Multifunctional DNA Nanoflower Applied for High Specific Photodynamic Cancer Therapy In Vivo.

Photodynamic therapy (PDT) is a newly emerged strategy for disease treatment. One challenge of the application of PDT drugs is the side-effect caused by the non-specificity of the photosensitive molecules. Most of the photosensitizers may invade not only the pathogenic cells but also the normal cells. In recent, people tried to use special cargoes to deliver the drugs into target cells. DNA nanoflowers (NFs) are a kind of newly-emerged nanomaterial which constructed through DNA rolling cycle amplification (RCA) reaction. It is reported that the DNA NFs were suitable materials which have been widely applied as nanocargos for drug delivery in cancer chemotherapeutic treatment. In this paper, we have introduced a new multifunctional DNA NF which could be prepared through an one-pot RCA reaction. This proposed DNA NF contained a versatile AS1411 G-quadruplex moiety, which plays key roles not only for specific recognition of cancer cells but also for near-infrared ray based photodynamic therapy when conjugating with a special porphyrin molecule. We demonstrated that the DNA NF showed good selectivity toward cancer cells, leading to highly efficient photo-induced cytotoxicity. Moreover, the in vivo experiment results suggested this DNA NF is a promising nanomaterial for clinical PDT.

Cationic porphyrins with large side arm substituents as resonance light scattering ratiometric probes for specific recognition of nucleic acid G-quadruplexes.

Specific G-quadruplex-probing is crucial for both biological sciences and biosensing applications. Most reported probes are focused on fluorescent or colorimetric recognition of G-quadruplexes. Herein, for the first time, we reported a new specific G-quadruplex-probing technique-resonance light scattering (RLS)-based ratiometric recognition. To achieve the RLS probing of G-quadruplexes in the important physiological pH range of 7.4-6.0, four water soluble cationic porphyrin derivatives, including an unreported octa-cationic porphyrin, with large side arm substituents were synthesized and developed as RLS probes. These RLS probes were demonstrated to work well for ratiometric recognition of G-quadruplexes with high specificity against single- and double-stranded DNAs, including long double-stranded ones. The working mechanism was speculated to be based on the RLS signal changes caused by porphyrin protonation that was promoted by the end-stacking of porphyrins on G-quadruplexes. This work adds an important member in G-quadruplex probe family, thus providing a useful tool for studies on G-quadruplex-related events concerning G-quadruplex formation, destruction and changes in size, shape and aggregation. As a proof-of-concept example of applications, the RLS probes were demonstrated to work well for label-free and sequence-specific sensing of microRNA. This work also provides a simple and useful way for the preparation of cationic porphyrins with high charges.

Chiral Interaction Is a Decisive Factor To Replace d-DNA with l-DNA Aptamers.

Nucleic acid aptamers have been widely used in various fields such as biosensing, DNA chip, and medical diagnosis. However, the high susceptibility of nucleic acids to ubiquitous nucleases reduces the biostability of aptamers and limits their applications in biological contexts. Therefore, improving the biostability of aptamers becomes an urgent need. Herein, we present a simple strategy to resolve this problem by directly replacing the d-DNA-based aptamers with left-handed l-DNA. By testing several reported aptamers against respective targets, we found that our proposed strategy stood up well for nonchiral small molecule targets (e.g., Hemin and cationic porphyrin) and chiral targets whose interactions with aptamers are chirality-independent (e.g., ATP). We also found that the l-DNA aptamers were indeed endowed with greatly improved biostability due to the extraordinary resistance of l-DNA to nuclease digestion. With respect to other small-molecule targets whose interactions with aptamers are chirality-dependent (e.g., kanamycin) and biomacromolecules (e.g., tyrosine kinase-7), however, the proposed strategy was not entirely effective likely due to the participation of the DNA backbone chirality into the target recognition. In spite of this limitation, this strategy indeed paves an easy way to screen highly biostable aptamers important for the applications in many fields.

