Gold Nanomaterials-Implemented CRISPR-Cas Systems for Biosensing

Due to their superiority in the simple design and precise targeting, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have attracted significant interest for biosensing. On the one hand, CRISPR-Cas systems have the capacity to precisely recognize and cleave specific DNA and RNA sequences. On the other hand, CRISPR-Cas systems such as orthologs of Cas9, Cas12, and Cas13 exhibit cis-cleavage or trans-cleavage activities after recognizing the target sequence. Owing to the cleavage activities, CRISPR-Cas systems can be designed for biosensing by degrading tagged nucleic acids to produce detectable signals. To meet the requirements of point-of-care detection and versatile signal readouts, gold nanomaterials with excellent properties such as high extinction coefficients, easy surface functionalization, and biocompatibility are implemented in CRISPR-Cas-based biosensors. In combination with gold nanomaterials such as gold nanoparticles, gold nanorods, and gold nanostars, great efforts are devoted to fabricating CRISPR-Cas-based biosensors for the detection of diverse targets. This review focuses on the current advances in gold nanomaterials-implemented CRISPR-Cas-based biosensors, particularly the working mechanism and the performance of these biosensors. CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a are discussed and highlighted. Meanwhile, prospects and challenges are also discussed in the design of biosensing strategies based on gold nanomaterials and CRISPR-Cas systems.     

A Simplified Amplification-Free Strategy with Lyophilized CRISPR-CcrRNA System for Drug-Resistant Salmonella Detection

Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drug-resistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for the detection of drug-resistant Salmonella has an ultra-sensitive detection limit of 84 CFU mL⁻¹ within 30 min, with good linearity in the range of 10²–10⁶ CFU mL⁻¹. In real-world applications, spiked recoveries range from 76.22% to 145.91%, with a coefficient of variation less than 10.59%. AFC-TRFIA offers a cost-effective, sensitive, and virtually equipment-independent platform for preventing foodborne illnesses, screening for drug-resistant Salmonella, and guiding clinical use.     

Cascade DNA Circuits Mediated CRISPR-Cas12a Fluorescent Aptasensor based on Multifunctional Fe3O4@hollow-TiO2@MoS2 Nanochains for Tetracycline Determination

Herein, for the first time, the CRISPR-Cas12a system is combined with aptamer, cascaded dynamic DNA network circuits, and Fe₃O₄@hollow-TiO₂@MoS₂ nanochains (Fe₃O₄@h-TiO₂@MoS₂ NCs) to construct an efficient sensing platform for tetracycline (TC) analysis. In this strategy, specific recognition of the target is transduced and amplified into H1-H2 duplexes containing the specific sequence of Cas12a-crRNA through an aptamer recognition module and the dual amplification dynamic DNA network. Subsequently, the obtained activated Cas12a protein non-specifically cleaves the adjacent reporter gene ssDNA-FAM to dissociate the FAM molecule from the quencher Fe₃O₄@h-TiO₂@MoS₂ NCs, resulting in the recovery of the fluorescence signal and further signal amplification. Particularly, the synthesized multifunctional Fe₃O₄@h-TiO₂@MoS₂ NCs composites also exhibit superb magnetic separability and photocatalytic degradation ability. Under optimal conditions, the aptasensor displays a distinct linear relationship with the logarithm of TC concentration, and the limit of detection is as low as 0.384 pg mL⁻¹. Furthermore, the results of spiked recovery confirm the viability of the proposed aptasensor for TC quantification in real samples. This study extends the application of the CRISPR-Cas12a system in the field of analytical sensing and contributes new insights into the exploration of reliable tools for monitoring and treating hazards in food and environment.     

