DNA Carrier Research Articles

Desiccated Cyanobacteria Serve As Efficient Plasmid DNA Carriers in Space Flight.

Effective transport of biological systems as cargo during space travel is a critical requirement to use synthetic biology and biomanufacturing in outer space. Bioproduction using microbes will drive the extent to which many human needs can be met in environments with limited resources. Vast repositories of biological parts and strains are available to meet this need, but their on-site availability requires effective transport. Here, we explore an approach that allows DNA plasmids, ubiquitous synthetic biology parts, to be safely transported to the International Space Station and back to the Kennedy Space Center without low-temperature or cryogenic stowage. Our approach relied on the cyanobacterium Nostoc punctiforme PC73102, which is naturally tolerant to prolonged desiccation. Desiccated N. punctiforme was able to carry the non-native pSCR119 plasmid as intracellular cargo safely to space and back. Upon return to the laboratory, the extracted plasmid showed no DNA damage or additional mutations and could be used as intended to transform the model synbio host Escherichia coli to bestow kanamycin resistance. This proof-of-concept study provides the foundation for a ruggedized transport host for DNA to environments where there is a need to reduce equipment and infrastructure for biological parts stowage and storage.

Mirror-Image DNA Nanobox for Enhancing Environment Resistance of Nucleic Acid Probes.

Degradation and interference of the nucleic acid probes in complex biological environments like cytoplasm or body fluid can cause obvious false-positive signals and inefficient bioregulation in biosensing and biomedicine. To solve this problem, here, we proposed a universal strategy, termed L-DNA assembly mirror-image box-based environment resistance (L-AMBER), to protect nucleic acid probes from degradation and maintain their responsive activity in complex biological environments. Strand displacement reaction (SDR), aptamer, or DNAzyme-based D-DNA probes were encapsulated into an L-DNA box by using an L-D-L block DNA carrier strand to construct different kinds of L-AMBER probes. We proved that the L-DNA box could effectively protect the encapsulated D-DNA probes by shielding the interference of complex biological environments and only allowing small target molecules to enter for recognition. Compared with the D-AMBER probes, the L-AMBER probes can realize DNase I-assisted amplification detection of biological samples, low false-positive bioimaging, and highly efficient miRNA silence in living cells. Therefore, L-AMBER provided a universal and effective strategy for enhancing the resistance to environmental interference of nucleic acid probes in biosensing and biomedicine applications.

Unlocking the potential of beta-glucans: a comprehensive review from synthesis to drug delivery carrier potency.

Modernization and lifestyle changes have resulted in a number of diseases, including cancer, that require complicated and thorough treatments. One of the most important therapies is the administration of antibiotics and medicines. This is known as chemotherapy for cancer, and it is a regularly utilised treatment plan in which the medications used have negative side effects. This has resulted in extensive research on materials capable of delivering pharmaceuticals to particular targets over an extended period of time. Biopolymers have often been preferred as effective drug delivery carriers. Of these, β-glucan, a natural polysaccharide, has not been extensively studied as a drug delivery carrier, despite its unique properties. This review discusses the sources, extraction techniques, structures, and characteristics of β-glucan to provide an overview. Furthermore, the different methods employed to encapsulate drugs into β-glucan and its role as an efficient drug, SiRNA and Plasmid DNA carrier have been elaborated in this article. The capacity of β-glucan-based to specifically target and alter tumour-associated macrophages, inducing an immune response ultimately resulting in tumour suppression has been elaborated. Finally, this study aims to stimulate further research on β-glucan by thoroughly describing its many characteristics and demonstrating its effectiveness as a drug delivery vehicle.

Development of Bivalent Aptamer-DNA Carrier-Doxorubicin Conjugates for Targeted Killing of Esophageal Squamous Cell Carcinoma Cells.

