Nucleic Acid Aptamers Research Articles

Size and Charge Dual‐Switchable Nanoparticles for Achieving Chemosensitization and Immune Infiltration against Pancreatic Ductal Adenocarcinoma

AbstractPancreatic ductal adenocarcinoma (PDAC), one of the most challenging cancers, is uniquely characterized by a biological barrier composed of multiple components in the extracellular matrix, preventing penetration of chemotherapeutic agents and hindering clinical drug treatment. Collagen I arrangement is critical. In this study, gemcitabine is chosen as the chemotherapeutic agent and dasatinib as the collagen‐I‐disrupting agent. When collagen I arrangement is disrupted, not only the chemotherapeutic drug enters the tumor area better, but the infiltration of CD8+ T cells into the tumor also increases. Therefore, a hypoxia‐responsive linker‐crosslinked polymer, DGD/L@GBI, is designed, which can be simultaneously loaded with two therapeutic agents, gemcitabine and dasatinib. The polymer is encapsulated by nucleic acid aptamers that target the PDAC stromal microenvironment and shield the surface. The aptamer is removed upon arrival at the PDAC hypoxic microenvironment, exposing the positively charged core to achieve charge reversal. Particle size transformation is achieved using linkers that respond to a hypoxic microenvironment. Increased intratumoral penetration is achieved using the dual transformation capabilities of the formulation. Pairing these two agents can result in a powerful efficacy in chemotherapy sensitization and immune infiltration.

Targeting glutamine synthetase with AS1411-modified exosome-liposome hybrid nanoparticles for inhibition of choroidal neovascularization.

Choroidal neovascularization (CNV) is a leading cause of visual impairment in wet age-related macular degeneration (wAMD). Recent investigations have validated the potential of reducing glutamine synthetase (GS) to inhibit neovascularization formation, offering prospects for treating various neovascularization-related diseases. In this study, we devised a CRISPR/Cas9 delivery system employing the nucleic acid aptamer AS1411 as a targeting moiety and exosome-liposome hybrid nanoparticles as carriers (CAELN). Exploiting the binding affinity between AS1411 and nucleolin on endothelial cell surfaces, the delivery system was engineered to specifically target the glutamine synthetase gene (GLUL), thereby attenuating GS levels and continuously suppressing CNV. CAELN exhibited spherical and uniform dispersion. In vitro cellular investigations demonstrated gene editing efficiencies of CAELN ranging from 42.05 to 55.02% and its capacity to inhibit neovascularization in HUVEC cells. Moreover, in vivo pharmacodynamic studies conducted in CNV rabbits revealed efficacy of CAELN in restoring the thickness of intra- and extranuclear tissues. The findings suggest that GS is a novel target for the inhibition of pathological CNV, while the development of AS1411-modified exosome-liposome hybrid nanoparticles represents a novel delivery method for the treatment of neovascular-related diseases.

Programmed DNA tile response system enabled accurate detection of ATP for clinical diagnosis

Adenosine triphosphate (ATP), as a crucial signaling molecule, is of high potential diagnosis value for multiple diseases. However, current technologies for ATP detection struggle to satisfy the harsh conditions required for biospecimen tests, existing several limitations for clinical practical application. In this study, the DNA tile response system was designed and constructed by integrating nucleic acid aptamer, hybrid chain reaction (HCR), and DNA tile self-assembly technology to achieve accurate detection of ATP in body fluid specimens. The DNA tile response system, possessing high-order structure characteristic, could remove HCR false-positive products and conquer the high noise background to enhance the detection sensitivity. Under the optimal reaction parameters, this system achieved the low limit of detection (LOD) of 189.23 nM over a wide linear range and splendid detection specificity faced with multiple interference terms. Furthermore, this system, possessing simple storage conditions, long-term detection stability, and superior anti-interference performance, was sufficient to satisfy the detection requirements for complicated biological specimens in multi-scenarios. Ultimately, the DNA tile response system showed excellent diagnostic efficiency that could easily distinguish urinary tract infection patients and healthy controls through ATP quantification, as well as hypertension patients and healthy ones. This system provides a high-potential rapid detection tool for early diagnosis and dynamic monitoring of urinary tract infections and hypertension, holding enormous potential for clinical translation due to the superiorities of simple operation, general applicability, and low instrument requirements.

