Goran Pljevaljčić

Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA)

A new concept for sequence‐specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S‐adenosyl‐L‐methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase‐catalyzed sequence‐specific coupling with DNA. Sequence‐specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.

Deeply Inverted Electron-Hole Recombination in a Luminescent Antibody-Stilbene Complex

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel π-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody.

Efficient Synthesis of S‐Adenosyl‐L‐Homocysteine Natural Product Analogues and Their Use to Elucidate the Structural Determinant for Cofactor Binding of the DNA Methyltransferase M·HhaI

5′-Acetylthio-5′-deoxy-2′,3′-O-isopropylideneadenosine (8) was directly prepared from commercially available 2′,3′-O-isopropylideneadenosine (7) and thioacetic acid under Mitsunobu conditions in almost quantitative yield. In situ cleavage of the acetylthio function of 8 followed by coupling with different alkyl bromides proceeded with high yields. Deprotection of the obtained 5′-thionucleosides yielded the S-adenosyl-L-homocysteine analogues decarboxylated AdoHcy (11), deaminated AdoHcy (14) and 5′-[3-(cyano)propylthio]-5′-deoxyadenosine (16) in good overall yields. Direct deprotection of the thionucleoside 8 delivered 5′-thio-5′-deoxyadenosine (18) in excellent yield. In addition, binding constants of these AdoHcy analogues and the DNA methyltransferase M·HhaI were determined in a fluorescence assay.

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR.

Significance Activation of G protein-coupled receptors (GPCRs) by agonists is the first step of eukaryotic cellular signal transduction. Because GPCRs are expressed in almost all human tissues and play a key role in human physiology, they are the targets for more than 30% of pharmaceutical drugs. Binding of ligands on the extracellular surface of a GPCR induces a conformational change on the cytoplasmic surface, which is recognized by G proteins or other cellular effectors. Here we show that the β2-adrenergic receptor, a prototypical GPCR, naturally fluctuates between inactive and active conformations, and that agonist or inverse agonist ligands modulate the conformational exchange kinetics in distinct ways, explaining their different pharmacological efficacies. These insights should assist in the design of improved GPCR-targeting drugs. Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

Quantitative Labeling of Long Plasmid DNA with Nanometer Precision

Sequence-specific labeling of native DNA is of major interest for functional studies of DNA and DNA-modifying enzymes as well as for nanobiotechnology and medical diagnostics. Bearing in mind the size of DNA and the recurrence of only four major building blocks, sequence-specific chemical modification is a very challenging task. DNA sequence recognition can be achieved by using engineered zinc-finger proteins, hairpin polyamides, triplex-forming oligodeoxynucleotides (TFOs), or peptide nucleic acids. TFOs have been used for covalent DNA labeling, but they generally recognize rare homopurine– homopyrimidine tracks, which limits a broad applicability for sequence-specific DNA labeling. Most interestingly, sequence-specific DNA modification is already performed by nature. DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet or SAM, 1) to adenine or cytosine residues within specific DNA sequences that range between two and eight base pairs (Scheme 1). By replacing the methionine side chain of AdoMet with an aziridine group, DNA MTases can be tricked to couple the whole cofactor N-adenosylaziridine (2a, R=H) sequence-specifically with DNA. This system has been used to deliver functional groups to short duplex oligodeoxynucleotides that were then coupled with suitable functionalized reporter groups in a second step. However, this two-step procedure only gave moderate labeling yields. Quantitative labeling of long plasmid DNA was achieved with the DNA MTase M.TaqI, and direct attachment of the fluorescent dansyl group to the 8-position of the aziridine cofactor via a flexible linker. We have extended this work and synthesized the biotinylated aziridine cofactor 2b (R=NH ACHTUNGTRENNUNG(CH2)4NH-biotin; Scheme 2). The primary amino group of 8-amino ACHTUNGTRENNUNG[1’’-(4’’-aminobutyl)]-2’,3’O-isopropylideneadenosine was protected as trifluoroacetyl amide, the 5’ position was activated as mesyl ester, and the isopropylidene protecting group was removed under acidic conditions. Nucleophilic substitution of the mesylate with aziridine, removal of the trifluoroacetyl protecting group under basic aqueous work up, and coupling of the resulting primary amine with N-hydroxysuccinimidyl biotin (NHS–biotin) furACHTUNGTRENNUNGnished the desired N-adenosylaziridine derivative 2b (for details see the Supporting Information). Compared to the previously employed photocleavable 6-nitroveratryloxocarbonyl protecting group the trifluoroacetyl group proved superior because of its facile removal. The introduction of the reporter group in the last step by its NHS ester offers a versatile approach to aziridine cofactors with various commonly used labels. Aziridine cofactor 2b comprises three functions for DNA MTase-mediated coupling with DNA: the aziridine ring acts as an electrophilic reactive group after protonation, the adenosyl Scheme 1. Reactions catalyzed by the DNA methyltransferase M.TaqI. Nucleophilic attack of the exocyclic amino group of adenine onto the activated methyl group of S-adenosyl-l-methionine (AdoMet, 1) leads to methyl group transfer to adenine in the 5’-TCGA-3’ recognition sequence (left), and nucleophilic ring opening of the aziridine group in cofactor 2 results in sequence-specific coupling with DNA (right).

