Endonuclease-rolling circle amplification-based method for sensitive analysis of DNA-binding protein

A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA （ssDNA） were spotted on a microarray. The endonuclease recognition site （ERS） and the DNA-binding sites （DBS） were arranged side by side within the duplex region. The working principle of the detection system is described as follows： when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification （RCA）. When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.

Rolling Circle Amplification on Biotin-Streptavidin Complexes Immobilized to Activated Cyclic Polyolefin Surfaces

Cyclic polyolefin (COP) is an inexpensive hydrophobic material with low auto-fluorescence, high light transmittance and thermal stability, broad chemical resistance and no non-specific protein binding. Here, the hydrophobic alkane COP was modified to have carbonyl functionalities through oxygen plasma and chemical etching treatments to increase usefulness for chemical and biochemical applications. Then, biotin-hydrazide was used to create biotinylated surfaces that bound streptavidin. A biotinylated target oligonucleotide was subsequently bound to the immobilized biotin-streptavidin and ligation mediated rolling circle amplification-based (L-RCA) SNP detection was demonstrated.

RNA SNP Detection Method With Improved Specificity Based on Dual-competitive-padlock-probe

Objective The detection of RNA single nucleotide polymorphism(SNP)is of great importance due to their association with protein expression related to various diseases and drug responses.At present,splintR ligase-assisted methods are important approaches for RNA direct detection,but its specificity will be limited when the fidelity of ligases is not ideal.The aim of this study was to create a method to improve the specificity of splintR ligase for RNA detection.Methods In this study,a dualcompetitive-padlock-probe(DCPLP)assay without the need for additional enzymes or reactions is proposed to improve specificity of splintR ligase ligation.To verify the method,we employed dual competitive padlock probe-mediated rolling circle amplification(DCPLP-RCA)to genotype the CYP2C9 gene.Results The specificity was well improved through the competition and strand displacement of dual padlock probe,with an 83.26%reduction in nonspecific signal.By detecting synthetic RNA samples,the method demonstrated a dynamic detection range of 10 pmol/L-1 nmol/L.Furthermore,clinical samples were applied to the method to evaluate its performance,and the genotyping results were consistent with those obtained using the qPCR method.Conclusion This study has successfully established a highly specific direct RNA SNP detection method,and provided a novel avenue for accurate identification of various types of RNAs.

High-throughput quantitative detection of triple-negative breast cancer-associated expressed miRNAs by rolling circle amplification on fluorescence-encoded microspheres

Compared with other types of breast cancer,triple-negative breast cancer(TNBC)has the characteristics of a high degree of malignancy and poor prognosis.Early diagnosis of TNBC through biological markers and timely development of effective treatment methods can reduce its mortality.Many Research experiments have confirmed that some specific mi RNA expression profiles in TNBC can used as markers for early diagnosis.However,detecting the expression profiles of multiple groups of miRNAs according to traditional detection methods is complicated and consumes many samples.To address this issue,we developed a method for high-throughput,high-sensitivity quantitative detection of multiple sets of miRNAs(including mi R-16,mi R-21,mi R-92,mi R-199,and mi R-342)specifically expressed in TNBC by rolling circle amplification(RCA)on fluorescence-encoded microspheres.Through the optimization of reaction system conditions,the developed method showed an extensive linear dynamic range and high sensitivity for all five miRNAs with the lowest limit of detection of 2 fmol/L.Meanwhile,this high-throughput detection method also appeared reasonable specificity.Only in the presence of a specific target miRNA,the fluorescence signal on the correspondingly encoded microspheres is significantly increased,while the fluorescence signal on other non-correspondingly encoded microspheres is almost negligible.Furthermore,this process exhibited good recovery and reproducibility in serum.The advantages of this method allow us to more conveniently obtain the expression profiles of multiple groups of TNBC-associated mi RNAs,which is beneficial for the early detection of TNBC.