Metal organic frameworks (MOFs)-based fluorescent CA125 analysis: A comparative study of the quenching effects of MIL-101, Cu-MOF, ZIF-8, UiO-66

Metal-organic frameworks (MOFs) are a class of materials with highly ordered pore structures. Due to their unique physical and chemical properties, they show great potential in the field of biosensors. Some MOFs can adsorb fluorescently labeled nucleic acid aptamers through various interaction mechanisms (such as electrostatic interactions, π-π stacking, hydrogen bonding, etc.). These interactions not only ensure the stable binding of the aptamers but also allow for their controlled release under specific conditions (such as changes in pH, temperature, or the presence of specific molecules). This mechanism provides multiple possibilities for the design of biosensors. Herein, we have systematically compared the quenching effects of widely used MOFs that can bind to aptamers, i.e., Fe-MOF (MIL-101), Cu-MOF, Zn-MOF (ZIF-8) and Zr-MOF (UiO-66). The study on the kinetics, quenching efficiency, and influencing factors such as ionic strength pH and temperature is performed. Interestingly, Cu-MOF exhibits superior quenching abilities to the other three materials in both the quenching efficiency and kinetics. Thus, a Cu-MOF based fluorescent sensor is reported to detect the ovarian cancer marker carbohydrate antigen 125 (CA125), which provides convenient detection performance (assay time about 10 min), and a detection range from 0.1 to 400 ng/mL. Moreover, it is designed in a simple mix-and-detect format and can be directly applied to clinical sample detection. This work may offer guidance for the choice of MOFs and elaborate design of biosensors.

Aptasensor based on cascaded fluorescent sensor assisted by exonuclease III for ultra-sensitive detection of Hg2.

Construct a sensitive and rapid detection fluorescence biosensor with the assistance of exonuclease III to achieve efficient detection of Hg2+. Nucleic acid aptamer H0 specifically recognizes Hg2+, and under the action of exonuclease III, H0 bound to Hg2+ is hydrolyzed, which in turn fails to trigger the catalytic hairpin self-assembly amplification reaction, resulting in a decrease in the number of double-stranded DNA bound to the fluorescent dye SYBR Green I in the solution, and a decrease in the fluorescence intensity. The results showed that the concentration of Hg2+ showed a good linear relationship with the fluorescence intensity of the sensing system within the range of 3.8-10pmol/L, and the detection limit was 0.53pmol/L. The recoveries of Xiangjiang River water used for the analysis of the actual samples were in the range of 99.57-103.58%, and the relative standard deviations of the determined values were 2.4-3.7%. The method is simple, sensitive, specific, cost-effective and can be applied to water samples.

"Explosive" CaCO3 microcapsules driven by EDTA combined with a "signal booster" semiconductor Zn0.995Ce0.005O SERS substrate for ultra-sensitive SERS detection of chloramphenicol