Isothermal cross-boosting extension-nicking reaction mediated exponential signal amplification for ultrasensitive detection of polynucleotide kinase.

A novel nucleic acid-based isothermal signal amplification strategy, named cross-boosting extension-nicking reaction (CBENR) is developed and successfully used for rapid and ultrasensitive detection of polynucleotide kinase (PNK) activity. Only two simple oligonucleotides (recognition substrate (RS) and TaqMan probe) are applied to construct the PNK-sensing platform. In the presence of PNK, the 3'-phosphate end of RS will be converted to the 3'-hydroxyl one, and then extended to a long poly-adenine (poly-A) sequence under the catalysis of terminal deoxynucleotidyl transferase (TdT). The poly-A sequence provides multiple binding sites for the TaqMan probe to form multiple DNA duplexes. Subsequently, ribonuclease HII (RNase HII) cuts the TaqMan probe into two parts at the pre-set uracil site, generating a fluorescence signal and providing new substrates for TdT elongation. The TdT-catalyzed substrate extension and RNase HII-catalyzed probe nicking are boosted by each other, resulting in persistent enlargement of these two reactions and thus giving ultrahigh signal amplification efficiency. Utilizing the CBENR-based PNK sensor, ultrasensitive detection of PNK activity was achieved with a detection limit as low as 3.0 × 10-6 U mL-1. Quantification of endogenous PNK activity at the single-cell level and the screening/evaluation of PNK inhibitors were also achieved.

Nanolantern-Based DNA Probe and Signal Amplifier for Tumor-Related Biomarker Detection in Living Cells.

The introduction of nanotechnology can overcome some inherent drawbacks of traditional DNA probes, thus promoting their applications in living cells. Herein, a three-dimensional DNA nanostructure, a DNA nanolantern, was prepared via simple nucleotide hybridization of four short-stranded oligonucleotides and successfully applied to the construction of a novel DNA probe and signal amplifier. Compared to most reported DNA nanostructures, a DNA nanolantern shows the distinct advantages of low cost, easy design and preparation, more and arbitrary adjusted probe numbers, and high fluorescence resonance energy transfer (FRET) signal readout. Compared to traditional DNA probes, the constructed nanolantern-based one has improved cell internalization efficiency, enhanced biostability, accelerated reaction kinetics, excellent biocompatibility, and greatly reduced false-positive output and was demonstrated to work well for probing the expression level of tumor-related mRNA and microRNA in living cells. The DNA nanolantern can also be easily integrated with some reported signal amplification strategies, e.g., isothermal hybridization chain reaction (HCR), and the obtained signal amplifier combines the advantages of the DNA nanolantern and the HCR, enabling sensitive imaging detection of ultralow abundance targets in living cells. This work demonstrated that this simple DNA nanostructure can not only improve the performance of traditional DNA probes but can also be easily integrated with reported DNA-based strategy and technology, thus showing a broad application prospect.

Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase.

As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3'-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.

Recent Advances in Constructing Higher-Order DNA Structures.

Molecular self-assembly is widely used in the fields of biosensors, molecular devices, efficient catalytic materials, and medical biomaterials. As the carrier of genetic information, DNA is a kind of biomacromolecule composed of deoxyribonucleotide units. DNA nanotechnology extends DNA of its original properties as a molecule that stores and transmits genetic information from its biological environment by taking advantage of its unique base pairing and inherent biocompatibility to produce structurally-defined supramolecular structures. With the continuously development of DNA technology, the assembly method of DNA nanostructures is not only limited on the basis of DNA hybridization but also other biochemical interactions. In this review, we summarize the latest methods used to construct higher-order DNA structures. The problems of DNA nanostructures are discussed and the future directions in this field are provided.

DNA nanostructure-based nucleic acid probes: construction and biological applications.

