Biosensing with DNAzymes.

This article provides a comprehensive review of biosensing with DNAzymes, providing an overview of different sensing applications while highlighting major progress and seminal contributions to the field of portable biosensor devices and point-of-care diagnostics. Specifically, the field of functional nucleic acids is introduced, with a specific focus on DNAzymes. The incorporation of DNAzymes into bioassays is then described, followed by a detailed overview of recent advances in the development of in vivo sensing platforms and portable sensors incorporating DNAzymes for molecular recognition. Finally, a critical perspective on the field, and a summary of where DNAzyme-based devices may make the biggest impact are provided.

Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets.

Beyond its extraordinary genome editing ability, the CRISPR-Cas systems have opened a new era of biosensing applications due to its high base resolution and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic-acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic-acid targets, we herein report CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detection. The sensors are based on the Cas12a-dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA), and its single-stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA molecules that can lock a DNA activator for Cas12a-crRNA, preventing the ssDNA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by commercially available portable fluorimeters. Using this method, ATP and Na+ have been detected quantitatively under ambient temperature (25 °C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system significantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanalytical and biomedical applications.

Two-in-One Chemogene Assembled from Drug-Integrated Antisense Oligonucleotides To Reverse Chemoresistance.

Combinatorial chemo and gene therapy provides a promising way to cure drug-resistant cancer, since the codelivered functional nucleic acids can regulate drug resistance genes, thus restoring sensitivity of the cells to chemotherapeutics. However, the dramatic chemical and physical differences between chemotherapeutics and nucleic acids greatly hinder the design and construction of an ideal drug delivery system (DDS) to achieve synergistic antitumor effects. Herein, we report a novel approach to synthesize a nanosized DDS using drug-integrated DNA with antisense sequences (termed "chemogene") to treat drug-resistant cancer. As a proof of concept, floxuridine (F), a typical nucleoside analog antitumor drug, was incorporated in the antisense sequence in the place of thymine (T) based on their structural similarity. After conjugation with polycaprolactone, a spherical nucleic acid (SNA)-like two-in-one chemogene can be self-assembled, which possesses the capabilities of rapid cell entry without the need for a transfection agent, efficient downregulation of drug resistance genes, and chronic release of chemotherapeutics for treating the drug-resistant tumors in both subcutaneous and orthotopic liver transplantation mouse models.

Metal-Dependent DNAzymes for the Quantitative Detection of Metal Ions in Living Cells: Recent Progress, Current Challenges, and Latest Results on FRET Ratiometric Sensors.

Many different metal ions are involved in various biological functions including metallomics and trafficking, and yet there are currently effective sensors for only a few metal ions, despite the first report of metal sensors for calcium more than 40 years ago. To expand upon the number of metal ions that can be probed in biological systems, we and other laboratories employ the in vitro selection method to obtain metal-specific DNAzymes with high specificity for a metal ion and then convert these DNAzymes into fluorescent sensors for these metal ions using a catalytic beacon approach. In this Forum Article, we summarize recent progress made in developing these DNAzyme sensors to probe metal ions in living cells and in vivo, including several challenges that we were able to overcome for this application, such as DNAzyme delivery, spatiotemporal control, and signal amplification. Furthermore, we have identified a key remaining challenge for the quantitative detection of metal ions in living cells and present a new design and the results of a Förster resonance energy transfer (FRET)-based DNAzyme sensor for the ratiometric quantification of Zn2+ in HeLa cells. By converting existing DNAzyme sensors into a ratiometric readout without compromising the fundamental catalytic function of the DNAzymes, this FRET-based ratiometric DNAzyme design can readily be applied to other DNAzyme sensors as a major advance in the field to develop much more quantitative metal-ion probes for biological systems.

A Crosslinked Nucleic Acid Nanogel for Effective siRNA Delivery and Antitumor Therapy.

Functional siRNAs are employed as cross-linkers to direct the self-assembly of DNA-grafted polycaprolactone (DNA-g-PCL) brushes to form spherical and nanosized hydrogels via nucleic acid hybridization in which small interfering RNAs (siRNAs) are fully embedded and protected for systemic delivery. Owing to the existence of multivalent mutual crosslinking events inside, the crosslinked nanogels with tunable size exhibit not only good thermostability, but also remarkable physiological stability that can resist the enzymatic degradation. As a novel siRNA delivery system with spherical nucleic acid (SNA) architecture, the crosslinked nanogels can assist the delivery of siRNAs into different cells without any transfection agents and achieve the gene silencing effectively both in vitro and in vivo, through which a significant inhibition of tumor growth is realized in the anticancer treatment.

