Danqing Lu

Photon-manipulated drug release from a mesoporous nanocontainer controlled by azobenzene-modified nucleic acid.

Herein a photon-manipulated mesoporous release system was constructed based on azobenzene-modified nucleic acids. In this system, the azobenzene-incorporated DNA double strands were immobilized at the pore mouth of mesoporous silica nanoparticles. The photoisomerization of azobenzene induced dehybridization/hybridization switch of complementary DNA, causing uncapping/capping of pore gates of mesoporous silica. This nanoplatform permits holding of guest molecules within the nanopores under visible light but releases them when light wavelength turns to the UV range. These DNA/mesoporous silica hybrid nanostructures were exploited as carriers for the cancer cell chemotherapy drug doxorubicin (DOX) due to its stimuli-responsive property as well as good biocompatibility via MTT assay. It is found that the drug release behavior is light-wavelength-sensitive. Switching of the light from visible to the UV range uncapped the pores, causing the release of DOX from the mesoporous silica nanospheres and an obvious cytotoxic effect on cancer cells. We envision that this photocontrolled drug release system could find potential applications in cancer therapy.

Targeted Bioimaging and Photodynamic Therapy Nanoplatform Using an Aptamer‐Guided G‐Quadruplex DNA Carrier and Near‐Infrared Light

Photodynamic therapy (PDT) has recently emerged as an effective, noninvasive, and economical treatment for diseases including cancers. Traditional PDT suffers mainly from having an insufficient number of photons penetrating the tissue and preferentially targeting cancerous tissues with photosensitizers. Therefore, it is necessary to construct a method for controllable singlet-oxygen (O2) generation (SOG) with high selectivity and accurate localization to provide more efficient PDTwith fewer side effects. Several research groups have developed novel selective PDT agents, such as peptide or protein conjugates, and photosensitizer encapsulated nanocarriers. In recent years, single-stranded oligonucleotides called aptamers have emerged as a novel class of molecules which rival antibodies in both therapeutic and diagnostic applications. Compared to antibodies, aptamers offer significant advantages, such as flexible design, synthetic accessibility, easy modification, chemical stability, and rapid tissue penetration. Therefore, aptamers can potentially endow traditional PDT with high selectivity and accurate localization. The formation of a G-quadruplex (GQ) structure is results from a type of DNA self-assembly mode. This structure can be stabilized by a central monovalent metal cation such as potassium or sodium ions and small-molecule ligands including porphyrins. Cationic porphyrins usually bind to G-quadruplexes through p–p interactions with G-quartets and through electrostatic interactions with the anionic phosphate groups on G-quadruplexes. According to previous studies, some photosensitizers are porphyrin derivatives and are broadly used in PDT, such as 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4). Therefore, the G-quadruplex DNA sequence can be a carrier for photosensitizers with porphyrin molecular structures. By taking advantage of the loading function of the G-quadruplex structure and the recognition function of aptamers, the photosensitizer can be delivered to a target cell with high affinity and selectivity. Up to now, most photosensitizers have been activated by visible light. As a consequence, the shallow penetration depth of incident light has limited their otherwise wide applications. If the photosensitizers are linked to a visible-light generator and the generator is remotely controlled (turned on/off) by near-infrared (NIR) light, then the problem of limited depth penetration by visible light could be easily overcome. To solve the problem of limited depth penetration of current PDT techniques, we used upconversion nanoparticles (UCNPs), in particular, lanthanide-doped rare-earth nanocrystals. These nanomaterials are able to emit shorter-wavelength photons under excitation by NIR light, thus making them good visiblelight generators with the ability to be remotely controlled by NIR light. Furthermore, owing to the ladderlike arrangement of energy levels in lanthanide ions, UCNPs show high efficiency of photon upconversion with a distinct set of sharp emission peaks under moderate excitation densities, thereby enabling targeted bioimaging when functionalized with biomolecular recognition moieties. Herein, we report a specific aptamer-guided G-quadruplex DNA nanoplatform for targeted bioimaging and PDT, and it is capable of selective recognition and imaging of cancer cells, controllable and effective activation of the photosensitizer, and improvement of the therapeutic effect. In particular, a guanine-rich DNA segment is linked to an aptamer to form a bifunctional DNA sequence, termed a G4aptamer. The G4-aptamer not only loads the photosensitizer but also specifically recognizes target cells. As shown in Scheme 1, the G4-aptamer is bioconjugated to a UCNP, thus placing the photosensitizer TMPyP4 at position near the UCNP for energy transfer between the UCNP and TMPyP4. Once the nanoplatform is delivered into cancer cells, the UCNPs are excited by NIR light to emit visible light to image cancer cells and, in turn, to activate TMPyP4, which, finally, generates sufficient ROS to efficiently kill cancer cells. [*] Prof. Q. Yuan, Y. Wu, D. Lu, Dr. Z. Zhao, T. Liu, Prof. X. Zhang, Prof. W. Tan Molecular Science and Biomedicine Laboratory State Key Laboratory for Chemo/Bio-Sensing and Chemometrics College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Research Center for Chemistry and Molecular Medicine Hunan University, Changsha 410082 (China) E-mail: tan@chem.ufl.edu Prof. Q. Yuan, J. Wang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education) College of Chemistry and Molecular Sciences Wuhan University, Wuhan 430072 (China) [] These authors contributed equally to this work.

