Allosteric Nucleic Acid Enzyme: A Versatile Stimuli-Responsive Tool for Molecular Computing and Biosensing Nanodevices.

Allostery is a naturally occurring mechanism in which effector binding induces the modulation and fine control of a related biomolecule function. Deoxyribozyme (DNAzyme) with catalytic activity and substrate recognition ability is ideal to be regulated by allosteric strategies. However, the current regulations frequently confront various obstacles, such as severe activity decay, signal leakage, and limited effectors. In this work, a rational regulation strategy for developing versatile effectors-responsive allosteric nucleic acid enzyme (ANAzyme) by introducing an allosteric domain in response to diverse effectors is established. The enzyme-like activity of this re-engineered ANAzyme can be modulated in a more predictable and fine way compared with the previous DNAzyme regulation strategies. Based on the allosteric strategy, the construction of allosterically coregulatory nanodevices and a series of basic logic gates and logic circuits are achieved, demonstrating that the proposed ANAzyme-regulated strategy showed great potential in molecular computing. Given these facts, the rational design of ANAzyme with the allosteric domain presented here can expand the available toolbox to develop a variety of stimuli-responsive allosteric DNA materials, including molecular machines, computing systems, biosensing platforms, and gene-silencing tools.

Self-Assembly Nanostructure Induced by Regulation of G-Quadruplex DNA Topology via a Reduction-Sensitive Azobenzene Ligand on Cells.

The control of DNA assembly systems on cells has increasingly shown great importance for precisely targeted therapies. Here, we report a controllable DNA self-assembly system based on the regulation of G-quadruplex DNA topology by a reduction-sensitive azobenzene ligand. Specifically, three azobenzene multiamines are developed, and AzoDiTren is identified as the best G4 binder, which displays high affinity and specificity for G4 DNA. Moreover, the reduction-sensitive nature of the azobenzene scaffold allows AzoDiTren to induce a complete change of the G4 topology in a tissue-specific manner, even at high metal cation concentrations. On this basis, the AzoDiTren-induced G4 conformational switch achieves control of the self-assembly of G4-functionalized DNAs on cells. This strategy enables the regulation of G4 and DNA self-assembly by the bioreductant-responsive ligand.

A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs.

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited.

Cell-Trappable BODIPY-NBD Dyad for Imaging of Basal and Stress-Induced H2S in Live Biosystems.

H2S is a gaseous signaling molecule that is involved in many physiological and pathological processes. In general, the level of intracellular H2S (<1 µM) is much lower than that of GSH (∼1-10 mM), leading to the remaining challenge of selective detection and differentiation of endogenous H2S in live biosystems. To this end, we quantitatively demonstrate that the thiolysis of NBD amine has much higher selectivity for H2S over GSH than that of the reduction of aryl azide. Subsequently, we developed the first NBD-based cell-trappable probe 1 (AM-BODIPY-NBD) for highly selective and ultrasensitive imaging of intracellular H2S. Probe 1 demonstrates a 207-fold fluorescence enhancement at 520 nm after reaction with H2S/esterase to produce a bright BODIPY (quantum yield 0.42) and a detection limit of 15.7 nM. Probe 1 is water-soluble, cell-trappable, and not cytotoxic. Based on this excellent chemical tool, relative levels of basal H2S in different cell lines were measured to reveal a positive correlation between endogenous H2S and the metastatic potential of colon and breast cancer cells. In addition, H2S biogenesis in vivo was also validated by probe 1 both in tobacco leaves under viral infection and in zebrafish after tail amputation. It is anticipated that probe 1 will have widespread applications in imaging and for investigating different H2S-related biological processes and diseases.

Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.

FTO demethylates internal N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

Tetrazine-Induced Bioorthogonal Activation of Vitamin E-Modified siRNA for Gene Silencing.