Research progress and prospects of nucleic acid isothermal amplification technology

Nucleic acid(DNA and RNA)detection and quantification methods play vital roles in molecular biology.With the development of molecular biology,isothermal amplification of DNA/RNA,as a new molecular biology technology,can be amplified under isothermal condition,it has the advantages of high sensitivity,high specificity,and high efficiency,and has been applied in various fields of biotechnology,including disease diagnosis,pathogen detection,food hygiene and safety detection and so on.This paper introduces the progress of isothermal amplification technology,including rolling circle amplification(RCA),nucleic acid sequence-dependent amplification(NASBA),strand displacement amplification(SDA),loop-mediated isothermal amplification(LAMP),helicase-dependent amplification(HDA),recombinase polymerase amplification(RPA),cross-priming amplification(CPA),and its principle,advantages and disadvantages,and application development are briefly summarized.

A Programmable Hybrid DNA Nanogel for Enhanced Photodynamic Therapy of Hypoxic Glioma

Photodynamic therapy(PDT)is a promising cancer therapy due to the evident advantages of a rapid curative eff ect,minimal or non-invasiveness,and circumvention of drug resistance.However,the hydrophobicity of photosensitizers and the hypoxic tumor microenvironment in solid tumors reduce the therapeutic eff ect of PDT immensely.Herein,we construct a programmable hybrid mesoporous silica nanoparticle/DNA nanogel(H-DNA nanogel)for enhanced PDT.The H-DNA nanogel is constituted with a virus-like mesoporous silica nanoparticle(VMSN)as the core to provide an appropriate nano-interface and a self-assembly programmable DNA hydrogel layer based on rolling circle amplifi cation(RCA)as the shell.Two kinds of G-quadruplex structures inserted with a hemin and zinc phthalocyanine(ZnPc)photosensitizer are introduced into the H-DNA nanogel by base pairing.The two modules of G-quadruplex structure work as an oxygen supplement in the hypoxic tumor microenvironment and increase the yield of singlet oxygen,respectively.Our hybrid DNA nanogel system provides a modular platform for effi cient cancer PDT and has great potential in the broader biomedical fi eld.

Self-assembly of DNA Origami Using Rolling Circle Amplification Based DNA Nanoribbons

During the development of structural DNA nanotechnology,the emerging of scaffolded DNA origami is marvelous.It utilizes DNA double helix inherent specificity of Watson-Crick base pairing and structural features to create self-assembling structures at the nanometer scale exhibiting the addressable character.However,the assembly of DNA origami is disorderly and unpredictable.Herein,we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers.Firstly,long single-stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs.Subsequently,the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons.By mixing them up,we illustrate the one-dimensional even two-dimensional assembly of DNA origami with good orientation.

Ultrasensitive detection of DNA and protein markers in cancer cells

Cancer cells differ from normal cells in various parameters, and these differences are caused by genomic mutations and consequential altered gene expression. The genetic and functional heterogeneity of tumor cells is a major challenge in cancer research, detection, and effective treatment. As such, the use of diagnostic methods is important to reveal this heterogeneity at the single-cell level. Droplet microfluidic devices are effective tools that provide exceptional sensitivity for analyzing single cells and molecules. In this review, we highlight two novel methods that employ droplet microfluidics for ultrasensitive detection of nucleic acids and protein markers in cancer cells. We also discuss the future practical applications of these methods.

Dual Rolling Circle Amplification- Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Exosomes secreted by tumor cells carry abundant molecular biomarkers that reflect the status of their originating cells.These tumor-derived exosomes(TDEs)have emerged as attractive diagnostic targets.However,the identification and characterization of highly heterogeneous TDEs remain practically challenging.Here,we report a dual rolling circle amplification(DRCA)-assisted approach for the selective encapsulation of single TDEs for fluorescence microscopic and flow cytometric analysis.TDEs have been targeted by aptamers that recognized their surface tumor marker and exosomal marker CD63,following DRCA that produced entangling polymeric DNA chains,resulting in facile particle enlargement that allows single-particle fluorescence profiling of exosome heterogeneity.We have demonstrated the use of a dual-marker positive ratio for exosome differentiation and applied division and multiplication operations for normalized andmagnified marker heterogeneity analysis.We further applied this assay to distinguish lung adenocarcinoma and pulmonary nodule patients and found an accuracy of 90%.We anticipate promising transformations of this straightforward assay into clinically implantable diagnostic methods.