Esophageal cancer ranks the seventh in cancer incidence and the sixth in cancer death. Esophageal squamous cell carcinoma (ESCC) accounts for approximately 90% of the total cases of esophageal cancer. Chemotherapy is the most effective drug-based method for treatment of esophageal cancer. However, severe side effects of traditional chemotherapy limit its treatment efficacy. Targeted chemotherapy can deliver chemotherapeutic drugs to cancer cells and specifically kill these cells with reduced side effects. In the work, the bivalent aptamer-DNA carrier (BAD) was designed by using an ESCC cell-specific aptamer as the recognition molecule and a GC base-rich DNA sequence as the drug carrier. With doxorubicin (Dox) as chemotherapeutic drugs, the bivalent aptamer-DNA-Dox conjugate (BADD) was constructed for targeted killing of ESCC cells. Firstly, the truncated A2(35) aptamer with a retained binding ability was obtained through optimization of an intact A2(80) aptamer and was used to fuse with DNA carrier sequences for constructing the BAD through simple DNA hybridization. The results of gel electrophoresis and flow cytometry analysis showed that the BAD was successfully constructed and had a stronger binding affinity than monovalent A2(35). Then, the BAD was loaded with Dox drugs to construct the BADD through noncovalent intercalation. The results of fluorescence spectra and flow cytometry assays showed that the BADD was successfully constructed and can bind to target cells strongly. Confocal imaging further displayed that the BADD can be specifically internalized into target cells and release Dox. The results of CCK-8 assays, Calcein AM/PI staining, and wound healing assays demonstrated that the BADD can specifically kill target cells, but not control cells. Our results demonstrate that the developed BADD can specifically deliver doxorubicin to target ESCC cells and selectively kill these cells, offering a potentially effective strategy for targeted chemotherapy of ESCC.

Purinosomes and Purine Metabolism in Mammalian Neural Development: A Review.

Neural stem/progenitor cells (NSPCs) in specific brain regions require precisely regulated metabolite production during critical development periods. Purines-vital components of DNA, RNA, and energy carriers like ATP and GTP-are crucial metabolites in brain development. Purine levels are tightly controlled through two pathways: de novo synthesis and salvage synthesis. Enzymes driving de novo pathway are assembled into a large multienzyme complex termed the "purinosome." Here, we review purine metabolism and purinosomes as spatiotemporal regulators of neural development. Notably, around postnatal day 0 (P0) during mouse cortical development, purine synthesis transitions from the de novo pathway to the salvage pathway. Inhibiting the de novo pathway affects mTORC1 pathway and leads to specific forebrain malformations. In this review, we also explore the importance of protein-protein interactions of a newly identified NSPC protein-NACHT and WD repeat domain-containing 1 (Nwd1)-in purinosome formation. Reduced Nwd1 expression disrupts purinosome formation, impacting NSPC proliferation and neuronal migration, resulting in periventricular heterotopia. Nwd1 interacts directly with phosphoribosylaminoimidazole-succinocarboxamide synthetase (PAICS), an enzyme involved in de novo purine synthesis. We anticipate this review will be valuable for researchers investigating neural development, purine metabolism, and protein-protein interactions.

Combining the potency of a neoantigen-expressing DNA vector, an anti-CTLA-4 antibody, and an attenuated poxvirus in a personalized immunization platform: Preclinical studies of ODI-2001.

