Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.     

Site-specific chemical labeling of long RNA molecules

Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range of functional groups.     

Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase

We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.     

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.     

Site-specific covalent labeling of proteins inside live cells using small molecule probes

The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.     

Combinatorial discovery of fluorophores and fluorescent probes

The properties of organic fluorophores are difficult to predict, even in simple cases. Fluorescent probes--which combine fluorescent properties with the equally challenging problem of molecular recognition--are even more difficult to develop. Combinatorial approaches to the development of such molecules are a new but promising endeavor, and reviewing the state of the art delineates the near-and long-term possibilities.     

Site-specific labeling of supercoiled DNA

Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since SfiI requires simultaneous interaction with two DNA recognition sites for stable binding, this requirement is satisfied by providing an isolated recognition site in the DNA target and an additional short DNA duplex also containing the recognition site. The SfiI/DNA complexes were visualized with AFM and the specificity of the labeling was confirmed by the length measurements. Using this approach, two sites in plasmid DNA were labeled in the presence of a large excess of the helper duplex to compete with the formation of looped structures of the intramolecular synaptic complex. We show that the labeling procedure does not interfere with the superhelical tension-driven formation of alternative DNA structures such as cruciforms. The complex is relatively stable at low and high pH (pH 5 and 9) making the developed approach attractive for use at conditions requiring the pH change.     

Novel fluorophores for labeling of nucleosides and oligonucleotides

Five novel fluorophores have been synthesized and their comparative fluorescence has been studied in different organic solvents and aqueous solutions of inorganic ions. Out of these, two highly sensitive fluorophores have been used to label phosphoramidites and oligodeoxyribonucleotides. The fluorescently labelled amidites and oligodeoxyribonucleotides showed good fluorescence signals.     

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.     

Diazirine-containing tag-free RNA probes for efficient RISC-loading and photoaffinity labeling of microRNA targets

We designed and synthesized a photo-reactive and tag-free RNA probe for the identification of microRNA (miRNA) targets. To synthesize the RNA probe, we designed a novel nucleoside analog 1-O-[3-ethynyl-5-(3-trifluoromethyl-3H-diazirine-3-yl)]benzyl-β-d-ribofuranose containing aryl trifluoromethyl diazirine and ethynyl moieties. The RNA probe containing this analog was observed to form crosslinks with complementary RNA by UV irradiation and was rapidly tagged by Cu-catalyzed azide alkyne cycloaddition (CuAAC). In addition, the tag-free and photo-reactive miRNA-145 probe showed comparable gene silencing activity to that of unmodified miRNA-145. Therefore, miRNA probes containing the nucleoside analog are promising candidates for the identification of target mRNAs of miRNAs.     

Site-specific labeling of proteins with cyclen-bound transition metal ions

A general procedure for site-specific and reversible labeling of proteins with transition metal ions is described. The method is based on the use of the novel ligand 1-(2-thioethyl)-1,4,7,10-tetraazacyclododecane (TETAC), which specifically and readily reacts with thiol groups. Synthesis of TETAC from 1,4,7,10-tetraazacyclododecane (cyclen) and ethylene disulfide yielded a mixture of products, including TETAC and its oxidized disulfide in 56.4% yield. The procedure for labeling proteins with TETAC is straightforward and led to separation of the TETAC-containing product mixture through gel-filtration chromatography. The resulting protein-TETAC adducts were shown to contain a single TETAC group which bound transition metal ions. Protein-TETACCu²⁺ had a UV-Vis spectrum similar to that of Cu²⁺(cyclen) while the protein-TETACCo²⁺ complex had a different spectrum to that of the cobalt-containing cyclen. This is because attachment to the protein prevented the Co²⁺-containing TETAC from dimerising and binding O₂, which the cobalt-containing cyclen is able to do. The proteins used to develop this labeling procedure were the DNase domain of colicin E9 and its inhibitor protein Im9. Unlike Im9, the DNase does not contain a cysteine residue but the Ser30Cys variant of the DNase was prepared by site-directed mutagenesis. Both Im9 and the Ser30Cys DNase were modified with TETAC and the modifications shown to be structurally and functionally benign through NMR spectroscopy of the modified Im9 and fluorescence spectroscopy binding assays in which DNase-Im9 complexes were formed. The simplicity of the method, and its general application to any protein through the introduction of cysteine by site-directed mutagenesis, suggests it will be of wide use in protein chemistry applications.     

Site-specific derivatization of RNA with photocrosslinkable groups

Derivatization of RNA with heterobifunctional photocrosslinking reagents becomes an increasingly popular method for the analysis of structural properties of ribonucleoprotein complexes. This article describes a simple chemical modification-derivatization strategy used to introduce selected chemical groups at specific internal positions within the RNA ribose backbone. The strategy is based on the coupling of a haloacetyl adduct to a thiol residue in the phosphodiester bond. The use of a number of RNA probes derivatized with several different photoreactive groups can provide invaluable information on the structural distribution of components in complex ribonucleoprotein assemblies.     