The temporal activation of siRNA provides a valuable strategy for the regulation of siRNA activity and conditional gene silencing. The bioorthogonal bond-cleavage reaction of benzonorbonadiene and tetrazine is a promising trigger in siRNA temporal activation. Here, we developed a new method for the bio-orthogonal chemical activation of siRNA based on the tetrazine-induced bond-cleavage reaction. Small-molecule activatable caged siRNAs were developed with the 5'-vitamin E-benzonobonadiene-modified antisense strand targeting the green fluorescent protein (GFP) gene and the mitotic kinesin-5 (Eg5) gene. The addition of tetrazine triggered the reaction with benzonobonadiene linker and induced the linker cleavage to release the active siRNA. Additionally, the conditional gene silencing of both exogenous GFP and endogenous Eg5 genes was successfully achieved with 5'-vitamin E-benzonobonadiene-caged siRNAs, which provides a new uncaging strategy with small molecules.

Reversible On-Off Photoswitching of DNA Replication Using a Dumbbell Oligodeoxynucleotide.

In most organisms, DNA extension is highly regulated; however, most studies have focused on controlling the initiation of replication, and few have been done to control the regulation of DNA extension. In this study, we adopted a new strategy for azODNs to regulate DNA extension, which is based on azobenzene oligonucleotide chimeras regulated by substrate binding affinity, and the conformation of the chimera can be regulated by a light source with a light wavelength of 365 nm. The results showed that the primer was extended with Taq DNA polymerase after visible light treatment, and DNA extension could be effectively hindered with UV light treatment. We also verify the reversibility of the photoregulation of primer extension through photoswitching of dumbbell asODNs by alternate irradiation with UV and visible light. Our method has the advantages of fast and simple, green response and reversible operations, providing a new strategy for regulating gene replication.

Photoregulation of Gene Expression with Ligand-Modified Caged siRNAs through Host/Guest Interaction.

Small interfering RNA (siRNA) can effectively silence target genes through Argonate 2 (Ago2)-induced RNA interference (RNAi). It is very important to control siRNA activity in both spatial and temporal modes. Among different masking strategies, photocaging can be used to regulate gene expression through light irradiation with spatiotemporal and dose-dependent resolution. Many different caging strategies and caging groups have been reported for light-activated siRNA gene silencing. Herein, we describe a novel caging strategy that increases the blocking effect of RISC complex formation/process through host/guest (including ligand/receptor) interactions, thereby enhancing the inhibition of caged siRNA activity until light activation. This strategy can be used as a general approach to design caged siRNAs for the photomodulation of gene silencing of exogenous and endogenous genes.

Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation*.

There is an urgent need to develop antiviral drugs and alleviate the current COVID-19 pandemic. Herein we report the design and construction of chimeric oligonucleotides comprising a 2'-OMe-modified antisense oligonucleotide and a 5'-phosphorylated 2'-5' poly(A)4 (4A2-5 ) to degrade envelope and spike RNAs of SARS-CoV-2. The oligonucleotide was used for searching and recognizing target viral RNA sequence, and the conjugated 4A2-5 was used for guided RNase L activation to sequence-specifically degrade viral RNAs. Since RNase L can potently cleave single-stranded RNA during innate antiviral response, degradation efficiencies with these chimeras were twice as much as those with only antisense oligonucleotides for both SARS-CoV-2 RNA targets. In pseudovirus infection models, chimera-S4 achieved potent and broad-spectrum inhibition of SARS-CoV-2 and its N501Y and/or ΔH69/ΔV70 mutants, indicating a promising antiviral agent based on the nucleic acid-hydrolysis targeting chimera (NATAC) strategy.

Bioorthogonal SERS Nanotags as a Precision Theranostic Platform for in Vivo SERS Imaging and Cancer Photothermal Therapy.