BackgroundChloramphenicol (CAP) is a broad-spectrum antibiotic, and its continuous use in human medicine, livestock has resulted disturbances in ecosystem stability. The complex background and low concentration of CAP in aquatic environments present significant scientific challenges for its sensitive detection. Currently detection techniques such as high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are hindered by their complex procedures and high costs. Surface-enhanced Raman spectroscopy (SERS), due to its unique ability for fingerprint recognition, has emerged as a powerful tool for analysis and detection. However, it is often limited by the low kurtosis expression of the target. ResultThe water-soluble explosive polyethylenimide grafted calcium carbonate (PEI@CaCO3) microcapsule was utilized to encapsulate the signal probe Ag@4-NTP and combine it with a semiconductor SERS substrate Zn0.995Ce0.005O, enabling ultra-sensitive detection of CAP through a signal attenuation strategy. Moreover, magnetic molecularly imprinted polymers (MMIP) and nucleic acid aptamers were employed as capture probes for achieving rapid and direct magnetic separation, followed by layer-by-layer assembly to construct the sensor probe. In the presence of CAP, the aptamers and rMMIPs selectively recognized the target, forming a PEI@CaCO3@Ag@4-NTP@Apt@CAP@rMMIPs "sandwich" structure (Microcapsule@Apt@rMMIPs). After multiple magnetic separations, the "igniter" EDTA solution was added, causing the CaCO3 capsules to rapidly "explode" and release a significant amount of Ag@4-NTP. This was then applied to the "signal booster" semiconductor SERS substrate. Under optimal conditions, this method exhibited a detection range of 1.0 × 10−5 M to 1.0 × 10−15 M, with a detection limit of 6.84 × 10−16 M. SignificantThe unite of magnetic MIP and nucleic acid aptamer to form a “sandwich” structure, in combination with a semiconductor SERS substrate, not only enhances the sensor's sensitivity but also offers significant economic advantages. Moreover, this innovative technology holds great promise for accurate and highly sensitive detection of pollutants in complex environments.

Construction of fluorescent aptasensor based on graphene oxide for the detection of metronidazole in food

Aiming at the detection of metronidazole (MNZ) residues in food, a high sensitivity fluorescent aptasensor was designed, which carboxyfluorescein (FAM)-labeled nucleic acid aptamer (FAM-MNZ) as the core probe and graphene oxide (GO) as an efficient fluorescence quencher. The principle is that GO adsorbs the aptamer onto the surface, causing short-range contact between the fluorescent group of the aptamer and GO. Due to its sp2 hybridized domains, GO undergoes fluorescence resonance energy transfer (FRET). Thus, the fluorescence is quenched. When MNZ is added, it preferentially forms a stable complex with the aptamer without FRET. Under optimized conditions, milk samples can be accurately detected with a detection limit of 0.033 μM. In addition, molecular docking, lumo electron orbital simulations, and experimental methods were used to explore the effect of different average radial sizes of single-layer GO on sensing performance. It is shown that GO with a larger average size makes it easy for aptamers to be regularly arranged on its surface. This also provides a basis for electron transfer, resulting in higher fluorescence quenching ability through the FRET mechanism. Therefore, the constructed fluorescent aptamer sensor has a significant application value in food detection.

Pattern recognition-based aptasensor array for discrimination of matrix effect

Nucleic acid aptamers have attracted considerable attention in recent years, particularly in the fields of disease diagnosis and treatment and biosensing. However, complex matrix effects have severely hindered their progress towards practical application. In this study, the novel strategy of a pattern recognition-based aptasensor array was introduced for target recognition based on the predictable selectivity capability of aptamers by circumventing the challenge of the interference of the matrix effect. As one proof of concept, we chose lactoferrin as the target protein, three selected aptamers with differential affinity as its recognition probes, eleven matrix samples of milk powder as the pattern discrimination model, and AuNPs color as the response signals. In the AuNPs colorimetric arrays, the lactoferrin-added milk powder matrix induced different degrees of protection on the surface of AuNPs particles due to different degrees of binding of lactoferrin to the three aptamer sequences and therefore exhibited differentiated red-to-blue color change along with salt-induced aggregation behaviors. The difference in color responses exhibited with and without lactoferrin addition was considered as the fingerprint pattern of the lactoferrin in each milk powder matrix, followed by the supervised machine learning analysis of linear discriminant analysis (LDA) for better orthogonality to complete the identification and prediction of the unknown samples. By this strategy, eleven kinds of milk powder at the added concentration of lactoferrin (50 nmol/L) presented a distinct response pattern and have been identified and classified with accuracies of 100 % via LDA patterns (three aptamers × eleven milk powder matrices × five replicates) with three canonical factors (91.2 %, 8.58 %, and 0.22 %). Furthermore, the accuracy of 22 unknown samples was 100 %, indicating the achievement in differentiating between different milk powder matrices and the identification of each matrix. The proposed aptasensor array complements the traditional “lock-and-key” single aptasensor and opens a new direction for developing sensitive sensing array systems circumventing complex matrix effects.