A Molecular Recognition Approach To Synthesize Nucleoside Analogue Based Multifunctional Nanoparticles for Targeted Cancer Therapy.

Tumor-targeted drug delivery with simultaneous cancer imaging is highly desirable for personalized medicine. Herein, we report a supramolecular approach to design a promising class of multifunctional nanoparticles based on molecular recognition of nucleobases, which combine excellent tumor-targeting capability via aptamer, controlled drug release, and efficient fluorescent imaging for cancer-specific therapy. First, an amphiphilic prodrug dioleoyl clofarabine was self-assembled into micellar nanoparticles with hydrophilic nucleoside analogue clofarabine on their surface. Thereafter, two types of single-stranded DNAs that contain the aptamer motif and fluorescent probe Cy5.5, respectively, were introduced onto the surface of the nanoparticles via molecular recognition between the clofarabine and the thymine on DNA. These drug-containing multifunctional nanoparticles exhibit good capabilities of targeted clofarabine delivery to the tumor site and intracellular controlled drug release, leading to a robust and effective antitumor effect in vivo.

DNA Trojan Horses: Self-Assembled Floxuridine-Containing DNA Polyhedra for Cancer Therapy.

Based on their structural similarity to natural nucleobases, nucleoside analogue therapeutics were integrated into DNA strands through conventional solid-phase synthesis. By elaborately designing their sequences, floxuridine-integrated DNA strands were synthesized and self-assembled into well-defined DNA polyhedra with definite drug-loading ratios as well as tunable size and morphology. As a novel drug delivery system, these drug-containing DNA polyhedra could ideally mimic the Trojan Horse to deliver chemotherapeutics into tumor cells and fight against cancer. Both in vitro and in vivo results demonstrate that the DNA Trojan horse with buckyball architecture exhibits superior anticancer capability over the free drug and other formulations. With precise control over the drug-loading ratio and structure of the nanocarriers, the DNA Trojan horse may play an important role in anticancer treatment and exhibit great potential in translational nanomedicine.

Spatial imaging of glycoRNA in single cells with ARPLA.

Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization methods. We report sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA) to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggest that surface glycoRNA is inversely associated with tumor malignancy and metastasis. Investigation of the relationship between glycoRNAs and monocyte-endothelial cell interactions suggests that glycoRNAs may mediate cell-cell interactions during the immune response.

Monitoring leaching of Cd2+ from cadmium-based quantum dots by an Cd aptamer fluorescence sensor.

Quantum Dots (QDs) have been demonstrated with outstanding optical properties and thus been widely used in many biological and biomedical studies. However, previous studies have shown that QDs can cause cell toxicity, mainly attributable to the leached Cd2+. Therefore, identifying the leaching kinetics is very important to understand QD biosafety and cytotoxicity. Toward this goal, instrumental analyses such as inductively coupled plasma mass spectrometry (ICP-MS) have been used, which are time-consuming, costly and do not provide real-time or spatial information. To overcome these limitations, we report herein a fast and cost-effective fluorescence sensor based a Cd2+-specific aptamer for real-time monitoring the rapid leaching kinetics of QDs in vitro and in living cells. The sensor shows high specificity towards Cd2+ and is able to measure the Cd2+ leached either from water-dispersed CdTe QDs or two-layered CdSe/CdS QDs. The sensor is then used to study the stability of these two types of QDs under conditions to mimic cellular pH and temperature and the results from the sensor are similar to those obtained from ICP-MS. Finally, the sensor is able to monitor the leaching of Cd2+ from QDs in HeLa cells. The fluorescence aptamer sensor described in this study may find many applications as a tool for understanding biosafety of numerous other Cd-based QDs, including leaching kinetics and toxicity mechanisms in living systems.

Efficient delivery of a DNA aptamer-based biosensor into plant cells for glucose sensing through thiol-mediated uptake.