e14674 Background: Immune checkpoint inhibitors (ICI) have transformed cancer treatment. Their impressive effectiveness is crucially dependent on pre-existing neoepitope-specific immune responses. ODI-2001 aims at increasing the immune response against tumors by combining a DNA expressing multiple neoepitopes of the tumor, a MVA vaccinia virus as physiologic adjuvant and DNA carrier, and an anti-CTLA4 antibody as amplifier of the immune response. Methods: ODI-2001 was administered by repeated subcutaneous injections after tumor cells implantation in mice melanoma model B16F10 and colorectal cancer model CT26, and both before and after implantation in breast cancer model 4T1. In B16F10 model combination of ODI-2001 with anti-PD1 was assessed also. Neoepitopes were chosen from literature in B16F10 and CT26 models. In 4T1 model they were predicted using a dedicated neoepitope prediction algorithm (ImmunoEngine, myNEO, Gent, Belgium). Tumor volume and mice survival were assessed over time. Tumors were also analysed by immunofluorescence for immune cell infiltration. Each experiment received approval from the ethics committee for animals. Results: A significant improvement in survival was observed in mice treated with ODI-2001 in B16F10, and CT26 syngeneic mice models. In B16F10 the combination of ODI-2001 with anti-PD1 antibody demonstrated synergistic activity and a significant improvement in survival, while anti-PD1 alone or combined with anti-CTLA-4 failed to show any therapeutic effect (ODI-2001 + anti-PD1 vs. anti-CTLA-4 + anti-PD1 p<0.01). ODI-2001 treatment was associated with tumor rejections in the three animal models. In 4T1 model the ImmunoEngine epitope prediction algorithm demonstrated an improved efficacy as compared to a set of neoepitopes chosen from the literature. In B16F10, following immunization with ODI-2001, an increased CD8 immune cell infiltration of the tumor was documented. ODI-2001 was well tolerated by the treated animals. Conclusions: These data demonstrate a significant antitumoral activity of ODI-2001 as a neoantigen immunization platform across different preclinical cancer models, both in monotherapy and combined with anti-PD1. ODI-2001 represents a promising option to induce de novo cancer-specific immune responses, potentially increasing the proportion of patients responding to immunotherapy treatments. Based on this preclinical program a phase I with ODI-2001 in patients with advanced colorectal carcinoma is currently under preparation.

A phase II study evaluating the effect of IMNN-001 on second-look laparoscopy (SLL) when administered in combination with bevacizumab (BEV) and neoadjuvant chemotherapy (NACT) in patients newly diagnosed with advanced epithelial ovarian cancer (EOC).

TPS5633 Background: IMNN-001, an interleukin-12 (IL-12) DNA plasmid formulated with a DNA carrier, polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, has been shown to exhibit a favorable benefit/risk ratio when administered intraperitoneally (IP), in combination with intravenous platinum-taxane chemotherapy, up to doses of 100 mg/m2 in women with advanced EOC in earlier Phase I & II studies. The 201-21-202 study (NCT05739981) is a multi-center, randomized, open-label phase I/II study which hypothesizes the addition of IMNN-001 to neoadjuvant & adjuvant carboplatin-paclitaxel and bevacizumab (chemo + BEV) reduces the risk of histologically documented minimal residual disease (MRD) as determined by SLL by 50%. Methods: Patients with newly diagnosed stage III and IV high grade serous EOC who are candidates for NACT will be enrolled into the trial. Initial safety lead-in will evaluate at least 6 patients randomized to the experimental arm. Upon confirmation of safety by the DSMB, a target of ~50 patients will be randomized 1:1 to receive either chemo + BEV with or without IMNN-001. Patients receive at least 4 cycles of NACT followed by interval cytoreductive surgery (ICS), and 3 additional adjuvant cycles of study treatments will be administered. BEV will be included with each cycle except cycle 1, the last cycle of NACT immediately preceding ICS, and the first cycle of adjuvant chemotherapy. In the experimental arm, IMNN-001 will be administered on C1D15 and continue weekly though the last cycle of adjuvant therapy. Following adjuvant therapy, all patients will undergo SLL with peritoneal washing and multiple biopsies. Pathologic detection of residual tumor in any biopsies or peritoneal cytology constitutes presence of MRD. All patients will receive maintenance therapy with Olaparib + BEV, IMNN-001 + BEV, or BEV alone based on their homologous recombination deficiency status and treatment randomization. Translational:All subjects will undergo biospecimen collection and organoid preparation prior to treatment, at the time of ICS and SLL for comprehensive longitudinal genomic, circulating tumor DNA, immune, and microbiome profiling. Subjects in the experimental arm will also undergo peritoneal lavage and collection of fluid and cells for assessment of the peritoneal tumor environment. Endpoints: The primary endpoint is the rate of MRD at SLL. Secondary endpoints include progression-free survival, overall survival, objective response rate, chemotherapy response score, surgical response, and serologic response. All patients will be evaluated for safety. Status: The study is actively enrolling and is in the safety lead-in portion. We plan to enlist approximately 4 sites in USA. Clinical trial information: NCT05739981 .