Single-Molecule adenosine detection and chiral selectivity by DNA aptamer conformational changes using α-Hemolysin nanopore

Nucleic acid aptamers, which are short single-stranded RNA or DNA molecules, undergo conformational changes upon recognizing small molecules. However, their structural characterization is challenging due to the flexibility of single-stranded oligonucleotides. Nanopores provide a sensitive method for single-molecule analysis of molecular conformational changes as they transport through the pore. In this study, we employed α-hemolysin (α-HL) nanopore with high spatiotemporal resolution to investigate the conformational changes of DNA aptamer (ADO-23) upon binding to adenosine (Ado) molecules. Unlike the events generated by the aptamer alone, the DNA-Ado complex produced two distinct new characteristic events, indicating the formation of two unique secondary structures. By analyzing current events, the structures formed by the DNA-Ado complex were speculated to be hairpin DNA and G-quadruplex (G4) DNA. Furthermore, we demonstrated the potential of the aptamer as a chiral selector within the nanopore. The results showed that the aptamer specifically bound to D-Ado and not to L-Ado. Finally, we realized specific and sensitive detection of small molecule Ado by α-HL pore, with a detection limit as low as 10.5 nM, and successfully applied it to the detection of real human serum samples. The nanopore platform provides a powerful tool for studying small molecule-biomolecule conformational changes, and we constructed sensors for detecting adenosine through the aptamer. This work also offers a promising new approach to the recognition of enantiomers at the single-molecule level.

Cellular fibronectin-targeted fluorescent aptamer probes for early detection and staging of liver fibrosis

Liver fibrosis is a key process in the progression of chronic liver disease to cirrhosis. Currently, early diagnosis and precise staging of liver fibrosis remain great challenges. Extracellular matrix (ECM) molecules expressed specifically during liver fibrosis are ideal targets for bioimaging and detection of liver fibrosis. Here, we report that fluorescent probes based on a nucleic acid aptamer (ZY-1) targeting cellular fibronectin (cFN), a critical ECM molecule significantly accumulating during liver fibrosis, are promising bioimaging agents for the staging of liver fibrosis. In the work, the outstanding binding affinity of ZY-1 to cFN was validated through an in vitro model of human-derived hepatic stellate cells (HSCs). Subsequently, we constructed different ZY-1-based fluorescent probes and explored the real-time imaging performance of these fluorescent probes in CCl4-induced mouse models of different liver fibrosis stages. The ZY-1-based fluorescent probes, for the first time, effectively identified and distinguished early-stage liver fibrosis (stage 3 of Ishak 6) from advanced liver fibrosis (stage 5 of Ishak 6). The proof-of-concept study provides compelling evidences that ZY-1-based probes are a promising tool for the early diagnosis and staging of liver fibrosis and paves the way for further development of clinical-related diagnosis strategies for fibrotic diseases of the liver and other organs. Statement of significanceCurrently, early diagnosis and accurate staging of liver fibrosis continue to present significant challenges. This study demonstrates that fluorescent probes based on the nucleic acid aptamer ZY-1, which targets cellular fibronectin (cFN)—a crucial extracellular matrix (ECM) molecule that significantly accumulates during liver fibrosis—are promising bioimaging agents for staging liver fibrosis. The ZY-1-based fluorescent probes effectively identified and differentiated early-stage liver fibrosis from advanced liver fibrosis. This proof-of-concept study not only provides compelling evidence that ZY-1-based probes show promise for the early diagnosis and staging of liver fibrosis but also paves the way for further investigations into the use of ZY-1 in detecting other diseases associated with cFN.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

Multiplex Determination of Glycan Profiles on Urinary Prostate-Specific Antigen by Quartz-Crystal Microbalance Combined with Surface-Enhanced Raman Scattering.