Molecular Beacon-Based Junction Probes for Efficient Detection of Nucleic Acids via a True Target-Triggered Enzymatic Recycling Amplification

This work reports the development of a new molecular beacon-based junction sensing system with highly sensitive DNA detection and a strong capability to identify SNPs. The single linear probe typically labels the midsection of the oligonucleotide, but our next-generation junction sensing system uses a hairpin-structured MB with labels on each end of the oligonucleotide to maintain the cleaving activity of our newly designed ssDNA-cleaved endonuclease, Nt.BbvCI, rather than the typical dsDNA-cleaved endonuclease. These design improvements guarantee a true and efficient target-triggered enzymatic recycling amplification process in our sensing system. They also afford a faster and more sensitive response toward target DNA than the first-generation junction sensing system.

Localizable and Photoactivatable Fluorophore for Spatiotemporal Two-Photon Bioimaging

Photoactivatable probe-based fluorescent imaging has become an efficient and attractive technique for spatiotemporal microscopic studies of biological events. However, almost all previously reported photoactivatable organic probes have been based on hydrosoluble precursors, which have produced water-soluble active fluorophores able to readily diffuse away from the photocleavage site, thereby dramatically reducing spatial resolution. Hydroxyphenylquinazolinone (HPQ), a small organic dye known for its classic luminescence mechanism through excited-state intramolecular proton transfer (ESIPT), shows strong light emission in the solid state, but no emission in solution. In this work, HPQ was employed as a precursor to develop a localizable, photoactivatable two-photon probe (PHPQ) for spatiotemporal bioimaging applications. After photocleavage, PHPQ releases a precipitating HPQ fluorophore which shows both one-photon and two-photon excited yellow-green fluorescence, thereby producing a localizable fluorescence signal that affords high spatial resolution for bioimaging, with more than 200-fold one-photon and 150-fold two-photon fluorescence enhancement.

Molecular engineering of a mitochondrial-targeting two-photon in and near-infrared out fluorescent probe for gaseous signal molecules H2S in deep tissue bioimaging

Hydrogen sulfide (H2S), one of the biologically important gaseous signal molecules, plays an essential role in diverse normal biochemical functions and pathological processes. Herein, an efficient two-photon in and near-infrared out mitochondria-targeting dye has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (C˭C) of 4-hydroxybenzaldehyde and 6-(diethylamino)-1,2,3,4-tetrahydroxanthylium (mitochondria-targeting), which possesses large two-photon action absorption cross-section ~160g and high fluorescence quantum yield ~0.15. Encouraged by the results, we proceeded to conjugate this new dye with a H2S recognition moiety (4-dinitrobenzene-ether, DNB), on the basis of the intramolecular charge transfer (ICT) strategy, to construct a novel H2S fluorescent probe (TP-NIR-HS), which shows a targeting ability with high sensitivity and selectivity, and low cytotoxicity. This new probe was then applied for two-photon imaging of living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ~350µm, thus demonstrating its practical application in biological systems, and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of H2S.

Aptamer-assembled nanomaterials for fluorescent sensing and imaging

Abstract Aptamers, which are selected in vitro by a technology known as the systematic evolution of ligands by exponential enrichment (SELEX), represent a crucial recognition element in molecular sensing. With advantages such as good biocompatibility, facile functionalization, and special optical and physical properties, various nanomaterials can protect aptamers from enzymatic degradation and nonspecific binding in living systems and thus provide a preeminent platform for biochemical applications. Coupling aptamers with various nanomaterials offers many opportunities for developing highly sensitive and selective sensing systems. Here, we focus on the recent applications of aptamer-assembled nanomaterials in fluorescent sensing and imaging. Different types of nanomaterials are examined along with their advantages and disadvantages. Finally, we look toward the future of aptamer-assembled nanomaterials.