Site-specific modification or rabbit muscle aldolase with fluorescent probes

The site-specific modification of rabbit muscle aldolase A by labeling of thiol residues of Cys-289 with 5-(2-((iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid and Cys-239 with 5-iodoacetamidofluorescein or 4-dimethylamino-phenylazophenyl-4'-maleimide has been described. The method is based on the differences in kinetics of the chemical modification of aldolase thiols with the above reagents either in the presence or in the absence of a competitive inhibitor. The spectral properties of the doubly labeled aldolase derivatives were compared with those of the singly labeled enzyme. The doubly labeled aldolase derivatives exhibited full catalytic activity.     

Use of nonradiative decays of extrinsic fluorophores as structural and dynamical probes in protein environments: Fluorescence quenching

In this work a combined pulsed-laser, time-resolved photoacoustic calorimetry (PAC) and fluorescence study is presented on two widely used covalent protein probes, fluorescein-5-isothiocyanate (FITC) and 6-acryloyl-2-dimethylaminonaphtalene (acrylodan). Three proteins that contain a single free thiol, namely carbonic anhydrase, bovine serum albumin (BSA) and papain, have been selectively labelled with FITC and acrylodan, and their fluorescence emission was quenched with KI. Nonradiative decays of the excited states of FITC are used to complement the information usually obtained by monitoring the quenching of fluorescence emssion. Data analysis evidences the dependence of the nonradiative quenching constants on the exposure of the dye to the solvent, and shows the involvement of a triplet state of FITC in the non radiative deexcitation. The shielding of the binding sites from the solvent is demonstrated also by the fluorescence emission of acrylodan and by the Stern-Volmer analysis of fluorescence quenching by KI. From photoacoustic data, an estimate of the fluorescent quantum yield of bound FITC is obtained. This work demonstrates the complete equivalence of quenching data obtained by fluorescence and photoacoustics measurements and shows that this combined approach allows a better control of the photophysics of the dyes involved in the quenching process.     

Site-specific independent double labeling of proteins with reporter atoms.

Many types of physical, spectroscopic, and biological studies of proteins and other macromolecules are facilitated by the incorporation of reporter groups. In many cases these are single atom substitutes, for example isotopes (13C for C), or light (F for H) and heavy (Se for S) atom homologs. In some circumstances the incorporation of two different labels in the same molecule would be greatly desirable. Commonly used protein engineering methods for incorporating them can rarely cope with differential double labeling, and have other limitations such as universal, non-specific, or random incorporation. Although de novo peptide synthesis has the power to achieve highly specific labeling, the difficulties inherent in creating long sequences lead us to propose protein semisynthesis as the most practical approach. By ligating combinations of natural and labeled synthetic fragments to reform holoproteins, we can overcome any of the limitations discussed. Using cytochrome c as a model protein we show that two reporter atoms, selenium and bromine, can be simultaneously and site-specifically incorporated without significant consequences to structure and (or) function. This capability opens up the prospect of advances in a number of areas in structural biology.     

Site-specific labeling of proteins with small molecules in live cells

The principal bottleneck for the utilization of small-molecule probes in live cells is the shortage of methodologies for targeting them with very high specificity to biological molecules or compartments of interest. Recently developed methods for labeling proteins with small-molecule probes in cells employ special protein or peptide handles that recruit small-molecule ligands, harness enzymes to catalyze small-molecule conjugation or hijack the cell's protein translation machinery.     

Site-specific protein labeling with amine-containing molecules using Lactobacillus plantarum sortase

Modification of proteins with small molecules is a widely used and powerful tool in biological research. Enzymatic approaches are particularly promising because substrate specificity allows for site-specific modification. Sortase A, a transpeptidase from Staphylococcus aureus, cleaves between the T and G residues in the sequence LPXTG, and subsequently links the carboxyl group of the T residue to an amino group of N-terminal glycine oligomers by a native peptide bond. Although Gram-positive bacteria have several kinds of sortases, there are few reports concerning their expression and substrate specificity. Here, we demonstrate site-specific protein modification with primary amine-containing molecules catalyzed by Lactobacillus plantarum sortase. Enhanced green fluorescent protein (EGFP) was employed as a model protein, and an amine-containing biotin molecule was site-specifically conjugated with LPQTSEQ-tagged EGFP. We developed a novel Lactobacillus plantarum sortase that has different substrate specificity compared to Staphylococcus aureus sortase. Amine-directed protein modification was achieved using the Lactobacillus plantarum sortase 'LPQTSEQ' sequence original recognition tag. Our results demonstrate a promising method for expanding the capabilities of site-specific protein-small molecule modification.