In recent years, DNA has been widely noted as a kind of material that can be used to construct building blocks for biosensing, in vivo imaging, drug development, and disease therapy because of its advantages of good biocompatibility and programmable properties. However, traditional DNA-based sensing processes are mostly achieved by random diffusion of free DNA probes, which were restricted by limited dynamics and relatively low efficiency. Moreover, in the application of biosystems, single-stranded DNA probes face challenges such as being difficult to internalize into cells and being easily decomposed in the cellular microenvironment. To overcome the above limitations, DNA nanostructure-based probes have attracted intense attention. This kind of probe showed a series of advantages compared to the conventional ones, including increased biostability, enhanced cell internalization efficiency, accelerated reaction rate, and amplified signal output, and thus improved in vitro and in vivo applications. Therefore, reviewing and summarizing the important roles of DNA nanostructures in improving biosensor design is very necessary for the development of DNA nanotechnology and its applications in biology and pharmacology. In this perspective, DNA nanostructure-based probes are reviewed and summarized from several aspects: probe classification according to the dimensions of DNA nanostructures (one, two, and three-dimensional nanostructures), the common connection modes between nucleic acid probes and DNA nanostructures, and the most important advantages of DNA self-assembled nanostructures in the applications of biosensing, imaging analysis, cell assembly, cell capture, and theranostics. Finally, the challenges and prospects for the future development of DNA nanostructure-based nucleic acid probes are also discussed.

An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity.

DNA methylation is a significant epigenetic mechanism involving processes of transferring a methyl group onto cytosine or adenine. Such DNA modification catalyzed by methyltransferase (MTase) plays important roles in the modulation of gene expression and other cellular activities. Herein, we develop a simple and sensitive biosensing platform for the detection of DNA MTase activity by using only two oligonucleotides. The fluorophore labeled molecular beacon (MB) can be methylated by MTase and subsequently cleaved by endonuclease DpnI at the stem, giving a shortened MB. The shortened MB can then hybridize with a primer DNA, initiating a cycle of strand displacement amplification (SDA) reactions. The obtained SDA products can unfold new MB and initiate another cycle of SDA reaction. Therefore, continuous enlargement of SDA and exponential amplification of the fluorescence signal are achieved. Because the triple functions of substrate, template and probe are elegantly integrated in one oligonucleotide, only two oligonucleotides are necessary for multiple amplification cycles, which not only reduces the complexity of the system, but also overcomes the laborious and cumbersome operation that is always a challenge in conventional methods. This platform exhibits an extremely low limit of detection of 3.3 × 10-6 U mL-1, which is the lowest to our knowledge. The proposed MTase-sensing platform was also demonstrated to perform well in a real-time monitoring mode, which can achieve a further simplified and high-throughput detection. The sensing strategy might be extended to the activity detection of other enzymes, thus showing great application potential in bioanalysis and clinical diagnosis.

Terminal Deoxynucleotidyl Transferase-Catalyzed Preparation of pH-Responsive DNA Nanocarriers for Tumor-Targeted Drug Delivery and Therapy.

Developing a highly efficient carrier for tumor-targeted delivery and site-specific release of anticancer drugs is a good way to overcome the side effects of traditional cancer chemotherapy. Benefiting from the nontoxic and biocompatible characteristics, DNA-based drug carriers have attracted increasing attention. Herein, we reported a novel and readily manipulated strategy to construct spherical DNA nanocarriers. In this strategy, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA extension reaction is used to prepare a thick DNA layer on a gold nanoparticle (AuNP) surface by extending long poly(C) sequences from DNA primers immobilized on AuNPs. The poly(C) extension products can then hybridize with G-rich oligonucleotides to give CG-rich DNA duplexes (for loading anticancer drug doxorubicin, Dox) and multiple AS1411 aptamers. Via synergic recognition of multiple aptamer units to nucleolin proteins, biomarker of malignant tumors, Dox-loaded DNA carrier can be efficiently internalized in cancer cells and achieve burst release of drugs in acidic organelles because of i-motif formation-induced DNA duplex destruction. An as-prepared pH-responsive drug carrier was demonstrated to be promising for highly efficient delivery of Dox and selective killing of cancer cells in both in vitro and in vivo experiments, thus showing a huge potential in anticancer therapy.