Synthesis and Properties of Dextran-Nalidixic Acid Ester as a Colon-Specific Prodrug of Nalidixic Acid

Dextran-nalidixic acid ester (dextran-NA) with a varied degree of substitution (DS) was synthesized as a colon-specific prodrug of nalidixic acid (NA). Solubility in water (mg/ml) of dextran-NA with DS (mg NA/100 mg dextran-NA) of 7, 19, or 32 was 57.57 (equivalent to 4.00 mg NA/ml), 0.53 (equivalent to 0.10 mg NA/ml), or 0.03 (equivalent to 0.01 mg NA/ml), respectively, and that for NA was 0.03 at 25°C. To ensure the chemical stability of dextran-NA at conditions similar to those of the stomach and small intestine, dextran-NA was placed in a solution of pH 1.2 hydrochloric acid buffer or pH 6.8 phosphate buffer and incubated at 37°C; no NA was detected during the 6 h of the incubation period, which indicated that dextran-NA might be chemically stable during the transit through the gastrointestinal tract. Degree of depolymerization (%) by dextranase determined by the 2,4-dinitrosalicylic acid (DNS) method at 37°C for dextran-NA with DS of 7, 19, or 32 was 81, 68, or 8, respectively, in 8 h, and that for dextran was 91. When dextran-NA (equivalent to 50 μg of NA) with a DS of 7 or 17 was incubated with cecal contents (100 mg) of rats at 37°C, the extent of NA released in 24 h was 41% or 32% of the dose, respectively. NA was not liberated from the incubation of dextran-NA with the homogenate of tissue and contents of the small intestine.     

Synthesis and Properties of Dextran–Nalidixic Acid Ester as a Colon-Specific Prodrug of Nalidixic Acid

Dextran–nalidixic acid ester (dextran-NA) with a varied degree of substitution (DS) was synthesized as a colon-specific prodrug of nalidixic acid (NA). Solubility in water (mg/ml) of dextran-NA with DS (mg NA/100 mg dextran-NA) of 7, 19, or 32 was 57.57 (equivalent to 4.00 mg NA/ml), 0.53 (equivalent to 0.10 mg NA/ml), or 0.03 (equivalent to 0.01 mg NA/ml), respectively, and that for NA was 0.03 at 25°C. To ensure the chemical stability of dextran-NA at conditions similar to those of the stomach and small intestine, dextran-NA was placed in a solution of pH 1.2 hydrochloric acid buffer or pH 6.8 phosphate buffer and incubated at 37°C; no NA was detected during the 6 h of the incubation period, which indicated that dextran-NA might be chemically stable during the transit through the gastrointestinal tract. Degree of depolymerization (%) by dextranase determined by the 2,4-dinitrosalicylic acid (DNS) method at 37°C for dextran-NA with DS of 7, 19, or 32 was 81, 68, or 8, respectively, in 8 h, and that for dextran was 91. When dextran-NA (equivalent to 50 μg of NA) with a DS of 7 or 17 was incubated with cecal contents (100 mg) of rats at 37°C, the extent of NA released in 24 h was 41% or 32% of the dose, respectively. NA was not liberated from the incubation of dextran-NA with the homogenate of tissue and contents of the small intestine.     

Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change. Two different systems are chosen, DNase I (a DNA endonuclease) and 8-17 (a Pb(2+)-depedent RNA-cleaving DNAzyme), to demonstrate the utility of our assay for the detection of metal ions and sensing enzyme activities. Compared with previous studies in which AuNP aggregates are converted into dispersed AuNPs by enzymatic cleavage of DNA crosslinkers, the present assay is technically simpler. Moreover, the accessibility of DNA to biomolecular recognition elements (e.g. enzymes) on well-dispersed AuNPs in our assay appears to be higher than that embedded inside aggregates. This biosensing system should be readily adaptable to other enzymes or substrates for detection of analytes such as small molecules, proteases and their inhibitors.     

