Reversible RNA Acylation Using Bio-Orthogonal Chemistry Enables Temporal Control of CRISPR-Cas9 Nuclease Activity.

The CRISPR-Cas9 system is a widely popular tool for genome engineering. There is strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this work, we describe a bio-orthogonal chemistry-based approach to control nuclease activity with temporal precision. We report a trans-cyclooctene (TCO)-acylimidazole reagent that acylates 2'-OH groups of RNA. Poly acylation ("cloaking") of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine, which removes the TCO moieties via a two-step process ("uncloaking"). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of an organism's development.

Bio-Orthogonal Chemistry Conjugation Strategy Facilitates Investigation of N-methyladenosine and Thiouridine Guide RNA Modifications on CRISPR Activity.

The CRISPR-Cas9 system is an important genome editing tool that holds enormous potential toward the treatment of human genetic diseases. Clinical success of CRISPR technology is dependent on the incorporation of modifications into the single-guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. We developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 4-thiouridine (s4U). The impact of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells.

Recent Development of Prodrugs of Gemcitabine.

Gemcitabine is a nucleoside analog that has been used widely as an anticancer drug for the treatment of a variety of conditions, including ovarian, bladder, non-small-cell lung, pancreatic, and breast cancer. However, enzymatic deamination, fast systemic clearance, and the emergence of chemoresistance have limited its efficacy. Different prodrug strategies have been explored in recent years, seeking to obtain better pharmacokinetic properties, efficacy, and safety. Different drug delivery strategies have also been employed, seeking to transform gemcitabine into a targeted medicine. This review will provide an overview of the recent developments in gemcitabine prodrugs and their effectiveness in treating cancerous tumors.

Non-Chromatographic Purification of Synthetic RNA Using Bio-Orthogonal Chemistry.

Solid-phase synthesis of RNA oligonucleotides over 100 nt in length remains challenging due to the complexity of purification of the target strands from the failure sequences. This article describes a non-chromatographic procedure that will enable routine solid-phase synthesis and purification of long RNA strands. The optimized five-step process is based on bio-orthogonal inverse electron demand Diels-Alder chemistry between trans-cyclooctene (TCO) and tetrazine (Tz), and entails solid-phase synthesis of RNA on a photo-labile support. The target oligonucleotide strands are selectively tagged with Tz while on-support. After photocleavage from the solid support, the target oligonucleotide strands can be captured and purified from the failure sequences using immobilized TCO. The approach can be applied for purification of 76-nt long tRNA and 101-nt long sgRNA for CRISPR experiments. Purity of the isolated oligonucleotides should be evaluated using gel electrophoresis, while functional fidelity of the sgRNA should be confirmed using CRISPR-Cas9 experiments. © 2021 Wiley Periodicals LLC. Basic Protocol: Five-step non-chromatographic purification of synthetic RNA oligonucleotides Support Protocol 1: Synthesis of the components that are required for the non-chromatographic purification of long RNA oligonucleotides. Support Protocol 2: Solid-phase RNA synthesis.

Chemiluminescent probe for the detection of inverse electron demand Diels-Alder reaction between tetrazine and trans-Cyclooctene.

A chemiluminescent probe has been developed, consisting of phenoxy-dioxetane moiety covalently attached to trans-cyclooctene. The inverse electron demand Diels-Alder reaction with tetrazine produces a cycloaddition product which undergoes a series of spontaneous rearrangements resulting in emission of green light. The chemiluminescent probe can be applied to study bioconjugation chemistry with tetrazine-modified biomaterials, which have recently been shown to have great potential for anticancer drug delivery. This work describes in vitro studies, including NMR and spectroscopic investigation of chemiluminescence, which will pave way for future in vivo bioconjugation experiments.

Correction: Click activated protodrugs against cancer increase the therapeutic potential of chemotherapy through local capture and activation.

[This corrects the article DOI: 10.1039/D0SC06099B.].

Click activated protodrugs against cancer increase the therapeutic potential of chemotherapy through local capture and activation.