Self-assembly of a AuNPs/Ti3C2 MXene hydrogel for cascade amplification of microRNA-122 biosensing.

A three-dimensional (3D) self-assembled AuNPs/Ti3C2 MXene hydrogel (AuNPs/Ti3C2 MXH) nanocomposite was prepared for the fabrication of a novel microRNA-122 electrochemical biosensor. The 3D hydrogel structure was gelated from two-dimensional MXene nanosheets with the assistance of graphite oxide and ethylenediamine. MXene hydrogels supported the in situ formation of Au nanoparticles (AuNPs) that predominantly exploring the (111) facet, and these AuNPs are utilized as carriers for hairpin DNA (hpDNA) probes, facilitating DNA hybridization. MXene acted as both a reductant and stabilizer, significantly improving the electrochemical signal. In addition, the conjugation of PAMAM dendrimer-encapsulated AuNPs and H-DNA worked as an ideal bridge to connect targets and efficient electrochemical tags, providing a high amplification efficiency for the sensing of microRNA-122. A linear relationship between the peak currents and the logarithm of the concentrations of microRNA-122 from 1.0 × 10-2 to 1.0 × 102 fM(I = 1.642 + 0.312 lgc, R2 = 0.9891), is obtained. The detection limit is 0.8 × 10-2 fM (S/N = 3). The average recovery for human serum detection ranged from 97.32 to 101.4% (RSD < 5%).

A Novel Carrier Based on DNA and Exosome to Sustained Release of 5‐Fluorouracil Anticancer Drug

AbstractThe development of effective drug delivery systems is crucial for improving cancer treatment outcomes. A promising carrier using a combination of DNA and exosomes (EXO) was synthesized to sustain the release of the powerful anticancer drug 5‐fluorouracil (5‐FU). The carrier was prepared by encapsulating 5‐FU‐loaded exosomes (5‐FU.EXO) in the heated DNA carriers (5‐FU.EXO@DNA). Alteration of the DNA structure was studied using molecular dynamics. The physicochemical properties of the carriers were characterized using fourier‐transform infrared spectroscopy (FTIR), field‐emission scanning electron microscopy (FESEM), and dynamic light scattering (DLS). The 5‐FU.EXO@DNA carrier exhibited good stability and biocompatibility, with a uniform size distribution (709±269 nm) and a zeta potential of +312 mV. The interaction between 5‐FU and DNA was confirmed using molecular docking. In vitro release studies showed that both 5‐FU@DNA and 5‐FU.EXO@DNA carriers achieved sustained release of 5‐FU for 576 h, with cumulative releases of approximately 83 % and 89 %, respectively. The 5‐FU release profile showed 40 % release over 86 h. The release rate and other kinetic parameters were also determined. Furthermore, the release kinetics followed by the Korsmeyer‐Peppas model and the diffusion mechanism plays a significant role in controlling the release process.

Oligosarcosine Conjugation of Arginine-Rich Peptides Improves the Intracellular Delivery of Peptide/pDNA Complexes.