Colorimetric and electrochemical dual-signal detection of uracil-DNA glycosylase using functionalized pure DNA hydrogel on paper-based analytical devices

The development of simple and accurate detection of uracil-DNA glycosylase(UDG)is of great significance for early clinical diagnosis and biomedical research.Here,we on the first effort introduced the uracil bases into the rolling circle amplification(RCA)reaction to produce the functionalized pure DNA hydrogel(PDH)for UDG detection.During RCA process,methylene blue(MB)molecules as the indicators were encapsulated into PDH.The addition of UDG can remove the uracil bases of PDH to generate abasic sites,which are further cleaved with the assistance of apurinic/apyrimidinic endonuclease(APE),thus resulting in the dissociation of PDH to release blue MB.By combining with the paper analytical devices as the signal readout platform,a colorimetric and electrochemical dual-signal biosensor was constructed for convenient and accurate detection of UDG.The proposed MB@PDH-based dual-signal sensing system exhibited good selectivity and high sensitivity with a detection limit of 6.4104 U/mL(electrochemical method).It was also demonstrated that this sensing system showed excellent performance in UDG inhibitor screening,thus providing great potential in UDG-related disease diagnosis and drug discovery.

Special RCA based sensitive point-of-care detection of HPV mRNA for cervical cancer screening

Developing the sensitive point-of-care testing(POCT)of oncogenic nucleic acids from human papillomavirus(HPV)infection is essential in preventing cervical can-cer,especially in resource-limited settings.Rolling circle amplification(RCA)is attractive in achieving POCT via nucleic acid-based aggregation under isothermal conditions.However,the influence of RCA product structure on the aggregation remains unexplored resulting in limited sensitivity.Here,a minimum secondary structured RCA technique(MSS-RCA)is developed by designing a unique circu-lar template,demonstrating significantly enhanced detection sensitivity with only one amplification step and one primer under isothermal conditions.The amplifi-cation efficiency of MSS-RCA could be kinetically manipulated by controlling the secondary structure of the circular template.Introducing the invertase probe to MSS-RCA,HPV16 E6/E7 nucleic acid target was detected with a personal glucose meter(PGM)with a sensitivity of 5 fM(50 zmol in 10µL).This integrated MSS-RCA-PGM detection system was successfully applied to detect HPV16 E6/E7 mRNA extracted from 54 cervical swab samples reaching a positive predictive value of 100.00%and negative predictive values of 96.00%(77.77%to 99.40%,95%CI).MSS-RCA-PGM provides a sensitive POCT platform for the detection of nucleic acid biomarkers for screening of cervical cancer or other diseases.

A sensitive one-pot ROA assay for rapid miRNA detection

MicroRNAs(miRNAs)and short RNA fragments(18–25 nt)are crucial biomarkers in biological research and disease diagnostics.However,their accurate and rapid detection remains a challenge,largely due to their low abundance,short length,and sequence similarities.In this study,we report on a highly sensitive,one-step RNA O-circle amplification(ROA)assay for rapid and accurate miRNA detection.The ROA assay commences with the hybridization of a circular probe with the test RNA,followed by a linear rolling circle amplification(RCA)using dUTP.This amplification process is facilitated by U-nick reactions,which lead to an exponential amplification for readout.Under optimized conditions,assays can be completed within an hour,producing an amplification yield up to the microgram level,with a detection limit as low as 0.15 fmol(6 pM).Notably,the ROA assay requires only one step,and the results can be easily read visually,making it user-friendly.This ROA assay has proven effective in detecting various miRNAs and phage ssRNA.Overall,the ROA assay offers a user-friendly,rapid,and accurate solution for miRNA detection.

