibits unique altered pharmacological activities in comparison with the LNA ASO in terms of the relative effect between liver and skeletal muscle.

LNA-induced dynamic stability in a therapeutic aptamer: insights from molecular dynamics simulations

Modulation of structural and thermodynamic properties of nucleic acids with synthetic modifications is a promising area of research with possible applications in nanotechnology and nanotherapeutics. Locked nucleic acid (LNA) is one such modification in which the C4’ and O2’ atoms of the sugar moiety are connected through a methylene bridge. The LNA modified DNA aptamer RNV66, and its unmodified counterpart V7t1, both of which target the vascular endothelial growth factor (VEGF) implicated in oncogenic angiogenesis, have a G-rich tract that can fold into G-quadruplex structures. However, it is not understood why V7t1 has a polymorphic structure while its LNA modified counterpart RNV66 has a unique quadruplex fold with higher nuclease resistance, thermal stability and greater binding affinity for VEGF. In this work, we have performed extensive molecular dynamics simulations of RNV66 and V7t1 to study and compare the structural and dynamic consequences of the insertion of LNAs. It was observed that the increase in dynamic stability was significant in the presence of LNA residues and our protocol for combining different torsional parameters using OL15 for the DNA aptamer and parm99_LNA along with parmbsc0 and βOL15 for the LNAs nicely reproduced the experimentally observed conformational features of RNV66. Our observations would help in further theoretical studies in understanding the lack of frustration in the folding of the LNA modified aptamer and its higher affinity for VEGF. Communicated by Ramaswamy H. Sarma

Intraperitoneal administration of synthetic microRNA-214 elicits tumor suppression in an intraperitoneal dissemination mouse model of canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) has an extremely poor prognosis, making it necessary to develop new systemic treatment methods. MicroRNA-214 (miR-214) is one of many microRNAs (miRNA) that can induce apoptosis in HSA cell lines. Synthetic miR-214 (miR-214/5AE), which showed higher cytotoxicity and greater nuclease resistance than mature miR-214, has been developed for clinical application. In this study, we evaluated the effects of miR-214/5AE on stage 2 HSA in a mouse model. Mice intraperitoneally administered with miR-214/5AE (5AE group) had significantly fewer intraperitoneal dissemination tumor foci (median number: 72.5 vs. 237.5; p < 0.05) and a lower median foci weight (0.26g vs. 0.61g; p < 0.05). Mice in the 5AE group had increased expression of p53 and cleaved caspase-3, and a significantly lower proportion of Ki-67-positive cells, than those in the non-specific miR group. Notably, no significant side effects were observed. These results indicate that intraperitoneal administration of miR-214/5AE exhibits antitumor effects in an intraperitoneal dissemination mouse model of HSA by inducing apoptosis and suppressing cell proliferation. These results provide a basis for future studies on the antitumor effect of miR-214/5AE for HSA.

Acidic microenvironment triggered in situ assembly of activatable three-arm aptamer nanoclaw for contrast-enhanced imaging and tumor growth inhibition in vivo.

Rationale: Static assembled multivalent DNA nanotheranostics system have encountered some bottleneck problems in cancer imaging and therapy, such as poor penetration and high immunogenicity. Herein, we proposed an acidic tumor microenvironment triggered assembly of activatable multivalent nanodevice, called “three-arm aptamer nanoclaw” (TA-aptNC), assembled from three pH-responsive aptamer-decorated DNA monomers (pH-aptDMs) to facilitate their functions of imaging and therapy.Methods: The activated TA-aptNC was constructed by acidic microenvironment triggered in situ assembly of three pH-aptDMs. Designer pH-aptDM was established based on the combination of a pH-responsive i-motif switch and an assembly module with a cell membrane anchoring aptamer ligand. Acidic microenvironment-triggered the assembly of the TA-aptNC was characterized by electrophoresis and atomic force microscopic (AFM). The binding affinity and stability of the TA-aptNC, comparing the monovalent pH-aptDM, were studied via the flow cytometry and nuclease resistance assays. Acidic microenvironment-activated contrast-enhanced tumor imaging and significantly antitumor efficiency were evaluated in vitro and in vivo.Results: At physiological pH environment, the pH-aptDMs with excellent tissue permeability exited as inactivated and monodispersed small monomer. When encountering acidic microenvironment at the tumor site, pH-responsive i-motif switch liberated from the pH-aptDMs, and the three unconstrained DNA modules (DM1, DM2 and DM3) subsequently assembled in situ into the TA-aptNC. Compared with monovalent pH-aptDMs, the spontaneously formed activatable TA-aptNC afforded 2-fold enhanced binding ability via the multivalent effect, which further facilitated the selective tumor cell uptake capability, thus enabling a contrast-enhanced tumor imaging and significantly antitumor efficiency in vivo without systemic toxicity.Conclusions: The proposed strategy offers valuable insight into excavating an endogenous stimuli-triggered assembly of multivalent nanodevice for accurate diagnosis and efficient tumor therapy.