Cell-penetrating peptides (CPPs), for example, arginine (Arg) rich peptides, are used for the intracellular delivery of nucleic acids. In this study, oligosarcosine-conjugated Arg-rich peptides were designed as plasmid DNA (pDNA) carriers, and the physicochemical parameters and transfection efficiency of the peptide/pDNA complexes were evaluated. Oligosarcosine with different lengths were conjugated to a base sequence composed of arginine and α-aminoisobutyric acid (Aib) [(Aib-Arg-Arg)3]. Oligosarcosine conjugation inhibited the aggregation of the complexes after mixing with pDNA, shielded the positive charge of the complexes, and provided efficient pDNA transfection in cultured cells. The efficiency of the pDNA transfection was improved by varying the length of the oligosarcosine moiety (10-15 units were optimal). The cellular uptake efficiency and intracellular distribution of pDNA were the same regardless of oligosarcosine conjugation. These results implied that intracellular processes, including the decondensation of pDNA, contributed to the efficiency of the protein expression from pDNA. This study demonstrated the advantages of oligosarcosine conjugation to Arg-rich CPPs and provided valuable insight into the future design of CPPs.

Evaluation of DNA extraction methods for molecular traceability in cold pressed, solvent extracted and refined groundnut oils

Groundnut oil (GNO)/ peanut oil is one of the agro-food products with great economic value and hence an attractive target for adulteration and mislabeling. Simple Sequence Repeats (SSR) are markers of choice for DNA fingerprinting studies as they exhibit high polymorphism due to variable number of repeats. Hence, this study was designed to evaluate and optimized a method for DNA isolation from groundnut oil and study the possibility of using the isolated DNA for molecular traceability using SSR markers. Four methods to isolate DNA from groundnut oil were evaluated. All the four methods were modified CTAB protocols, but differed in procedures forextraction, buffer compositions, amount of oil used and DNA carriers. For molecular traceability of oils, extraction and recovery of DNA from edible oil is a key step, especially in refined oils. A method that employed DNA enrichment prior to extraction with CTAB buffer yielded amplifiable DNA from cold pressed GNO, crude hexaneextracted GNO and refined GNO. The optimized method for isolation of DNA from groundnut oil is simple, efficient, less costly and reproducible when compared to chromatography and spectroscopy based techniques.

Evaluation of the Antitumor Activity of Quaternary Ammonium Surfactants.

Quaternary ammonium surfactants, due to their diverse chemical structure and their biological properties, can be used in medicine as DNA carriers, disinfectants, and antimicrobial and antitumor agents. In this study, using melanoma A375, colon adenocarcinoma HT-29 and normal human dermal fibroblast (NHDF) cells, we tested the hypothesis that the quaternary ammonium surfactants 2-dodecanoyloxyethyl)trimethylammonium bromide (DMM-11), 2-dodecanoyloxypropyl)trimethylammonium bromide (DMPM-11) and 2-pentadecanoyloxymethyl)trimethylammonium bromide (DMGM-14) act selectively against cancer cells. The results showed that these compounds led to the initiation of the apoptotic process of programmed cell death, as evidenced by the ratio of the relative expression of Bax protein to Bcl-2. The encapsulation of surfactants in liposomes allowed lower concentrations to be used. Moreover, encapsulation reduced their toxicity towards non-cancerous cells. The anticancer efficiency and apoptotic effect of the liposomal formulations with surfactants (DMM-11, DMPM-11 and DMGM-14) were higher than those of surfactant-free liposomes. Therefore, quaternary ammonium surfactant-loaded liposomes show significant potential as delivery vehicles for the treatment of melanoma and colon cancers. The use of nano-formulations offers the advantage of optimizing quaternary ammonium surfactant delivery for improved anticancer therapy.

Salmon sperm DNA increases sample recovery from cotton swabs

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001–1–10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

DNA Carrier-Assisted Molecular Ping-Pong in an Asymmetric Nanopore.