Prostate cancer remains a major health concern, with prostate-specific antigen (PSA) being a key biomarker for its detection and monitoring. However, PSA levels often fall into a "gray zone", where PSA levels are not clearly indicative of cancer, thus complicating early diagnosis and treatment decisions. Glycosylation profiles, which often differ between healthy and diseased cells, have emerged as potential biomarkers to enhance the specificity and sensitivity of cancer diagnosis in these ambiguous cases. We propose the integration of two complementary techniques, namely quartz-crystal microbalance with dissipation (QCM-D) and surface-enhanced Raman scattering (SERS) to study PSA glycan profiles. QCM-D offers real-time operation, PSA mass quantification, and label-free detection with high sensitivity, as well as enhanced specificity and reduced cross-reactivity when using nucleic acid aptamers as capture ligands. Complementary SERS sensing enables the determination of the glycosylation pattern on PSA, at low concentrations and without the drawbacks of photobleaching, thereby facilitating multiplexed glycosylation pattern analysis. This integrated setup could retrieve a data set comprising analyte concentrations and associated glycan profiles in relevant biological samples, which may eventually improve early disease detection and monitoring. Prostate-specific antigen (PSA), a glycoprotein secreted by prostate epithelial cells, serves as our proof-of-concept analyte. Our platform allows multiplex targeting of PSA multiplex glycosylation profiles of PSA at "gray zone" concentrations for prostate cancer diagnosis. We additionally show the use of SERS for glycan analysis in PSA secreted from prostate cancer cell lines after androgen-based treatment. Differences in PSA glycan profiles from resistant cell lines after androgen-based treatment may eventually improve cancer treatment.

Mechanism of Dual-Site Recognition in a Classic DNA Aptamer.

Nucleic acid aptamers possess unique advantages in specific recognition. However, the lack of in-depth investigation into their dynamic recognition mechanisms has restricted their rational design and potential applications in fields such as biosensing and targeted therapy. We herein utilized enhanced sampling molecular dynamics to address affinities of adenosine monophosphate (AMP) to the dual binding sites in the DNA aptamer, focusing on the dynamic recognition mechanism and pathways. The present results indicate that in addition to the widely known intermolecular interactions, inequivalence of chemical environments of the two binding sites leads to slightly higher stability of AMP binding to the site proximal to the aptamer terminus. In the presence of two AMPs captured by the two sites, each binding free energy is enhanced. In particular, an additional hydrogen bond of AMP to A10 is introduced in the dual-site binding complex, which increases the binding energy from -4.25 ± 0.47 to -9.48 ± 0.33 kcal mol-1 in the site close to the loop. For the dual-site recognition process, the free energy landscape and minimum free energy pathway calculations elucidate the crucial role of electrostatic interactions between the AMP phosphate groups and Na+ ions in positively cooperative binding mechanisms.

Surface-enhanced Raman scatting microfluidic chip based on the identification competition strategy were used for rapid and simultaneous detection of liver cancer related proteins

Biomarker testing plays a crucial role in the early detection of liver cancer. Herein, we developed a dual-signal amplification approach utilizing magnetic aggregation and a recognition competition strategy for the simultaneous detection of alpha-fetoprotein (AFP) and manganese superoxide dismutase (MnSOD) in serum. 4-MBA@AuNPs@H1 and DTNB@AuNPs@H2 were synthesized by functionalizing Raman signaling molecules and aptamer complementary chains onto the surface of gold nanoparticles (AuNPs). The detection complex Raman signal molecule@AuNPs@H-Fe3O4@cDNA was assembled by conjugating 4-MBA@AuNPs@H1 and DTNB@AuNPs@H2 with two nucleic acid aptamers (cDNA1 and cDNA2) modified with Fe3O4 through partial base complementary pairing. The target protein exhibited specific binding with the aptamer, leading to the competitive displacement of 4-MBA@AuNPs@H1 and DTNB@AuNPs@H2 from the Fe3O4 array surface, consequently resulting in a reduction of the Surface-Enhanced Raman Spectroscopy (SERS) signal. Through this approach, the limit of detection (LOD) for AFP and MnSOD in serum was achieved at remarkably low levels of 5.89 pg/mL and 6.23 pg/mL, respectively, with a reaction incubation period of only 5 min. Finally, the platform was utilized for the quantification of AFP and MnSOD in the serum of a nude mouse hepatocellular carcinoma model. The results obtained through SERS were consistent with those from enzyme-linked immunosorbent assay (ELISA), validating its accuracy. This methodology presents a novel approach for the swift and concurrent detection of proteins, holding significant clinical promise for the early diagnosis of hepatocellular carcinoma.