A desired goal of targeted cancer treatments is to achieve high tumor specificity with minimal side effects. Despite recent advances, this remains difficult to achieve in practice as most approaches rely on biomarkers or physiological differences between malignant and healthy tissue, and thus benefit only a subset of patients in need of treatment. To address this unmet need, we introduced a Click Activated Protodrugs Against Cancer (CAPAC) platform that enables targeted activation of drugs at a specific site in the body, i.e., a tumor. In contrast to antibodies (mAbs, ADCs) and other targeted approaches, the mechanism of action is based on in vivo click chemistry, and is thus independent of tumor biomarker expression or factors such as enzymatic activity, pH, or oxygen levels. The CAPAC platform consists of a tetrazine-modified sodium hyaluronate-based biopolymer injected at a tumor site, followed by one or more doses of a trans-cyclooctene (TCO)-modified cytotoxic protodrug with attenuated activity administered systemically. The protodrug is captured locally by the biopolymer through an inverse electron-demand Diels-Alder reaction between tetrazine and TCO, followed by conversion to the active drug directly at the tumor site, thereby overcoming the systemic limitations of conventional chemotherapy or the need for specific biomarkers of traditional targeted therapies. Here, TCO-modified protodrugs of four prominent cytotoxics (doxorubicin, paclitaxel, etoposide and gemcitabine) are used, highlighting the modularity of the CAPAC platform. In vitro evaluation of cytotoxicity, solubility, stability and activation rendered the protodrug of doxorubicin, SQP33, as the most promising candidate for in vivo studies. In mice, the maximum tolerated dose (MTD) of SQP33 in combination with locally injected tetrazine-modified biopolymer (SQL70) was determined to be 19.1-times the MTD of conventional doxorubicin. Pharmacokinetics studies in rats show that a single injection of SQL70 efficiently captures multiple SQP33 protodrug doses given cumulatively at 10.8-times the MTD of conventional doxorubicin with greatly reduced systemic toxicity. Finally, combined treatment with SQL70 and SQP33 (together called SQ3370) showed antitumor activity in a syngeneic tumor model in mice.

Bio-orthogonal chemistry enables solid phase synthesis and HPLC and gel-free purification of long RNA oligonucleotides.

Solid phase synthesis of RNA oligonucleotides which are over 100-nt in length remains challenging due to the complexity of purification of the target strand from failure sequences. This work describes a non-chromatographic strategy that will enable routine solid phase synthesis of long RNA strands.

Bond-Breaking Bio-orthogonal Chemistry Efficiently Uncages Fluorescent and Therapeutic Compounds under Physiological Conditions.

Bond-breaking bio-orthogonal chemistry, consisting of a "click" reaction between trans-cyclooctene and tetrazine, followed by an intramolecular cyclization-driven uncaging step is described. The two-step process allows activation of caged compounds in biological media at neutral pH. The feasibility of this chemistry has been illustrated using NMR, while kinetics and pH-dependence were studied by fluorescence spectroscopy using caged coumarin. The practicality of the strategy is illustrated by activation of an anticancer drug, etoposide.

Titanium coating with mussel inspired polymer and bio-orthogonal chemistry enhances antimicrobial activity against Staphylococcus aureus.

Implant-associated infections present severe and difficult-to-treat complications after surgery, related to implant biofilm colonization. Systemic administration of antibiotics cannot reach sufficient concentrations at the infected site and may be toxic. Here we describe how mussel-inspired dendritic material coated on a titanium surface can locally activate a prodrug of daptomycin (pro-dapto) to treat methicillin-resistant Staphylococcus aureus. The mechanism of the prodrug activation is based on bio-orthogonal click chemistry between a tetrazine (Tz) and trans-cyclooctene (TCO). The former is attached to the dendritic polymer, while the later converts daptomycin into a prodrug. Characterization of the material's properties revealed that it is hydrophobic, non-toxic, and stable for a prolonged period of time. We envision that the titanium coated dendritic material will be able to improve the treatment of implant-associated infections by concentrating systemically administered antibiotic prodrugs, thus converting them into active localized medicines.