Dual‐Locking Nanoparticles Disrupt the PD‐1/PD‐L1 Pathway for Efficient Cancer Immunotherapy

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated (Cas) enzyme, Cas13a, holds great promise in cancer treatment due to its potential for selective destruction of tumor cells via collateral effects after target recognition. However, these collateral effects do not specifically target tumor cells and may cause safety issues when administered systemically. Herein, a dual‐locking nanoparticle (DLNP) that can restrict CRISPR/Cas13a activation to tumor tissues is described. DLNP has a core–shell structure, in which the CRISPR/Cas13a system (plasmid DNA, pDNA) is encapsulated inside the core with a dual‐responsive polymer layer. This polymer layer endows the DLNP with enhanced stability during blood circulation or in normal tissues and facilitates cellular internalization of the CRISPR/Cas13a system and activation of gene editing upon entry into tumor tissue. After carefully screening and optimizing the CRISPR RNA (crRNA) sequence that targets programmed death‐ligand 1 (PD‐L1), DLNP demonstrates the effective activation of T‐cell‐mediated antitumor immunity and the reshaping of immunosuppressive tumor microenvironment (TME) in B16F10‐bearing mice, resulting in significantly enhanced antitumor effect and improved survival rate. Further development by replacing the specific crRNA of target genes can potentially make DLNP a universal platform for the rapid development of safe and efficient cancer immunotherapies.     

Development and evaluation of active packaging for sliced mozzarella preservation

Antimicrobial films were formed by the incorporation of nisin (NI), natamycin (NA) and a combination of both (NI + NA) into cellulose polymer. Film efficacies were evaluated in vitro against Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 15313, Penicillium sp. and Geotrichum sp. The films were also evaluated on sliced mozzarella cheese against moulds and yeasts, Staphylococcus sp. and psychrotrophic bacteria. Mechanical and microscopic properties of the films and the diffusion of the antimicrobial agents from the film to the cheese were also evaluated. Films containing NI showed an antimicrobial effect in vitro against S. aureus and L. monocytogenes, while films containing NA were effective in vitro against Penicillium sp. and Geotrichum sp. By the ninth day of storage at 12 ± 2°C, the count of yeasts and moulds on cheese covered with films containing NA decreased 2 log10 units compared with the count on cheese with control films. NI film did not show an effect against Staphylococcus sp., but it was effective against psychrotrophic bacteria for 6 days of storage of the cheese. The incorporation of antimicrobial compounds decreased the resistance and elongation of the films and caused changes in their molecular conformation. NI diffusion from the films to the cheese was not detected; however, time‐dependent diffusion of NA from the film containing NI + NA was measured. The incorporation of NI and NA together in the films did not show an effect. The film containing NA showed potential for application as active food packaging for sliced mozzarella cheese. Copyright © 2008 John Wiley & Sons, Ltd.     

Typing of multiple single-nucleotide polymorphisms by a microsphere-based rolling circle amplification assay

The combination of suspension array with rolling circle amplification can lead to a sensitive and specific assay for single-nucleotide polymorphisms (SNPs) detection, as demonstrated in this study. A circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase on microspheres. The elongation products were labeled with fluorochrome-tagged probes and detected in a flow cytometer, indicating the mutation occurrence. As low as 10 amol of mutated strands was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, which could be attributed to the high amplification efficiency of Phi29, the high binding capacity of the microspheres, and the remarkable precision of DNA ligase in distinguishing mismatched bases at the ligation site. A novel design of using two differently labeled detection probes on the same microsphere to target both the wild-type and mutant samples allowed parallel determination of the heterozygosity for two SNPs (K-ras G12C and TP53 R273H) in PCR amplicons prepared from human genomic DNA extracts. This ability lays the groundwork for further enhancing the assay throughput by using multiple fluorophores and microspheres with distinct properties.     