Precise detection and effective treatment are crucial to prolong cancer patients' lives. Surface-enhanced Raman scattering (SERS) imaging coupled with photothermal therapy has been considered a precise and effective strategy for cancer theranostics. Nevertheless, Raman reporters employed in the literature usually possessed multiple shift peaks in the fingerprint region, which are overlapped with background signals from endogenous biological molecules. Herein, we fabricated a new kind of bioorthogonal Raman reporter and aptamer functionalized SERS nanotags. The SERS nanotags demonstrated a strong Raman signal at 2205 cm-1 in the biologically Raman-silent region and recognized MCF-7 breast cancer cells for Raman imaging with high specificity. Laser irradiation induced serious toxicity of MCF-7 cells due to the excellent photothermal capability of the SERS nanotags. After intravenous administration of the SERS nanotags, tumor Raman spectral detection and mapping in living mice were successfully achieved. Further in vivo antitumor experiments manifested that the aptamer-modified SERS nanotags significantly restrained tumor growth after laser irradiation with 99% inhibition rate and good biocompatibility. These results clearly revealed that the SERS nanotags could serve as a novel and precise theranostic platform for in vivo cancer diagnosis and photothermal therapy.

Photoregulation of Gene Expression with Amantadine-Modified Caged siRNAs through Host-Guest Interactions.

RNA interference is an essential and powerful tool for targeting and verifying specific gene functions. Conditional control of small interfering RNA (siRNA) activity, especially using light activation, is a potential method for regulating target gene expression and functions. In this study, a series of photolabile siRNAs with amantadine modification have been rationally designed and developed through host-guest interactions between amantadine and ß-cyclodextrin derivatives to enhance the blocking effect of siRNA binding and/or RNA-induced silencing complex processing. These caged siRNAs with amantadine modification at the 5' end of antisense-strand RNA were efficiently inactivated through the host-guest interactions between amantadine and ß-cyclodextrin. Photomodulation of the gene silencing activity of these amantadine-modified caged siRNAs targeting both exogenous and endogenous genes was successfully achieved, which indicates that host-guest interactions could be a new strategy for developing new caged siRNAs for gene photoregulation with low leaking activity.

Triton X-100-Modified Adenosine Triphosphate-Responsive siRNA Delivery Agent for Antitumor Therapy.

Modified polyethyleneimine (PEI) has been widely used as siRNA delivery agents. Here, a new Triton X-100-modified low-molecular-weight PEI siRNA delivery agent is developed together with the coupling of 4-carboxyphenylboronic acid (PBA) and dopamine grafted vitamin E (VEDA). Triton X-100, a nonionic detergent, greatly improves the cellular uptake of siRNA as well as the siRNA escape from endosome/lysosome because of its high transmembrane ability. In addition, the boronate bond between PBA and VEDA of the transfection agent can be triggered to release its entrapped siRNA because of the high level of adenosine triphosphate (ATP) in cancer cells. The transfection agent is successfully applied to deliver siRNAs targeting endogenous genes of epidermal growth factor receptor (EGFR) and kinesin-5 (Eg5) to cancer cells, showing good results on Eg5 and EGFR silencing ability and inhibition of cancer cell migration. Further in vivo study indicates that the Triton X-100-modified transfection agent is also efficient to deliver siRNA to cancer cells and shows significant tumor growth inhibition on mice tumor models. These results indicate that the Triton X-100-modified ATP-responsive transfection agent is a promising gene delivery vector for target gene silencing in vitro and in vivo.

Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA.

Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5' terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.

Multicolor Cocktail for Breast Cancer Multiplex Phenotype Targeting and Diagnosis Using Bioorthogonal Surface-Enhanced Raman Scattering Nanoprobes.