Small molecule-induced DNA hydrogel with encapsulation and release properties.

Hydrogels are networks of polymers that can be used for packaging different payload types. They are proven to be versatile materials for various biomedical applications. Implanted hydrogels with encapsulated drugs have been shown to release the therapeutic payloads at disease sites. Hydrogels are usually made through chemical polymerization reactions. Whereas, DNA is a naturally occurring biopolymer which can assemble into highly ordered structures through noncovalent interactions. Here, we have employed a small molecule, cyanuric acid (CA), to assemble polyA-tailed DNA motif into a hydrogel. Encapsulation of a small molecule chemotherapeutic drug, a fluorescent molecule, two proteins and several nanoparticle formulations has been studied. Release of doxorubicin, small fluorescent molecule and fluorescently-labeled antibodies has been demonstrated.

SNAP/CLIP-Tags and Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)/Inverse Electron Demand Diels-Alder (IEDDA) for Intracellular Orthogonal/Bioorthogonal Labeling.

Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Compared to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process. In the present study the orthogonality of the strain-promoted azide-alkyne cycloaddition (SPAAC) and the inverse electron demand Diels-Alder (IEDDA) reaction is corroborated in concurrent labeling of two different intracellular targets. An azido group and a strained alkene are first installed at specific subcellular locations via orthogonal enzymatic reactions of the genetically incorporated SNAP- and CLIP-tags. The subsequent bioorthogonal reactions with fluorophores carrying matching reactive functionalities result in simultaneous dual labeling. The two-step "orthogonal-bioorthogonal" labeling process would increase the utilities of SNAP/CLIP-tags and, as a consequence, would expand the capability of decorating biological specimens with functionalities beyond fluorophores to potentially include spin labels, radioactive tracers, or catalysts.

Switchable Fluorescence of Doxorubicin for Label-Free Imaging of Bioorthogonal Drug Release.

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans-cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond-cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA-MB-231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems.

High-resolution ion mobility spectrometry-mass spectrometry of isomeric/isobaric ribonucleotide variants.

In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (tD ) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the tD scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers thus substantiating the benefits of complementing unique tD information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomic studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species.

Three-Component Protein Modification Using Mercaptobenzaldehyde Derivatives.

A chemoselective primary amine modification strategy that enables the three-component, one-pot bioconjugation is described. The specifically designed, mercaptobenzaldehyde-based bifunctional linker achieves highly selective and robust amine labeling under biocompatible conditions. This linker demonstrates wide functional group tolerance and is simple to prepare, which allowed facile payload incorporation. Finally, our studies have shown that the introduction of linker does not impair the function of modified protein such as insulin.

Bio-Orthogonal Chemistry and Reloadable Biomaterial Enable Local Activation of Antibiotic Prodrugs and Enhance Treatments against Staphylococcus aureus Infections.

Systemic administration of antibiotics can cause severe side-effects such as liver and kidney toxicity, destruction of healthy gut bacteria, as well as multidrug resistance. Here, we present a bio-orthogonal chemistry-based strategy toward local prodrug concentration and activation. The strategy is based on the inverse electron-demand Diels-Alder chemistry between trans-cyclooctene and tetrazine and involves a biomaterial that can concentrate and activate multiple doses of systemic antibiotic therapy prodrugs at a local site. We demonstrate that a biomaterial, consisting of alginate hydrogel modified with tetrazine, is efficient at activating multiple doses of prodrugs of vancomycin and daptomycin in vitro as well as in vivo. These results support a drug delivery process that is independent of endogenous environmental markers. This approach is expected to improve therapeutic efficacy with decreased side-effects of antibiotics against bacterial infections. The platform has a wide scope of possible applications such as wound healing, and cancer and immunotherapy.

Controlled in-cell activation of RNA therapeutics using bond-cleaving bio-orthogonal chemistry.