Sequence-specific DNA labeling using methyltransferases.

Sequence-specific labeling of native deoxyribonucleic acid (DNA) still represents a more-or-less unsolved problem. Difficulties mainly arise from the necessity to combine two different functions: sequence-specific recognition of DNA and covalent bond formation between the label and DNA. DNA methyltransferases (MTases) naturally possess these two functions and transfer a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to adenine or cytosine residues within specific DNA sequences, typically ranging from two to eight base pairs. Unfortunately, the methyl group itself is a very limited reporter group and it would be desirable to transfer larger chemical entities with DNA MTases. Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to the synthetic cofactor N-adenosylaziridine, which is quantitatively, base- and sequence-specifically coupled with DNA in a DNA MTase-catalyzed reaction. By attaching interesting reporter groups to a suitable position of N-adenosylaziridine a large variety of new synthetic cofactors are obtained for sequence-specific labeling of DNA. This method is illustrated by coupling primary amino groups and biotin to short duplex oligodeoxynucleotides or plasmid DNA using the DNA MTase M.TaqI.

Single-Molecule Fluorescence Methods for the Analysis of RNA Folding and Ribonucleoprotein Assembly

Publisher Summary In addition to its role as an information carrier in gene expression, RNA is now recognized to carry out a broad range of biological functions, and novel activities continue to emerge on a regular basis. RNA molecules can act as catalysts in reversible phosphodiester-cleavage reactions, mediate splicing of premessenger RNA, silence specific genes, and act as molecular switches to sense metabolites and regulate the translation of genes into proteins, to name just a few important examples. Perhaps the most impressive (and unexpected) function of RNA is the ability to catalyze peptide bond formation on the ribosome. Similar to proteins, RNAs are synthesized as linear polymers that must fold into compact three-dimensional structures to attain their biological activity. However, there are fundamental differences between these two types of macromolecular folding processes. While small RNAs, including many ribozymes, can fold autonomously, most large RNA molecules require one or more protein chaperones for efficient and correct folding. Proteins can prevent misfolding of large RNAs and/or accelerate the escape from kinetic traps. Additionally, proteins may guide proper folding by manipulating the structure of the RNA chain. Chemical cross-linking can capture RNA folding intermediates, but the technique provides only limited structural information. Fluorescence spectroscopy can provide both structural and dynamic information and is emerging as a powerful tool for studies of RNA folding and RNP assembly processes. Fluorescence measurements can be performed in solution under physiologically relevant conditions, without restrictions arising from the size of the molecules under study. Moreover, the method provides dynamic information spanning a wide range of time scales, from picoseconds to minutes.

Analysis of RNA Folding and Ribonucleoprotein Assembly by Single-Molecule Fluorescence Spectroscopy

To execute their diverse range of biological functions, RNA molecules must fold into specific tertiary structures and/or associate with one or more proteins to form ribonucleoprotein (RNP) complexes. Single-molecule fluorescence spectroscopy is a powerful tool for the study of RNA folding and RNP assembly processes, directly revealing different conformational subpopulations that are hidden in conventional ensemble measurements. Moreover, kinetic processes can be observed without the need to synchronize a population of molecules. In this chapter, we describe the fluorescence spectroscopic methods used for single-molecule measurements of freely diffusing or immobilized RNA molecules or RNA-protein complexes. We also provide practical protocols to prepare the fluorescently labeled RNA and protein molecules required for such studies. Finally, we provide two examples of how these various preparative and spectroscopic methods are employed in the study of RNA folding and RNP assembly processes.