DNA aptamers have been widely used as biosensors for detecting a variety of targets. Despite decades of success, they have not been applied to monitor any targets in plants, even though plants are a major platform for providing oxygen, food, and sustainable products ranging from energy fuels to chemicals, and high-value products such as pharmaceuticals. A major barrier to progress is a lack of efficient methods to deliver DNA into plant cells. We herein report a thiol-mediated uptake method that more efficiently delivers DNA into Arabidopsis and tobacco leaf cells than another state-of-the-art method, DNA nanostructures. Such a method allowed efficient delivery of a glucose DNA aptamer sensor into Arabidopsis for sensing glucose. This demonstration opens a new avenue to apply DNA aptamer sensors for functional studies of various targets, including metabolites, plant hormones, metal ions, and proteins in plants for a better understanding of the biodistribution and regulation of these species and their functions.

A highly sensitive and selective fluoride sensor based on a riboswitch-regulated transcription coupled with CRISPR-Cas13a tandem reaction.

Nucleic acid sensors have realized much success in detecting positively charged and neutral molecules, but have rarely been applied for measuring negatively charged molecules, such as fluoride, even though an effective sensor is needed to promote dental health while preventing osteofluorosis and other diseases. To address this issue, we herein report a quantitative fluoride sensor with a portable fluorometer readout based on fluoride riboswitch-regulated transcription coupled with CRISPR-Cas13-based signal amplification. This tandem sensor utilizes the fluoride riboswitch to regulate in vitro transcription and generate full-length transcribed RNA that can be recognized by CRISPR-Cas13a, triggering the collateral cleavage of the fluorophore-quencher labeled RNA probe and generating a fluorescence signal output. This tandem sensor can quantitatively detect fluoride at ambient temperature in aqueous solution with high sensitivity (limit of detection (LOD) ≈ 1.7 µM), high selectivity against other common anions, a wide dynamic range (0-800 µM) and a short sample-to-answer time (30 min). This work expands the application of nucleic acid sensors to negatively charged targets and demonstrates their potential for the on-site and real-time detection of fluoride in environmental monitoring and point-of-care diagnostics.

pH-Responsive and Gemcitabine-Containing DNA Nanogel To Facilitate the Chemodrug Delivery.

Herein, we construct a structure-switchable gemcitabine (Ge)-containing DNA nanogel that can respond to the intracellular acidic environment, subsequently facilitating the chemodrug release inside the cells. Based on the structural similarity between Ge and deoxycytidine (dC), dC nucleotides in the component DNA strands used for nanogel assembly are fully replaced by Ge during their synthesis. By changing the designed sequences, two Ge-containing Y-shaped motifs with different sticky ends are first assembled and then associated together to form nanogel by sticky-end hybridizations. In particular, one of the sticky-end sequences is arbitrarily designed to be rich of Ge and the other is designed to be partially complementary to the first Ge-rich sticky end. At the neutral or basic condition, the Ge-rich sticky ends hybridize with the partially complementary sticky ends on the second Y motifs, keeping the assembled nanogel stable. Upon being exposed to the acidic condition, Ge-rich sticky ends intend to form intramolecular i-motif-like quadruplex structures, resulting in the disassembly of the nanogel. On the one hand, the nanosized feature enables the Ge-containing nanogel with rapid cellular uptake behavior. On the other hand, the pH-responsive feature endows the rapid disassembly of the nanogel to facilitate the enzymatic drug release inside the cell, resulting in the enhanced anticancer activity of the DNA-based drug delivery system.

Polygemcitabine nanogels with accelerated drug activation for cancer therapy.

To overcome the slow activation of gemcitabine, we synthesized a DNA-like polygemcitabine (Ge10) strand through solid-phase synthesis, which not only undergoes rapid intracellular degradation to generate active gemcitabine derivatives, but can also self-assemble into nanogels through molecular recognition, rendering them as promising self-delivered nanodrugs for cancer therapy.

Floxuridine-containing nucleic acid nanogels for anticancer drug delivery.

Herein, we report the self-assemblies of floxuridine-containing DNA and RNA nanogels with a precise drug loading ratio as effective drug delivery systems. Based on the structural similarity between the nucleoside analogue floxuridine (F) and the natural nucleoside thymidine (T), F can be incorporated into nucleic acid strands via either solid-phase synthesis or enzyme-mediated transcription. With the retained property of molecular recognition, the synthesized F-integrated DNA or RNA strands can be used as building units and further assembled into nucleic acid based spherical nanogels, which can be efficiently taken up by cancer cells and then release the therapeutic agents. As such, the drug-containing nucleic acid nanogels exhibit excellent inhibitory activity against cancer cells.