Selective and homogeneous fluorescent DNA detection by target-induced strand displacement using cationic conjugated polyelectrolytes

A new methodology has been developed for DNA detection that interfaces optical amplification properties of cationic conjugated polyelectrolytes with highly selective target-induced DNA strand displacement. The probe solution contains a cationic conjugated polyelectrolyte (CCP-1), partly hybridized duplex DNA labeled with a fluorescein at the 5'-terminus, and endonuclease Hae III. Excitation of the CCP-1 leads to efficient energy transfer from CCP-1 to fluorescein. In the presence of a complementary DNA strand to one strand of the probe duplex, a hairpin DNA with the recognition site of endonuclease Hae at the double-stranded stem is released following its cleavage by Hae III to generate short DNA fragment carrying fluorescein. The relatively weak electrostatic interactions between the DNA fragment and CCP-1 lead fluorescein far away from CCP-1 and inefficient energy transfer between them is present. Thus, the DNA can be detected by fluorescence spectra in view of the observed CCP-1 or fluorescein emission changes in aqueous solutions. To avoid utilizing unstable Hae III endonuclease, a new system based on RNA-cleaving DNAzyme was further developed. The protocol offers a convenient approach for homogeneous, selective, and sensitive DNA assay in aqueous solution without using any denaturation steps. Compared with previously reported DNA sensors based on conjugated polyelectrolytes, our new method is highly sequence specific and a single-nucleotide mismatch can be clearly detected in target DNA.     

Effects of source shape on the numerical aperture factor with a geometrical-optics model

We study the effects of an extended light source on the calibration of an interference microscope, also referred to as an optical profiler. Theoretical and experimental numerical aperture (NA) factors for circular and linear light sources along with collimated laser illumination demonstrate that the shape of the light source or effective aperture cone is critical for a correct NA factor calculation. In practice, more-accurate results for the NA factor are obtained when a linear approximation to the filament light source shape is used in a geometric model. We show that previously measured and derived NA factors show some discrepancies because a circular rather than linear approximation to the filament source was used in the modeling.     

Application of oligonucleotide as a template for the assembly of nucleoamphiphile bearing azobenzene at the air-water interface

In order to assemble amphiphilic adenine having azobenzene, C12AzoC5Ade (AzoAde), at the air-water interface based on the molecular recognition of DNA, we prepared aqueous linear oligothymidylic acids, dTn (n = 4, 6, 8, 10, 30), subphase as templates. Surface pressure-area (pi-A) isotherms and UV-Vis reflection absorption spectra of AzoAde were measured to investigate the effect of chain length of the oligothymidylic acid on the molecular recognition by forming a complementary A-T base pair. It was showed that AzoAde did not form a stable monolayer on the dT4 subphase and remained monomeric state. While AzoAde provided expanded monolayers and formed J-aggregates of azobenzene moieties on the dTn (n > 4) subphases even at the low molecular density. We also investigated the molecular recognition of template oligonucleotides by comparing dT30 with dA30, indicating that AzoAde had not a specific interaction with dA30 at the air-water interface due to base mismatching. The AzoAde monolayer on the dA30 subphase gave H-aggregate from monomeric state by compressing it. On the other hand, it remained J-aggregated state on dT30 subphase regardless of compression. It was, therefore, suggested that the linear oligothymidylic acids, dT, (n > 4), acted as templates for assembling AzoAde at the air-water interface.     

Probing biomolecular interactions with dual polarization interferometry: real-time and label-free coralyne detection by use of homoadenine DNA oligonucleotide