Chemiluminescence detection of kanamycin by DNA aptamer regulating peroxidase-like activity of Co3O4 nanoparticles.

Kanamycin (KAN) is widely used as a growth hormone analog and an antibacterial agent. However, abuse of this substance has resulted in the accumulation of excessive residue levels in foods of animal origin, which presents a significant risk to human health. A chemiluminescent aptasensor was constructed for the rapid quantitative detection of KAN by combining the properties of Co3O4 nanoparticles (Co3O4 NPs) nanozyme activity and DNA aptamer with high specificity. The DNA aptamer/Co3O4 NPs nanozyme regulated the chemiluminescence signal by exploiting the chemiluminescent properties of luminol oxidation by H2O2. Specific binding of KAN to the aptamer led to the formation of a steric hindrance block in the solution, which inhibited the activity of nanozyme and reduced signal intensity. The degree of signal reduction is related to the concentration of KAN. Under optimal conditions, there was good linearity between KAN concentration and chemiluminescence signal intensity in the range of 0.5-8.0 μΜ, with a detection limit of 0.26 μΜ. The detection system performed well in the presence of competing antibiotics and was virtually unaffected. The method was also suitable for the detection of KAN in milk samples with sample recoveries of 97.8%-99.1%. The chemiluminescence sensor has the advantages of low cost, specificity, and sensitivity, and does not require an external light source or modification of the nucleic acid aptamer which makes it a promising candidate for applications in the field of food detection.

Aptamer-Loop DNA Nanoflower Recognition and Multicolor Fluorescent Carbon Quantum Dots Labeling System for Multitarget Living Cell Imaging.

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Peptide-based CE-SELEX enables convenient isolation of aptamers specifically recognizing CD20-expressing cells

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with Kd estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds.

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3′→5′ exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5′-bond of phosphodiester from 3′ to 5′ end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3′-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Bacterial exonuclease III expands its enzymatic activities on single-stranded DNA.

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'→5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Detection of dual-methylated BRCA1/BRCA2 cell-free DNA for ovarian cancer on an aptamer-based integrated microfluidic system

A new screening strategy has been developed to select nucleic acid aptamer probes with an “in vivo-like” systematic evolution of ligands by exponential enrichment (SELEX) process featuring positive, negative selection and in vivo-like selection steps. One methylation-specific aptamer with high affinity and specificity was selected and it could capture methylated cell-free DNA (cfDNA) from unfiltered blood of ovarian cancer (OvCa) patients. This aptamer was conjugated onto magnetic beads and integrated into a new microfluidic chip for rapid and automatic detection and quantification of two prevalent methylated tumor suppressor genes, breast cancer type 1 and 2 susceptibility protein (BRCA 1/2), in the molecular profile of OvCa. The assay was conducted under physiological conditions that mimic the microenvironment of cancer patients at 25–37 °C, with a low pH value (5.5) and/or high blood ion levels, without requiring additional sample preparation steps. The results of this study could be extrapolated to the direct detection of methylated cfDNA at levels as low as 1 pM in blood or plasma. Not only was DNA methylation observed in OvCa patients, but it was also detected in many other types of cancer; it is therefore considered an important factor in carcinogenesis. Hence, it is inferred that this screened aptamer should not only be used for OvCa diagnosis but also for multiple cancer epigenetic detection and drug development research in the future.