Nanopore analysis relies on ensemble averaging of translocation signals obtained from numerous molecules, requiring a relatively high sample concentration and a long turnaround time from the sample to results. The recapture and subsequent re-reading of the same molecule is a promising alternative that enriches the signal information from a single molecule. Here, we describe how an asymmetric nanopore improves molecular ping-pong by promoting the recapture of the molecule in the trans reservoir. We also demonstrate that the molecular recapture could be improved by linking the target molecule to a long DNA carrier to reduce the diffusion, thereby achieving over 100 recapture events. Using this ping-pong methodology, we demonstrate its use in accurately resolving nanostructure motifs along a DNA scaffold through repeated detection. Our method offers novel insights into the control of DNA polymer dynamics within nanopore confinement and opens avenues for the development of a high-fidelity DNA detection platform.

Crosslinked chitosan microparticles as a safe and efficient DNA carrier for intranasal vaccination against cutaneous leishmaniasis

Intranasal (i.n.) vaccination with adjuvant-free plasmid DNA encoding the leishmanial antigen LACK (LACK DNA) has shown to induce protective immunity against both cutaneous and visceral leishmaniasis in rodents. In the present work, we sought to evaluate the safety and effectiveness of d,l-glyceraldehyde cross-linked chitosan microparticles (CCM) as a LACK DNA non-intumescent mucoadhesive delivery system. CCM with 5 μm of diameter was prepared and adsorbed with a maximum of 2.4 % (w/w) of DNA with no volume alteration. Histological analysis of mouse nostrils instilled with LACK DNA / CCM showed microparticles to be not only mucoadherent but also mucopenetrant, inducing no local inflammation. Systemic safeness was confirmed by the observation that two nasal instillations one week apart did not alter the numbers of bronchoalveolar cells or blood eosinophils; did not alter ALT, AST and creatinine serum levels; and did not induce cutaneous hypersensitivity. When challenged in the footpad with Leishmania amazonensis, mice developed significantly lower parasite loads as compared with animals given naked LACK DNA or CCM alone. That was accompanied by increased stimulation of Th1-biased responses, as seen by the higher T-bet / GATA-3 ratio and IFN-γ levels. Together, these results demonstrate that CCM is a safe and effective mucopenetrating carrier that can increase the efficacy of i.n. LACK DNA vaccination against cutaneous leishmaniasis.

Development of potent unmethylated CpG DNA hydrogel by introducing i-motifs into long single-stranded DNA

Unmethylated cytosine-phosphate-guanine (CpG) DNA is recognized by Toll-like receptor 9, expressed in the endosomes of immune cells, and induces the secretion of proinflammatory cytokines. CpG DNA is, therefore, expected to be used as vaccine adjuvants, but there are many obstacles for its therapeutic application, such as poor cellular uptake and biostability. Long single-stranded DNA (lssDNA) synthesized by rolling circle amplification can be a useful delivery carrier for CpG DNA because of its cellular uptake efficiency, but the immunostimulatory effect is transient because it is easily degraded in endosomes. To improve its stability, we constructed lssDNA which forms hydrogel by i-motifs in an acidic environment mimicking endosome, and incorporated CpG DNA into lssDNA (i-CpG-lssDNA). We synthesized lssDNA containing the optimized i-motif sequence, and confirmed the formation of a DNA hydrogel in an acidic environment. The i-CpG-lssDNA elicited a potent proinflammatory cytokine production in murine macrophages, compared to CpG DNA-containing lssDNA without i-motifs. Consistently, its intradermal administration induced potent inflammatory cytokines at the regional lymph nodes. These results suggested that i-CpG-lssDNA could serve as a novel type of adjuvant for the induction of a potent immune response.

Advancements in Nanogels for Enhanced Ocular Drug Delivery: Cutting-Edge Strategies to Overcome Eye Barriers.