Self-assembled multifunctional DNA nanoflowers for the circumvention of multidrug resistance in targeted anticancer drug delivery

Cancer chemotherapy has been limited by its side effects and multidrug resistance （MDR）, the latter of which is partially caused by drug efflux from cancer cells. Thus, targeted drug delivery systems that can circumvent MDR are needed. Here, we report multifunctional DNA nanoflowers （NFs） for targeted drug delivery to both chemosensitive and MDR cancer cells that circumvented MDR in both leukemia and breast cancer cell models. NFs are self-assembled via potential co-precipitation of DNA and magnesium pyrophosphate generated by rolling circle replication, during which NFs are incorporated using aptamers for specific cancer cell recognition, fluorophores for bioimaging, and doxorubicin （Dox）- binding DNA for drug delivery. NF sizes are tunable （down to N200 nm in diameter）, and the densely packed drug-binding motifs and porous intrastructures endow NFs with a high drug-loading capacity （71.4%, wt/wt）. Although the Dox- loaded NFs （NF-Dox） are stable at physiological pH, drug release is facilitated under acidic or basic conditions. NFs deliver Dox into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. NF-Dox induces potent cytotoxicity in both target chemosensitive cells and MDR cells, but not in nontarget cells, thus concurrently circumventing MDR and reducing side effects. Overall, these NFs are promising tools for circumventing MDR in targeted cancer therapy.

Coating with flexible DNA network enhanced T-cell activation and tumor killing for adoptive cell therapy

Adoptive cell therapy(ACT)is an emerging powerful cancer immunotherapy,which includes a complex process of genetic modification,stimulation and expansion.During these in vitro or ex vivo manipulation,sensitive cells are inescapability subjected to harmful external stimuli.Although a variety of cytoprotection strategies have been developed,their application on ACT remains challenging.Herein,a DNA network is constructed on cell surface by rolling circle amplification(RCA),and T cell-targeted trivalent tetrahedral DNA nanostructure is used as a rigid scaffold to achieve high-efficient and selective coating for T cells.The cytoprotective DNA network on T-cell surface makes them aggregate over time to form cell clusters,which exhibit more resistance to external stimuli and enhanced activities in human peripheral blood mononuclear cells and liver cancer organoid killing model.Overall,this work provides a novel strategy for in vitro T cell-selective protection,which has a great potential for application in ACT.

Recent advancements in DNA nanotechnology-enabled extracellular vesicles detection and diagnosis:A mini review

Extracellular vesicles(EVs)are cell-derived nanosized vesicles widely recognized for their critical roles in various pathophysiological processes.Molecular analysis of EVs is currently being considered an emerging tool for diseases diagnosis.However,the small size and heterogeneity of EVs has staggered the EVs research for diseases diagnosis.DNA nanotechnology enables self-assembly of versatile DNA nanostructures and has shown enormous potential in assisting EVs biosensing.In this review,we briefly introduce the recent advances in DNA nanotechnology approaches for EVs detection.The approaches were categorized based on the dimension of DNA nanostructures.We provide critical evaluation of these approaches,and summarize the pros and cons of specific methods.Further,we discuss the challenges and future perspectives in this field.

Signal Amplification for Highly Sensitive Immunosensing

To dissolve the bottleneck problem of life and biomedical science in detection of biomolecules with low abundance and acquisition of ultraweak biological signals,highly sensitive analytical methods coupling with the specificity of biological recognition events have been quickly developed by the exploring of signal amplification strategies.These strategies have extensively been introduced into the development of highly sensitive immunosensing methods by combining with highly specific immunological recognition.They can be divided into two groups.One group of strategies attempts to transfer the immunological recognition event into large number of reporter probes or signal probes for signal readout by employing nano/micro-materials as vehicles for multi-labeling and/or molecular biological amplification for increasing the abundance of the signal molecules.The other uses nanomaterials or enzyme mimics as catalytic tools/tags to obtain enhanced detection signal.This review focuses on the significant advances in design of signal amplification strategies for highly sensitive immunosensing.