Early precise diagnosis of cancers is crucial to realize more effective therapeutic interventions with minimal toxic effects. Cancer phenotypes may also alter greatly among patients and within individuals over the therapeutic process. The identification and characterization of specific biomarkers expressed on tumor cells are in high demand for diagnosis and treatment, but they are still a challenge. Herein, we designed three new bioorthogonal surface-enhanced Raman scattering (SERS) nanoprobes and successfully applied the cocktail of them for MDA-MB-231 and MCF-7 breast cancer multiplex phenotype detection. The SERS nanoprobes containing Raman reporters with diynl, azide, or cyano moieties demonstrated apparent Raman shift peaks in 2205, 2120, and 2230 cm-1, respectively, in the biologically Raman-silent region. Three target ligands, including oligonucleotide aptamer (AS1411), arginine-glycine-aspatic acid (RGD) peptide, and homing cell adhesion molecule antibody (anti-CD44), were separately conjugated to the nanoprobes for active recognition capability. The cocktail of the nanoprobes manifested minimal cytotoxicity and simultaneously multiplex phenotype imaging of MDA-MB-231 and MCF-7 cells. Quantitative measurement of cellular uptake by inductively coupled plasma mass spectrometry (ICPMS) verified that MDA-MB-231 cells harbored a much higher expression level of CD44 receptor than MCF-7 cells. For in vivo SERS detection, Raman shift peaks of 2120, 2205, and 2230 cm-1 in the micro-tumor were clearly observed, representing the existence of three specific biomarkers of nucleolin, integrin αvß3, and CD44 reporter, which could be used for early cancer phenotype identification. The biodistribution results indicated that target ligand modified nanoprobes exhibited much more accumulation in tumors than those nanoprobes without target ligands. The multicolor cocktail of bioorthogonal SERS nanoprobes offers an attractive and insightful strategy for early cancer multiplex phenotype targeting and diagnosis in vivo that is noninvasive and has low cross-talk, unique spectral-molecular signature, high sensitivity, and negligible background interference.

Multicolor Raman Beads for Multiplexed Tumor Cell and Tissue Imaging and in Vivo Tumor Spectral Detection.

Developing new nanomaterials with strong and distinctive Raman vibrations in the biological Raman-silent region (1800-2800 cm-1) were highly desirable for Raman hyperspectral detection and imaging in living cells and animals. Herein, polymeric nanoparticles with monomers containing alkyne, cyanide, azide, and carbon-deuterate were prepared as Raman-active nanomaterials (Raman beads) for bioimaging applications. Intense Raman signals were obtained due to the high density of alkyne, cyanide, azide, and carbon-deuterate in single nanoparticles, in absence of metal (such as Au or Ag) as Raman enhancers. We have developed a library of Raman beads for frequency multiplexing through the end-capping substitutions of monomers and demonstrated five-color SRS imaging of mixed nanoparticles with distinct Raman frequencies. In addition, with further surface functionalization of targeting moieties (such as nucleic acid aptamers and targeting peptides), targetable Raman beads were successfully used as probes for tumor targeting and Raman spectroscopic detection, including multicolor SRS imaging in living tumor cells and tissues with high specificity. Further in vivo studies indicated that Raman beads anchored with targeting moieties were successfully employed to target tumors in living mice after tail intravenous injection, and Raman spectral detection of tumor in live mice was achieved only through spontaneous Raman signal at the biological Raman-silent region without any signal enhancement due to a high density of Raman reporters in Raman beads. With further copolymerization of these monomers, Raman beads with supermultiplex barcoding could be readily achieved.

Dextran-Conjugated Caged siRNA Nanoparticles for Photochemical Regulation of RNAi-Induced Gene Silencing in Cells and Mice.

RNA interference (RNAi)-based gene therapy is a precision therapeutic approach for highly efficient sequence-specific gene silencing in vivo or in vitro. Caged RNAs featuring dextran conjugation of antisense and sense RNA strands using photolabile linker were rationally designed and self-assembled to form caged siRNA nanoparticles (Dex- p-siRNA) for photoregulation of target gene expression. The dextran-conjugated caged siRNA nanoparticles showed significant serum nuclease-resistance due to the formation of dextran-siRNA nanoparticles. Photomodulation of exogenous GFP and endogenous mitotic kinesin-5 ( Eg5) gene expression in cells was achieved using the prepared caged Dex- p-siRNA nanoparticles. The caged Dex- p-siRNA nanoparticles targeting GFP successfully photoregulated GFP expression in tumor-bearing mice via intratumoral injection. Caged siRNA nanoparticles with high serum stability not only show great promise for photoregulation of exogenous and endogenous gene expression for both in vitro and in vivo applications, but also provide a novel and convenient way to spatiotemporally control RNAi-induced gene silencing.