A small molecule nanodrug consisting of amphiphilic targeting ligand-chemotherapy drug conjugate for targeted cancer therapy.

Targeted drug delivery is a broadly applicable approach for cancer therapy. However, the nanocarrier-based targeted delivery system suffers from batch-to-batch variation, quality concerns and carrier-related toxicity issues. Thus, to develop a carrier-free targeted delivery system with nanoscale characteristics is very attractive. Here, a novel targeting small molecule nanodrug self-delivery system consisting of targeting ligand and chemotherapy drug was constructed, which combined the advantages of small molecules and nano-assemblies together and showed excellent targeting ability and long blood circulation time with well-defined structure, high drug loading ratio and on-demand drug release behavior. As a proof-of-concept, lactose (Lac) and doxorubicin (DOX) were chosen as the targeting ligand and chemotherapy drug, respectively. Lac and DOX were conjugated through a pH-responsive hydrazone group. For its intrinsic amphiphilic property, Lac-DOX conjugate could self-assemble into nanoparticles in water. Both in vitro and in vivo assays indicated that Lac-DOX nanoparticles exhibited enhanced anticancer activity and weak side effects. This novel active targeting nanodrug delivery system shows great potential in cancer therapy.

Host-guest binding motifs based on hyperbranched polymers.

Host-guest chemistry involves the binding of a substrate molecule (guest) to a receptor molecule (host). Various molecules, including crown ethers, cryptands, cyclophanes, calixarenes, cyclodextrins, and so on, have been used as molecular hosts. However, only limited small molecules or simple ions can be encapsulated in these hosts. Fortunately, as a class of unique host molecules, hyperbranched polymers (HBPs) can bind to numerous guests through topological entrapment, electrostatic bonding, hydrogen bonding or hydrophobic interactions in the core, at the branching points or at the periphery. Hence, hyperbranched polymeric hosts have received increasing attention in the past few decades because of their specific and unique properties. This review briefly summarizes these unique properties related to HBPs serving as hosts. In addition, HBP-based host-guest systems will be classified according to the types of guests encapsulated. Besides, the corresponding applications will be presented as well. We hope to motivate an increased understanding of molecular recognition in HBPs, and further facilitate the optimization of future host-guest systems.

Dendritic Polymers for Theranostics.

Dendritic polymers are highly branched polymers with controllable structures, which possess a large population of terminal functional groups, low solution or melt viscosity, and good solubility. Their size, degree of branching and functionality can be adjusted and controlled through the synthetic procedures. These tunable structures correspond to application-related properties, such as biodegradability, biocompatibility, stimuli-responsiveness and self-assembly ability, which are the key points for theranostic applications, including chemotherapeutic theranostics, biotherapeutic theranostics, phototherapeutic theranostics, radiotherapeutic theranostics and combined therapeutic theranostics. Up to now, significant progress has been made for the dendritic polymers in solving some of the fundamental and technical questions toward their theranostic applications. In this review, we briefly summarize how to control the structures of dendritic polymers, the theranostics-related properties derived from their structures and their theranostics-related applications.

Cancer Theranostic Nanoparticles Self-Assembled from Amphiphilic Small Molecules with Equilibrium Shift-Induced Renal Clearance.

Nano drug delivery systems have emerged as promising candidates for cancer therapy, whereas their uncertainly complete elimination from the body within specific timescales restricts their clinical translation. Compared with hepatic clearance of nanoparticles, renal excretion of small molecules is preferred to minimize the agent-induced toxicity. Herein, we construct in vivo renal-clearable nanoparticles, which are self-assembled from amphiphilic small molecules holding the capabilities of magnetic resonance imaging (MRI) and chemotherapy. The assembled nanoparticles can accumulate in tumor tissues for their nano-characteristics, while the small molecules dismantled from the nanoparticles can be efficiently cleared by kidneys. The renal-clearable nanoparticles exhibit excellent tumor-inhibition performance as well as low side effects and negligible chronic toxicity. These results demonstrate a potential strategy for small molecular nano drug delivery systems with obvious anticancer effect and low-toxic metabolism pathway for clinical applications.