Temporal control of siRNA activation is a major challenge for RNAi-based therapeutics. The majority of the reported siRNA delivery systems rely on environmental factors, such as differences in extracellular and intracellular redox potential, ATP concentration, or pH to activate an siRNA payload. However dynamic endogenous environments are far too complex to rely on for controllable siRNA release and can result in premature siRNA activation prior to reaching the intended biological target. In addition, there are uncertainties about timing, degree and rate of the siRNA activation with spontaneous release approaches. Herein we describe a bio-orthogonal chemistry approach to address this important challenge. With our approach we were able achieve two major goals: complete siRNA inactivation upon immobilization of the payload on the surface of iron oxide nanoparticles and controlled in-cell activation with the addition of a small non-toxic chemical trigger after sufficient cellular uptake of the nanoparticles was confirmed. We have demonstrated our in-cell activation approach using two siRNAs against green fluorescent protein (GFP) and cyclin dependent kinase 8 (CDK8) in GFP expressing MDA-MB-231 cell line. We anticipate that this methodology will potentially advance the clinical translation of RNAi-based therapeutics, as the described bio-orthogonal chemistry can be generalized for any siRNA of choice.

Single-trigger dual-responsive nanoparticles for controllable and sequential prodrug activation.

Here we have developed a novel approach where two synergistically acting drugs were completely inactivated upon chemical immobilization on a nanoparticle template and activated in response to a chemical stimulus. The activation rate of each drug payload is controlled using a biologically inert bioorthogonal chemistry approach. By exploiting the subtle differences in the 'click-to-release' bioorthogonal reaction, we engineered a single delivery platform capable of releasing the payloads in a time-staggered manner in response to a single dose of a highly specific, yet reactive, small molecule. Incorporation of both di-axial, 'fast release', and di-equatorial, 'slow release', TCO linkers into our nanodrug assembly inhibited the activity of the drug molecules and enabled us to control the timing and activation of each payload. This single-trigger dual-responsive nanoparticle construct and its release kinetics were characterized using two molecular fluorescent probes and tested in vitro for efficient delivery of molecular payloads. In this manuscript we show that this approach was also successful in the treatment of triple negative BT-20 breast cancer cells. Our nanodrug loaded with the slow-releasing doxorubicin and fast-releasing PAC-1 prodrugs displayed a greater therapeutic response than the nanodrug which released both payloads simultaneously.

Cobalt-based paramagnetic probe to study RNA-protein interactions by NMR.

Paramagnetic resonance enhancement (PRE) is an NMR technique that allows studying three-dimensional structures of RNA-protein complexes in solution. RNA strands are typically spin labeled using nitroxide reagents, which provide minimal perturbation to the native structure. The current work describes an alternative approach, which is based on a Co2+-based probe that can be covalently attached to RNA in the vicinity of the protein's binding site using 'click' chemistry. Similar to nitroxide spin labels, the transition metal based probe is capable of attenuating NMR signal intensities from protein residues localized <40Å away. The extent of attenuation is related to the probe's distance, thus allowing for construction of the protein's contact surface map. This new paradigm has been applied to study binding of HIV-1 nucleocapsid protein 7, NCp7, to a model RNA pentanucleotide.

Cu(II)-Based Paramagnetic Probe to Study RNA-Protein Interactions by NMR.

Paramagnetic NMR techniques allow for studying three-dimensional structures of RNA-protein complexes. In particular, paramagnetic relaxation enhancement (PRE) data can provide valuable information about long-range distances between different structural components. For PRE NMR experiments, oligonucleotides are typically spin-labeled using nitroxide reagents. The current work describes an alternative approach involving a Cu(II) cyclen-based probe that can be covalently attached to an RNA strand in the vicinity of the protein's binding site using "click" chemistry. The approach has been applied to study binding of HIV-1 nucleocapsid protein 7 (NCp7) to a model RNA pentanucleotide, 5'-ACGCU-3'. Coordination of the paramagnetic metal to glutamic acid residue of NCp7 reduced flexibility of the probe, thus simplifying interpretation of the PRE data. NMR experiments showed attenuation of signal intensities from protein residues localized in proximity to the paramagnetic probe as the result of RNA-protein interactions. The extent of the attenuation was related to the probe's proximity allowing us to construct the protein's contact surface map.