We incorporated the specific recognition of adenine-rich singled-stranded DNA (ssDNA) into dual polarization interferometry (DPI) measurements for direct, selective, and sensitive detection of the small molecule coralyne, and we simultaneously employed the real-time and label-free technique for detailed investigation of the interaction between coralyne and adenine-rich ssDNA. Data from UV-visible spectroscopy, circular dichroism (CD) spectroscopy, and DNA melting firmly confirmed that 48-mer homoadenine ssDNA oligonucleotide (A(48)) had highly specific recognition for coralyne, whereas 48-mer homothymine ssDNA oligonucleotide (T(48)) as the control had no such recognition. The immobilization of ssDNA (A(48) or T(48)) on a silicon oxynitride chip could be achieved through a preadsorbed poly(ethylenimine) (PEI) layer. Mass, thickness, and refractive index (RI) changes resolved by DPI during the whole process of ssDNA immobilization suggested that most ssDNA molecules were likely to lie on the PEI surface mainly in the form of a flat monolayer and insert themselves partly into the PEI layer. Qualitative and quantitative analysis of mass, thickness, and RI changes in A(48)/PEI layer upon addition of different concentrations of coralyne revealed that A(48) most likely underwent a conformational change from single-stranded to double-stranded structure. By evaluation of the binding curves from changes in mass, the association rate constant (k(a)), dissociation rate constant (k(d)), and association constant (K(A)) between coralyne and A(48) were determined to be 4.95 × 10(3) M(-1) s(-1), 0.031 s(-1), and 1.6 × 10(5) M(-1), respectively. Good linear correlations between coralyne concentrations ranging from 0.5 to 12 μM and three parameters (mass, thickness, and RI) resolved by the response to coralyne binding were obtained. The detection limits were 0.22 μM for mass calibration, 0.14 μM for thickness calibration, and 0.32 μM for RI calibration. The high selectivity of the biosensor to coralyne at the A(48)/PEI interface was successfully confirmed by using the other two interfaces (T(48)/PEI and PEI) and three typical intercalators (ethidium bromide, daunomycin, and methylene blue). It is expected that the biosensing platform may be extended to simultaneously detect and characterize the interactions of a variety of target molecules with functional DNA molecules with high sensitivity.     

Optimized nanoparticle-mediated delivery of CRISPR-Cas9 system for B cell intervention

B cells exert multiple effector functions, and dysfunctions of B cells often lead to initiation and progression of diseases, including autoimmune and inflammatory diseases. Therefore, B cell intervention may be an effective strategy to treat diseases involving B cells. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing system has been widely used for DNA deletion, insertion, and replacement. Nanocarriers have been developed as relatively mature systems and may be applied to deliver the CRISPR-Cas9 system to B cells in vivo. In this study, we created a library of nanoparticles (NPs) with different polyethylene glycol densities and zeta potentials and screened an optimal NP for in vivo B cell targeting. The selected NP could deliver the CRISPR-Cas9 system to B cells and induce Cas9 expression inside the cell environment. Injection of the NP encapsulated with Cas9/gB220 (NP_Cas9/gB220) into mice could disrupt B220 expression in B cells, suggestive of its applications to intervene the expression of the target molecule in B cells. Moreover, the treatment with NP_Cas9/gBAFFR could decrease the number of B cells and exert therapeutic effect in rheumatoid arthritis, as B-cell activating factor receptor (BAFFR) is vital for the survival and functions of B cells. In conclusion, we developed a carrier for the delivery of the CRISPR-Cas9 gene editing system for B cell intervention that could be used for the treatment of diseases related to B cell dysfunctions.

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Electrochemical assay of nonlabeled DNA chip and SNOM imaging by using streptavidin-biotin interaction

The assay of DNA biosensor-based nucleic acid recognition using microfabrication technology provides for high sensitivity, good surface coverage and reproducibility. We have achieved efficient immobilization and hybridization of nonlabeled DNA using cyclic voltammetry (CV), square wave voltammetry (SWV) and scanning near-field optical microscopy (SNOM) techniques. The increased electrochemical response observed following the immobilization of biotinlyated ssDNA probe suggests that nucleic acid is a somewhat better medium for electronic transfer. We demonstrated the high coverage of immobilized FITC-labeled biotinylated DNA probe on a streptavidin-modified surface using SNOM imaging. SNOM imaging of FITC-labeled complementary DNA also exhibited fluorescent light spots of hybridization distributed throughout. No fluorescent light was observed with the hybridization of non-complementary DNA.     

