RNA Ligation for Mono and Dually Labeled RNAs

Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3′-end labeling of in vitro transcribed RNAs. We synthesized novel adenosine 3′,5′-bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide-containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain-promoted alkyne-azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD-capped RNAs, and 5′-fluorescently labeled m⁷Gp₃A_m-capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence-based monitoring of decapping. This method will facilitate the use of various functionalized mRNA-based probes.     

2‐Oxazoline Formation for Selective Chemical Labeling of 5‐Hydroxylysine

Hydroxylation of lysine, one of posttranslational modifications of proteins, generates 5‐hydroxylysine (Koh) and plays a crucial role in regulating protein functions in cellular activity. We have developed a chemical labeling method of Koh. The 1,2‐aminoalcohol moiety of Koh in synthetic peptide sequences was trapped by an alkyne‐containing benzimidate to form a 2‐oxazoline ring. An additional ammonia treatment process removed the undesirable amidine residue formed between benzimidate and lysine. During the ammonia treatment, the oxazoline residue formed at Koh mainly remained intact, and the ring opening to the amide form was observed for only part of oxazoline, indicating that the chemical labeling is amino acid selective. Azide‐substituted biotin or fluorescent dye was attached to the peptide through Huisgen cycloaddition at Koh and converted into an alkyne‐labeled oxazoline form. The Koh‐labeling assay could provide a platform to enhance proteomic research of lysine hydroxylation.     

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.     

Strategies for the Design of Drugs Targeting RNA and RNA-Protein Complexes

In many steps of gene replication and expression, RNA molecules participate as key players, which renders them attractive targets for therapeutic intervention. While the function of nucleic acids as carriers of genetic material is based on their sequence, a number of important RNAs are involved in processes that depend on the defined three-dimensional structures of these molecules. As for proteins, numerous complex folds of RNA exist. The development of drugs that bind specifically to RNA folds opens exciting new ways to expand greatly the existing repertoire of protein-targeted therapeutics. Most functions of RNAs involve interactions with proteins that contain RNA-binding domains. Effector molecules targeted at RNA may either alter the functional three-dimensional structure of the nucleic acid, so the interaction with proteins is thereby inhibited or enhanced, or, as interface inhibitors, they may directly prevent the formation of competent RNA-protein complexes. While the same tools used for the design of protein-targeted drugs may be considered for studying effectors binding to nucleic acids, the differences between proteins and RNAs in the forces which dominate their three-dimensional folding call for novel drug design strategies. In the present review, I will outline how our rapidly expanding knowledge of RNA three-dimensional structure and function facilitates rational approaches to develop RNA-binding compounds. Putative RNA targets for therapeutic intervention will be discussed along with recent advances in understanding RNA-small molecule and RNA-protein interactions.     

A photo-oxidation driven proximity labeling strategy enables profiling of mitochondrial proteome dynamics in living cells

Mapping the proteomic landscape of mitochondria with spatiotemporal precision plays a pivotal role in elucidating the delicate biological functions and complex relationship with other organelles in a variety of dynamic physiological processes which necessitates efficient and controllable chemical tools. We herein report a photo-oxidation driven proximity labeling strategy to profile the mitochondrial proteome by light dependence in living cells with high spatiotemporal resolution. Taking advantage of organelle-localizable organic photoactivated probes generating reactive species and nucleophilic substrates for proximal protein oxidation and trapping, mitochondrial proteins were selectively labeled by spatially limited reactions in their native environment. Integration of photo-oxidation driven proximity labeling and quantitative proteomics facilitated the plotting of the mitochondrial proteome in which up to 310 mitochondrial proteins were identified with a specificity of 64% in HeLa cells. Furthermore, mitochondrial proteome dynamics was deciphered in drug resistant Huh7 and LPS stimulated HMC3 cells which were hard-to-transfect. A number of differential proteins were quantified which were intimately linked to critical processes and provided insights into the related molecular mechanisms of drug resistance and neuroinflammation in the perspective of mitochondria. The photo-oxidation driven proximity labeling strategy offers solid technical support to a highly precise proteomic platform in time and finer space for more knowledge of subcellular biology.

A visible light-dependent strategy with high spatiotemporal precision for subcellular proteomics reveals mitochondrial proteome dynamics in a non-perturbed state, and elucidates the potential mechanism of neuroinflammation mediated by mitochondria.     

A Multi‐responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super‐resolution Imaging

Understanding the biomolecular interactions in a specific organelle has been a long‐standing challenge because it requires super‐resolution imaging to resolve the spatial locations and dynamic interactions of multiple biomacromolecules. Two key difficulties are the scarcity of suitable probes for super‐resolution nanoscopy and the complications that arise from the use of multiple probes. Herein, we report a quinolinium derivative probe that is selectively enriched in mitochondria and switches on in three different fluorescence modes in response to hydrogen peroxide (H₂O₂), proteins, and nucleic acids, enabling the visualization of mitochondrial nucleoprotein dynamics. STED nanoscopy reveals that the proteins localize at mitochondrial cristae and largely fuse with nucleic acids to form nucleoproteins, whereas increasing H₂O₂ level leads to disassociation of nucleic acid–protein complexes.     