A universal aptameric biosensor: Multiplexed detection of small analytes via aggregated perylene-based broad-spectrum quencher

A universal aptameric system based on the taking advantage of double-stranded DNA/perylene diimide (dsDNA/PDI) as the signal probe was developed for multiplexed detection of small molecules. Aptamers are single-stranded DNA or RNA oligonucleotides which are selected in vitro by a process known as systematic evolution of ligands by exponential enrichment. In this work, we synthesized a new kind of PDI and reported this aggregated PDI could quench the double-stranded DNA (dsDNA)-labeled fluorophores with a high quenching efficiency. The quenching efficiencies on the fluorescence of FAM, TAMRA and Cy5 could reach to 98.3%±0.9%, 97.2%±0.6% and 98.1%±1.1%, respectively. This broad-spectrum quencher was then adopted to construct a multicolor biosensor via a label-free approach. A structure-switching-triggered enzymatic recycling amplification was employed for signal amplification. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity towards small analytes. For other targets, changing the corresponding aptamer can achieve the goal. The quencher did not interfere with the catalytic activity of nuclease. The biosensor could be manipulated with similar sensitivity no matter in pre-addition or post-addition manner. Moreover, simultaneous and multiplexed analysis of several small molecules in homogeneous solution was achieved, demonstrating its potential application in the rapid screening of multiple biotargets.

mRNA-Initiated, Three-Dimensional DNA Amplifier Able to Function inside Living Cells

DNA molecular machines show great promise in fields such as biomarker discovery and biological activity regulation, but operating DNA machines with specific functions within living systems remains extremely challenging. Although DNA machines have been engineered with exact molecular-level specifications, some intrinsic imperfections such as poor cell permeation and fragility in complex cytoplasmic milieu persist due to the well-established character of nucleic acid molecules. To circumvent these problems, we herein report a molecularly engineered, entropy-driven three-dimensional DNA amplifier (EDTD) that can operate inside living cells in response to a specific mRNA target. In particular, mRNA target/EDTD interaction can specifically initiate an autonomous DNA circuit inside living cells owing to the exclusive entropy-driven force, thus providing enormous signal amplification for ultrasensitive detection of the mRNA. Moreover, owing to molecular engineering of a unique DNA tetrahedral framework into the DNA amplifier, EDTD exhibits significantly enhanced biostability and cellular uptake efficiency, which are prerequisites for DNA machines used for in vivo applications. This programmable DNA machine presents a simple and modular amplification mechanism for the detection of intracellular biomarkers. Moreover, this study provides a potentially valuable molecular tool for understanding the chemistry of cellular systems and offers a design blueprint for further expansion of DNA nanotechnology in living systems.

Tetraphenylethene derivative modified DNA oligonucleotide for in situ potassium ion detection and imaging in living cells.

The monitoring of K+ is very important and emergency because of their unique relationship in various disease diagnosis and treatment. G-quadruplex analogue is a classical recognition unit for K+ detection and has been widely applied in K+ relevant research. Common fluorescent dyes were employed for design of G-quadruplex structure-based K+ probes which suffered from the aggregation-caused quenching effect, and possibly limited the biological applications in living systems. Herein, we report an aggregation-induced emission (AIE) effect-based fluorescent probe for cellular K+ analysis and imaging. Benefitting from the K+ triggered AIE phenomenon, the designed TPE derivative modified guanine (G)-rich oligonucleotide fluorescent probe (TPE-oligonucleotide probe) exhibits high sensitivity (∼10-fold higher than most reported G-quadruplex-based probes) with extended photostability which facilitates the prolonged fluorescence observations of K+ in living cells. On the basis of these advantages, the TPE-oligonucleotide probe serves as a promising candidate for the functional study and analysis of K+.

A label-free and sensitive photoluminescence sensing platform based on long persistent luminescence nanoparticles for the determination of antibiotics and 2,4,6-trinitrophenol

The rapid detection of pollutants with high sensitivity and selectivity is of considerable significance for security screening, environmental safety, and human health. In this study, we prepared persistent luminescence nanoparticles (PLNPs) and constructed a label-free sensor for sensitive and selective detection of pollutants in real samples and test papers. Following excitation, PLNPs could store absorbed light energy and release it in the form of luminescence. Compared with a fluorescence-based technique, a PLNPs-based measurement could effectively avoid background interference. Under optimal conditions, the limit of detection for TNP was found to be 10 nM, while for an antibiotic it was 5 nM. The nanoprobe was successfully applied for the detection of pollutants in real samples including milk and Dianchi Lake water samples. Due to the long-lasting afterglow nature of PLNPs, the signal-to-noise ratio could be greatly increased in complex real samples. By hand-writing with TNP solution as ink on filter paper, the photoluminescence (PL) of the part stained with TNP was immediately quenched. Moreover, after direct exposure under a UV lamp for 10 min and without further excitation, the luminescence of the test paper was investigated to avoid interferents. This PLNP material could be potentially employed as a multi-responsive luminescent sensor. In addition, these easy-to-use visual techniques could provide a powerful tool for a convenient POC assay of organic pollutants.