Friend or Foe? Evidence Indicates Endogenous Exosomes Can Deliver Functional gRNA and Cas9 Protein

The clustered regularly interspaced short palindromic repeats (CRISPR)/associated nuclease (Cas) system is an efficient gene editing tool. In this study, it is found that both single guide RNA (gRNA) and Cas9 protein could be exported from the CRISPR/Cas9‐expressing cells by endogenous exosomes independently. Further experiments demonstrate that these naturally produced endogenous exosomes could be used as a vehicle to deliver the functional Cas9 and hepatitis B virus (HBV)‐specific gRNA to cut HBV DNA transfected in HuH7 cells or human papilloma virus (HPV)‐specific gRNA to cut the integrated HPV DNA in HeLa cells, respectively. In conclusion, this study indicates the potential of endogenous exosomes as a safe and effective delivery vehicle of the functional gRNA and Cas9 protein. Meanwhile, the endogenous exosomes‐mediated delivery of gene editing activity to adjacent and distant cells or tissues may further complicate the off‐target and safety concerns about the CRISPR/Cas9 system.     

Periodic DNA nanotemplates synthesized by rolling circle amplification

Rolling circle amplification (RCA) is an elegant biochemical method by which long single-stranded DNA molecules with a repeating sequence motif can be readily synthesized. In RCA, small circular single-stranded oligonucleotides serve as templates for the polymerization of the complementary strand. A DNA polymerase with an efficient strand displacement activity can copy the circular template without stopping. This results in a long DNA strand with periodic sequence. We here demonstrate that this method, using DNA recognition and biotin-streptavidin binding, provides a simple procedure for DNA-directed nanoscale organization of matter. As an example, a 74 nucleotide (nt) long circular DNA molecule is amplified into a sequence-periodic single strand with a length up to several micrometers. Hybridization of this long periodic DNA template to the biotinylated complement of the sequence motif results in a long DNA duplex with a periodic arrangement of biotin binding sites. On this duplex, streptavidin-coated particles can be organized into one-dimensional arrays. The resulting DNA constructs are characterized by gel electrophoresis and atomic force microscopy.     

A Nonconventional Approach to Patterned Nanoarrays of DNA Strands for Template‐Assisted Assembly of Polyfluorene Nanowires

DNA molecules have been widely recognized as promising building blocks for constructing functional nanostructures with two main features, that is, self‐assembly and rich chemical functionality. The intrinsic feature size of DNA makes it attractive for creating versatile nanostructures. Moreover, the ease of access to tune the surface of DNA by chemical functionalization offers numerous opportunities for many applications. Herein, a simple yet robust strategy is developed to yield the self‐assembly of DNA by exploiting controlled evaporative assembly of DNA solution in a unique confined geometry. Intriguingly, depending on the concentration of DNA solution, highly aligned nanostructured fibrillar‐like arrays and well‐positioned concentric ring‐like superstructures composed of DNAs are formed. Subsequently, the ring‐like negatively charged DNA superstructures are employed as template to produce conductive organic nanowires on a silicon substrate by complexing with a positively charged conjugated polyelectrolyte poly[9,9‐bis(6′‐N,N,N‐trimethylammoniumhexyl)fluorene dibromide] (PF2) through the strong electrostatic interaction. Finally, a monolithic integration of aligned arrays of DNA‐templated PF2 nanowires to yield two DNA/PF2‐based devices is demonstrated. It is envisioned that this strategy can be readily extended to pattern other biomolecules and may render a broad range of potential applications from the nucleotide sequence and hybridization as recognition events to transducing elements in chemical sensors.     

Preparation and characterization of plasmid DNA network via both triple helix formation and photo-crosslinking

New self-assembled network of plasmid DNA was prepared via both photo-crosslinking and triple helix formations, and evaluated the conformation of DNA network by electrophoresis. Two kinds of TFO sequences were designed to form the triple helix with pUC19 at different positions. In addition to the triple helix formation, a photo-crosslinking and lectin-molecular recognition were applied to form pUC19 network. Psoralen, photoreagent, and biotin were attached to 5′- and 3′-ends of the TFO, respectively. The biotin–TFO–psoralen conjugate, pUC19, and SA were mixed in a buffer and stand at 4 °C. The results of electrophoresis study indicated that both triple helix formation and photo-crosslinking with pUC19 were necessary for the network formation. Therefore, this method can provide stable plasmid DNA network that may be useful to manipulate nanostructure using typical plasmid DNA conformational changes: supercoiled circular and open circular forms.