G-Quadruplex/Porphyrin Composite Photosensitizer: A Facile Way to Promote Absorption Redshift and Photodynamic Therapy Efficacy.

Photosensitizer is one of the most important elements of photodynamic therapy (PDT). Herein, we reported a novel strategy to prepare a new series of composite photosensitizers. The composite photosensitizer was prepared by simply mixing DNA G-quadruplexes with a hydrophilic porphyrin (TMPipEOPP)4+·4I-. Compared with the conventional porphyrin photosensitizers, the excitation wavelength of the composite one has been ∼50 nm redshifted (from 650 to 700 nm), which is beneficial to the penetration of the light. Moreover, the composite photosensitizer showed an about 7.4-fold increase of light absorption efficiency, thus greatly enhancing the singlet oxygen (1O2) generation capacity and PDT efficacy. What is more, the introduction of nucleic acids in the composite photosensitizer could also provide some extra charming properties, such as the targeted recognition ability conferred by aptamer and high capability to assemble with various drug carriers. We demonstrated that the composite photosensitizer could be easily assembled with MnO2 nanosheet. The obtained nanodevice integrated the merits of a composite photosensitizer and MnO2 nanosheet, thus showing strong near-infrared absorption, high 1O2 generation efficiency, avoidance of nonideal 1O2 consumption by glutathione, and in situ O2 generation to relieve tumor hypoxia. This nanodevice showed greatly improved PDT efficacy both in vitro and in vivo, presenting a huge potential for applications in clinical therapy for tumors.

A modified exponential amplification reaction (EXPAR) with an improved signal-to-noise ratio for ultrasensitive detection of polynucleotide kinase.

We applied a modified exponential amplification reaction (EXPAR) strategy to design a label-free and "one-pot" biosensor for ultrasensitive detection of polynucleotide kinase (PNK). This method was also successfully applied in the screening of PNK inhibitors and analysis of the endogenous PNK activity at the single-cell level.

Trifunctional integrated DNA-based universal sensing platform for detection of diverse biomolecules in one-pot isothermal exponential amplification mode.

A biosensor with all the advantages of ultra-high sensitivity, easy operation, straightforward signal output and universal applicability is introduced. The biosensor was demonstrated to work well in the detection of polynucleotide kinase and DAM methyltransferase, thus providing a powerful tool for clinical diagnosis, drug screening and disease therapeutic assay.

pH-Controlled Intracellular in Situ Reversible Assembly of a Photothermal Agent for Smart Chemo-Photothermal Synergetic Therapy and ATP Imaging.

To advance anti-tumor efficiency and lessen the adverse effect caused by nanodrug residues in the body, a smart nanoagent system is developed and successfully used in intracellular ATP imaging and in vivo chemo-photothermal synergetic therapy. The nanoagent system is facilely prepared using a DNA complex to modify gold nanoparticles (AuNPs). The DNA complex is formed by three oligonucleotides (ATP aptamer, rC-DNA, and rG-DNA). The CG-rich structure in a ternary DNA complex could be exploited for payload of chemotherapeutic medicine doxorubicin (DOX), thus making efficient DOX transport into the tumor site possible. In tumor cells, especially in acidic organelles (e.g., endosome and lysosome), DOX could be rapidly released via the dual stimuli of overexpressed ATP and pH. What is more, the specific recognition of a fluorescently labeled aptamer strand to ATP can achieve the intracellular ATP imaging. pH-controlled reversible folding and unfolding of intermolecular i-motif formed by C-rich strands can lead to intracellular in situ assembly of AuNP aggregates with high photothermal conversion efficiency and promote relatively facile renal clearance of AuNPs through the disassociation of the aggregates in extracellular environments. Experiments in vivo and vitro present feasibility for a synergetic chemo-photothermal therapy. Such an in situ reversible assembly strategy of a chemo-photothermal agent also presents a new paradigm for a smart and highly efficient disease treatment with reduced side effects.