A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

ATP analogues have been powerful compounds for the study of kinase‐catalyzed phosphorylation. However, the cell impermeability of ATP analogues has largely limited their use to in vitro lysate‐based experiments. Herein, we report the first cell‐permeable ATP analogue, ATP–polyamine–biotin (APB). APB is shown to promote biotin labeling of kinase substrates in live cells and has future applications in phosphoprotein purification and analysis. More generally, these studies provide a foundation for the development of additional cell‐permeable ATP analogues for cell‐signaling research.     

Recent advances in the study of biopolymers

In describing some of the macromolecular characteristics of the nucleic acids, particularly DNA, and of representative proteins, the following aspects are discussed: computer-assisted modeling as a useful tool for depicting the three-dimensional structures and macromolecular characteristics of biopolymers; conformational flexibility in biopolymers; parameters determining protein recognition; and the importance of the three-dimensional structure of antibodies, enzymes, receptors, ion channels, and proteins interacting with nucleic acids, in determining their characteristic biological functions. Many of the problems raised by molecular biologists concerning the mode of action of biopolymers could be tackled by polymer chemists, because of their experience in dealing theoretically as well as experimentally with synthetic and native high molecular weight compounds.     

Peptide Nanofibers Modified with a Protein by Using Designed Anchor Molecules Bearing Hydrophobic and Functional Moieties

Self‐assembly of peptides and proteins is a key feature of biological functions. Short amphiphilic peptides designed with a β‐sheet structure can form sophisticated nanofiber structures, and the fibers are available as nanomaterials for arranging biomolecules. Peptide FI (H‐PKFKIIEFEP‐OH) self‐assembles into nanofibers with a coiled fine structure, as reported in our previous work. We have constructed anchor molecules that have both a binding moiety for the fiber structure and a functional unit capable of capturing target molecules, with the purpose of arranging proteins on the designed peptide nanofibers. Designed anchors containing an alkyl chain as a binding unit and biotin as a functional moiety were found to bind to peptide fibers FI and F2i (H‐ALEAKFAAFEAKLA‐NH₂). The surface‐exposed biotin moiety on the fibers could capture an anti‐biotin antibody. Moreover, hydrophobic dipeptide anchor units composed of iminodiacetate connected to Phe–Phe or Ile–Ile and a peptide composed of six histidine residues connected to biotin could also connect FI peptide fibers to the anti‐biotin antibody through the chelation of Ni²⁺ ions. This strategy of using designed anchors opens a novel approach to constructing nanoscale protein arrays on peptide nanomaterials.     

Peptide Nanomaterials Designed from Natural Supramolecular Systems

Natural supramolecular assemblies exhibit unique structural and functional properties that have been optimized over the course of evolution. Inspired by these natural systems, various bio‐nanomaterials have been developed using peptides, proteins, and nucleic acids as components. Peptides are attractive building blocks because they enable the important domains of natural protein assemblies to be isolated and optimized while retaining the original structures and functions. Furthermore, the peptide subunits can be conjugated with exogenous molecules such as peptides, proteins, nucleic acids, and metal nanoparticles to generate advanced functions. In this personal account, we summarize recent progress in the construction of peptide‐based nanomaterial designed from natural supramolecular systems, including (1) artificial viral capsids, (2) self‐assembled nanofibers, and (3) protein‐binding motifs. The peptides inspired by nature should provide new design principles for bio‐nanomaterials.     

Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition

The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO(2)H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.     

Stable isotope labeling for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay analysis of ribonucleic acids

Matrix-assisted laser desorption/ionization mass spectrometry is a powerful analytical tool for the structural characterization of oligonucleotides and nucleic acids. Here we report the application of stable isotope labeling for the simplified characterization of ribonucleic acids (RNAs). An (18)O label is incorporated at the 3'-phosphate of oligoribonucleotides during the enzymatic processing of intact RNAs. As implemented, a buffer solution containing a 50 : 50 mixture of H(2)O and (18)O-labeled H(2)O is used during endonuclease digestion. Upon digestion, characteristic doublets representative of the isotopic distribution of oxygen are noted for those products that contain 3'-phosphate groups. This approach is used to distinguish readily endonuclease digestion products from incomplete digestion products and non-specific cleavage products. In addition, RNase digestion products containing the characteristic isotopic doublet can be selected for further characterization by post-source decay (PSD) analysis. PSD products carrying the 3'-phosphate group will appear as a doublet, thereby simplifying fragment ion assignment.     

Site‐Specific N‐Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide

An efficient ligase with exquisite site‐specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase‐mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N‐terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields.     

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 Redox‐Activatable Fluorescent Sensor for the High‐Throughput Quantification of Cytosolic Delivery of Macromolecules

Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an “always on” reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal‐to‐noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox‐activatable sensor (qRAS) for the real‐time monitoring of cytosolic delivery of macromolecules. qRAS‐labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high‐throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications.     

A Rhizavidin Monomer with Nearly Multimeric Avidin‐Like Binding Stability Against Biotin Conjugates

Developing a monomeric form of an avidin‐like protein with highly stable biotin binding properties has been a major challenge in biotin‐avidin linking technology. Here we report a monomeric avidin‐like protein—enhanced monoavidin—with off‐rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head‐group‐biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24‐meric avidin probe by fusing eMA to a multimeric cage protein. The 24‐meric avidin and eMA were utilized to demonstrate how artificial clustering of cell‐surface proteins greatly enhances the internalization rates of assembled proteins on live cells.     

Phage display evolution of a peptide substrate for yeast biotin ligase and application to two-color quantum dot labeling of cell surface proteins

Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to two-color imaging and yeast-based applications.