Reversible Photocontrol of Thrombin Activity by Replacing Loops of Thrombin Binding Aptamer using Azobenzene Derivatives.

The photoisomerization of azobenzenes provides a general means for the photocontrol of many important biomolecular structures and organismal functions. For temporal and spatial control activity of thrombin binding aptamer (TBA) by light, azobenzene derivatives were carefully selected as light-triggered molecular switches to replace TT loops and the TGT loop of TBA to reversibly control enzyme activity. These molecules interconverted between the trans and cis states under alternate UV and visible light irradiation, which consequently triggered reversible formation of G-quadruplex morphology. In addition, we investigated the impact of three azobenzene derivatives on stability, thrombin binding ability, and anticoagulant properties. The result showed that 4,4'-bis(hydroxymethyl)azobenzene at the TGT loop position significantly photoregulated affinity to thrombin and blood clotting in human plasma, which provided a successful strategy to control blood clotting in human plasma and a further evidence for design of TBA analogues with pivotal positions of modifications.

Endogenous hydrogen sulfide sulfhydrates IKKß at cysteine 179 to control pulmonary artery endothelial cell inflammation.

BACKGROUND: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. METHODS: Purified recombinant human inhibitor of κB kinase subunit ß (IKKß) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. RESULTS: We showed that hydrogen sulfide (H2S) inhibited IKKß activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKß activity directly via sulfhydrating IKKß at cysteinyl residue 179 (C179) in purified recombinant IKKß protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKß inactivation. Furthermore, to demonstrate the significance of IKKß sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKß. In purified IKKß protein, C179S mutation of IKKß abolished H2S-induced IKKß sulfhydration and the subsequent IKKß inactivation. In human PAECs, C179S mutation of IKKß blocked H2S-inhibited IKKß activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKß abolished the inhibitory effect of H2S on IKKß activation and pulmonary vascular inflammation and remodeling. CONCLUSION: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKß via sulfhydrating IKKß at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

Selective and sensitive detection of cyanate using 3-amino-2-naphthoic acid-based turn-on fluorescence probe.

The cyanate anion (CNO-), formed spontaneously within cells from urea and carbamoyl phosphate, usually functions as a biomarker of some diseases such as chronic kidney disease. Therefore, accurate determination of CNO- is highly demanded. Herein, a 3-amino-2-naphthoic acid-based "turn-on" fluorescence probe was developed for specific detection of CNO-. Upon the addition of sodium cyanate, the weak-fluorescent 3-amino-2-naphthoic acid could react with CNO-, which triggered intense emission of green fluorescence. And up to 9-fold fluorescence enhancement was observed. The fluorescence enhancement ratios displayed a good linear relationship with the concentrations of CNO- in the range of 0.5-200 µM. The high selectivity and sensitivity for CNO- detection were investigated with the detection limit as low as 260 nM. The probe was further successfully applied to determine CNO- in real samples such as tap water, human urine and serum samples, which offered a promising approach in practical applications. Graphical abstract.

Chemical modifications of nucleic acid drugs and their delivery systems for gene-based therapy.

Gene-based therapy is one of essential therapeutic strategies for precision medicine through targeting specific genes in specific cells of target tissues. However, there still exist many problems that need to be solved, such as safety, stability, selectivity, delivery, as well as immunity. Currently, the key challenges of gene-based therapy for clinical potential applications are the safe and effective nucleic acid drugs as well as their safe and efficient gene delivery systems. In this review, we first focus on current nucleic acid drugs and their formulation in clinical trials and on the market, including antisense oligonucleotide, siRNA, aptamer, and plasmid nucleic acid drugs. Subsequently, we summarize different chemical modifications of nucleic acid drugs as well as their delivery systems for gene-based therapeutics in vivo based on nucleic acid chemistry and nanotechnology methods.