Fluorogenic Photo-Crosslinking of Glycan-Binding Protein Recognition Using a Fluorinated Azido-Coumarin Fucoside

Glycan recognition by glycan-binding proteins is central to the biology of all living organisms. The efficient capture and characterization of relatively weak non-covalent interactions remains an important challenge in various fields of research. Photoaffinity labeling strategies can create covalent bonds between interacting partners, and photoactive scaffolds such as benzophenone, diazirines and aryl azides have proved widely useful. Since their first introduction, relatively few improvements have been advanced and products of photoaffinity labeling remain difficult to detect. We report a fluorinated azido-coumarin scaffold which enables photolabeling under fast and mild activation, and which can leave a fluorescent tag on crosslinked species. Coupling this scaffold to an α-fucoside, we demonstrate fluorogenic photolabeling of glycan-protein interactions over a wide range of affinities. We expect this strategy to be broadly applicable to other chromophores and we envision that such “fluoro-crosslinkers” could become important tools for the traceable capture of non-covalent binding events.     

A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4-Hydroxyphenylpyruvate Dioxygenase in Plants

4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene-linked dibenzyl-cyclooctyne undergoes strain-promoted azide-alkyne cycloaddition with an azide-containing HPPD ligand. The activation-based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana, and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD-related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.     

Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.     

Fluorogenic tagging of peptides via Cys residues using thiol-specific vinyl sulfone affinity tags

Fluorescent tagging of Cys-containing peptides is presented herein. The procedure follows a two-step sequential reaction scheme using thiol specific bifunctional chemical reporters and fluorogenic labels. Vinyl-sulfone bearing chemical reporters have been synthesized and demonstrated to selectively modify cysteine under physiological conditions in the presence of other nucleophilic amino acids. Bifunctional chemical reporters decorated with a terminal alkyne moiety, suitable for modification with azide containing fluorogenic labels were also synthesized. Such fluorogenic (turn on) labels in combination with these new vinyl-sulfone tags can be used generally in fluorescent modulation schemes of thiol-bearing biomolecules.     

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.     

Site-Specific Fluorescent Labeling of RNA Interior Positions

The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo.     

Repurposing Antiviral Drugs for Orthogonal RNA-Catalyzed Labeling of RNA

In vitro selected ribozymes are promising tools for site-specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′-branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.     

水相合成荧光纳米晶与碱性蛋白酶的偶联及纯化

利用水相中合成的巯基丙酸包覆的Cd Te荧光纳米晶与碱性蛋白酶(Alcalase)进行偶联标记.荧光光谱分析表明,Cd Te纳米晶体具有较窄的粒度分布和较高的光稳定性,室温下30 d时荧光强度降低8.36%.透射电子显微镜结果显示,Cd Te纳米晶的直径约为3 nm;X射线衍射结果表明,Ce Te纳米晶具有闪锌矿结构.利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺磺酸钠盐(NHSS)偶联方法将Cd Te纳米晶与Alcalase进行偶联,通过超滤离心、凝胶柱层析及凝胶电泳技术对偶联后的Cd Te-Alcalase进行分离和纯化.荧光光谱显示偶联产物具有很好的光稳定性和较高的酶活力(61.06 U/μg).在荧光显微镜下可观察到标记蛋白的荧光,为研究酶反应的示踪及酶的构效关系等提供了实验基础.     

A New Bacterial Adenosine-Derived Nucleoside as an Example of RNA Modification Damage

The fields of RNA modification and RNA damage both exhibit a plethora of non-canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2-methylthio-1,N6-ethenoadenosine (ms²ϵA), which is related to 1,N6-ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms²ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2-methylthio-N6-isopentenyladenosine (ms²i⁶A). The relative abundance of ms²ϵA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage.     

Screening of Minimalist Noncanonical Sites in Duplex DNA and RNA Reveals Context and Motif-Selective Binding by Fluorogenic Base Probes

We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi-rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000× fluorescence enhancement over background. Binding is strongly context-dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine-rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi-rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.     

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

Functionalization of RNA at the 5′‐terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5′‐nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5′‐phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5′‐end of RNA.     

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

In vitro selected ribozymes are promising tools for site‐specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′‐branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.     

A TriPPPro-Nucleotide Reporter with Optimized Cell-Permeable Dyes for Metabolic Labeling of Cellular and Viral DNA in Living Cells

The metabolic labeling of nucleic acids in living cells is highly desirable to track the dynamics of nucleic acid metabolism in real-time and has the potential to provide novel insights into cellular biology as well as pathogen-host interactions. Catalyst-free inverse electron demand Diels–Alder reactions (iEDDA) with nucleosides carrying highly reactive moieties such as axial 2-trans-cyclooctene (2TCOa) would be an ideal tool to allow intracellular labeling of DNA. However, cellular kinase phosphorylation of the modified nucleosides is needed after cellular uptake as triphosphates are not membrane permeable. Unfortunately, the narrow substrate window of most endogenous kinases limits the use of highly reactive moieties. Here, we apply our TriPPPro (triphosphate pronucleotide) approach to directly deliver a highly reactive 2TCOa-modified 2′-deoxycytidine triphosphate reporter into living cells. We show that this nucleoside triphosphate is metabolically incorporated into de novo synthesized cellular and viral DNA and can be labeled with highly reactive and cell-permeable fluorescent dye-tetrazine conjugates via iEDDA to visualize DNA in living cells directly. Thus, we present the first comprehensive method for live-cell imaging of cellular and viral nucleic acids using a two-step labeling approach.     

Synthesis of novel C4-linked imidazole ribonucleoside phosphoramidites for probing general acid and base catalysis in ribozyme

We describe the synthesis of novel C4-linked imidazole ribonucleoside phosphoramidite (PA) 1a by which the imidazole moiety is incorporated into VS ribozyme to study its role in general acid and base catalysis. Investigation of protecting groups for the imidazole-N first indicated that pivaloyloxymethyl (POM) was adequate as an N-protecting group for the imidazole nucleoside, which could be readily removed under mild basic conditions. Further, the synthetic method was extended to synthesis of 2′-deoxy- and 2′-O-allyl nucleoside PAs 1b and 1c.     

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 °C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg²⁺ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.     

A Cleavable Crosslinking Strategy for Commodity Polymer Functionalization and Generation of Reprocessable Thermosets

Covalently crosslinked polymeric materials, known as thermosets, possess enhanced mechanical strength and thermal stability relative to the corresponding uncrosslinked thermoplastics. However, the presence of covalent inter-chain crosslinks that makes thermosets so attractive is precisely what makes them so difficult to reprocess and recycle. Here, we demonstrate the introduction of chemically cleavable groups into a bis-diazirine crosslinker. Application of this cleavable crosslinker reagent to commercial low-functionality polyolefins (or to a small-molecule model) results in the rapid, efficient introduction of molecular crosslinks that can be uncoupled by specific chemical inputs. These proof-of-concept findings provide one potential strategy for circularization of the thermoplastic/thermoset plastics economy, and may allow crosslinked polyolefins to be manufactured, used, reprocessed, and re-used without losing value. As an added benefit, the method allows the ready introduction of functionality into non-functionalized commodity polymers.