Nanomedicine in gel or particle formation holds considerable potential for enhancing passive and active targeting within ocular drug delivery systems. The complex barriers of the eye, exemplified by the intricate network of closely connected tissue structures, pose significant challenges for drug administration. Leveraging the capability of engineered nanomedicine offers a promising approach to enhance drug penetration, particularly through active targeting agents such as protein peptides and aptamers, which facilitate targeted release and heightened bioavailability. Simultaneously, DNA carriers have emerged as a cutting-edge class of active-targeting structures, connecting active targeting agents and illustrating their potential in ocular drug delivery applications. This review aims to consolidate recent findings regarding the optimization of various nanoparticles, i.e., hydrogel-based systems, incorporating both passive and active targeting agents for ocular drug delivery, thereby identifying novel mechanisms and strategies. Furthermore, the review delves into the potential application of DNA nanostructures, exploring their role in the development of targeted drug delivery approaches within the field of ocular therapy.

θ-Nanopipette for Single-Cell Resistive-Pulse Profiling of DNA Repair Proteins Accompanied by Drug Evaluation.

Single-cell analysis of the DNA repair protein is important but remains unachieved. Exploration of nanopipettte technologies in single-cell electroanalysis has recently seen rapid growth, while the θ-nanopipette represents an emerging technological frontier with its potential largely veiled. Here a θ-nanopipette is first applied for single-cell resistive-pulse sensing (RPS) of the important DNA repair protein O6-alkylguanine DNA alkyltransferase (hAGT). The removal of alkyl mutations by hAGT could restore the damaged aptamer linking with a structural DNA carrier, allowing the selective binding of the aptamer to thrombin with precisely matched size to produce distinct RPS signals when passing through the orifice. Kinetic analysis of hAGT repair was studied. Meanwhile, the device shows the simultaneous on-demand infusion of inhibitors to inactivate the hAGT activity, indicative of its potential in drug screening for enhanced chemotherapy. This work provides a new paradigm for θ-nanopipette-based single-cell RPS of a DNA repair protein accompanied by drug evaluation.

Synthesis of length-tunable DNA carriers for nanopore sensing.

Molecular carriers represent an increasingly common strategy in the field of nanopore sensing to use secondary molecules to selectively report on the presence of target analytes in solution, allowing for sensitive assays of otherwise hard-to-detect molecules such as small, weakly-charged proteins. However, existing carrier designs can often introduce drawbacks to nanopore experiments including higher levels of cost/complexity and carrier-pore interactions that lead to ambiguous signals and elevated clogging rates. In this work, we present a simple method of carrier production based on sticky-ended DNA molecules that emphasizes ease-of-synthesis and compatibility with nanopore sensing and analysis. In particular, our method incorporates the ability to flexibly control the length of the DNA carriers produced, enhancing the multiplexing potential of this carrier system through the separable nanopore signals they could generate for distinct targets. A proof-of-concept nanopore experiment is also presented, involving carriers produced by our method with multiple lengths and attached to DNA nanostructure targets, in order to validate the capabilities of the system. As the breadth of applications for nanopore sensors continues to expand, the availability of tools such as those presented here to help translate the outcomes of these applications into robust nanopore signals will be of major importance.

Translocation of Proteins through Solid-State Nanopores Using DNA Polyhedral Carriers.

The detection of biomolecules at the single molecule level has important applications in the fields of biosensing and biomedical diagnosis. The solid-state nanopore (SS nanopore) is a sensitive tool for detecting single molecules because of its unique label-free and low sample consumption properties. SS nanopore translocation of small biomolecules is typically driven by an electronic field force and is thus influenced by the charge, shape, and size of the target molecules. Therefore, it remains challenging to control the translocation of biomolecules through SS nanopores, particularly for different proteins with complex conformations and unique charges. Toward this problem, a DNA polyhedral carrier coating strategy to assist protein translocation through SS nanopores is developed, which facilitates target protein detection. The current signal-to-noise ratios are improved significantly using this DNA carrier loading strategy. The proposed method should aid the detection of proteins, which are difficult to translocate through nanopores. This coating-assisted method offers a wide range of applications for SS nanopore detection and promotes the development of single-molecule detection.