Modulation of an RNA-branching deoxyribozyme by a small molecule

We have engineered an RNA-branching deoxyribozyme to respond positively to ATP, resulting in modulated control of ligation activity that may be applicable to sensor and nanotechnology applications.     

Site-selective RNA cleavage by DNA bearing a base pair-mimic nucleoside

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.     

Investigation of a deoxyribozyme as a biofuel cell catalyst

We examined the ability of a previously identified peroxidase deoxyribozyme to be employed as a catalyst in biofuel cells, as a possible replacement for oxidoreductase proteins. We constructed a biocathode using a covalently linked version of the peroxidase deoxyribozyme-hemin complex and successfully paired it with a glucose dehydrogenase-modified bioanode for power production.     

Site-selective labeling strategies for screening by NMR

NMR based screening has become an important tool in the pharmaceutical industry. Methods that provide information on the location of small molecule binding sites on the surface of a drug target (e. g. SAR-by-NMR and related techniques) are of particular interest. In order to extend the applicability of such techniques to drug targets of higher molecular weight, selective labeling strategies may be employed. Dual-amino acid selective labeling and site directed non-native amino acid replacement (SNAAR) allow for the selective detection of NMR resonances of a specific amino acid residue. This results in significantly reduced spectral complexity, which not only enables application to higher molecular weight systems, but also eliminates the need for sequential resonance assignment in order to identify the binding site. Regio-selective (or segmental) labeling of an entire protein domain of a multi domain protein may also be achieved. Labeling only a selected part of a multi domain protein (e. g. a catalytic or ligand binding domain) is an attractive way to simplify the spectral interpretation without disturbing the system under study.     

Site-selective metal binding by designed alpha-helical peptides

It is known that the designed alpha-helical peptide family TRI [(Ac-G(LKALEEK)4G-CONH2)], containing single site substitution of a cysteine for a leucine, is capable of binding Cd(II) within a three-stranded coiled coil. The binding affinity of cadmium is dependent upon the site of substitution, with cysteine incorporated at the a site leading to cadmium complexes of higher affinity than when a d site was modified. In this work we have examined whether this differential binding affinity can be expressed in a di-cysteine-substituted peptide in order to develop site specificity within a designed system. The peptide TRI L9CL19C was used to determine whether significant differences in binding affinities at nearly proximal sites could be achieved in a short designed peptide. On the basis of 113Cd, 1H NMR, and circular dichroic spectroscopies, we have shown that 1 equiv of Cd(II) binds exclusively at the a site. Only after that position is filled does the second site become populated. Thus, the TRI system represents the first example where stoichiometrically equivalent peptides with different sequences form the framework for designing molecular assemblies that show site-specific ion recognition. We propose that the distinct metal affinities are due to the cysteine conformers at different substitution points along the peptide. Furthermore, we have shown that site selectivity in biomolecules can be encoded into relatively short peptides with helical sequences and, therefore, do not necessarily require the extensive protein scaffolds found in natural systems.     

Site-selective DNA hydrolysis induced by a metal-free peptide nucleic acid-cyclen conjugate

A metal-free artificial restriction DNA cutter which is composed of cyclen and classical peptide nucleic acid (PNA) was synthesized. Analysis of DNA cleavage products indicates the site-selective hydrolysis.     

Site-selective bromination of vancomycin

We report the site-selective bromination of vancomycin to produce, with substantial efficiency, previously unknown monobromovancomycins, a dibromovancomycin, and a tribromovancomycin. We document the inherent reactivity of native vancomycin toward N-bromophthalimide. We then demonstrate significant rate acceleration and perturbation of the inherent product distribution in the presence of a rationally designed peptide-based promoter. Alternative site selectivity is observed as a function of solvent and replacement of the peptide with guanidine.     

Conversion of a ribozyme to a deoxyribozyme through in vitro evolution

An RNA ligase ribozyme was converted to a corresponding deoxyribozyme through in vitro evolution. The ribozyme was prepared as a DNA molecule of the same sequence, and had no detectable activity. A population of randomized variants of this DNA was constructed and evolved to perform RNA ligation at a rate similar to that of the starting ribozyme. When the deoxyribozyme was prepared as an RNA molecule of the same sequence, it had no detectable activity. Thus, the evolutionary transition from an RNA to a DNA enzyme represents a switch, rather than a broadening, of the chemical basis for catalytic function. This transfer of both information and function is relevant to the transition between two different genetic systems based on nucleic acid-like molecules, as postulated to have occurred during the early history of life on Earth.     

Site-selective protein glycation and PEGylation

A two step protocol has been set up to selectively conjugate PEG to buried amino acids of proteins. The process involves site-specific glycation followed by PEGylation of the oxidized glycosides. Aimed at glycating the cysteine groups of proteins, two maleimide-glycosylic linkers have been synthesised: galactosyl-glucono-CO–NH–(CH₂)₁₂–NH–CO–(CH₂)₂-maleimide and maltosyl-glucono-CO–NH–(CH₂)₁₂–NH–CO–(CH₂)₂-maleimide. The linkers were extensively characterized by ¹H NMR, FT-IR, ESI–TOF mass spectrometry and colorimetric assays. Complete conjugation of the activated linkers to Cys³⁴ of human serum albumin was obtained in about 2 h. The selective oxidation of the galactosyl and maltosyl moieties by periodate treatment yielded two and three available aldehyde groups, respectively. The PEG-hydrazide conjugation to the aldehyde groups was found to be 100% in about 40 h, whereas less than 30% protein modification was obtained by direct conjugation of commercial PEG-maleimide to Cys³⁴. The pH dependent PEG-glycosyl hydrazone bond hydrolysis at various pH values was verified. PEG release was faster under mild acidic and basic conditions than at neutral pH. Furthermore, the maltosyl derivatives, by virtue of the higher number of coupled PEG chains, showed a slower protein release as compared to the galactosyl counterpart, indicating that the choice of the glycosylic linker allows for control of protein release kinetics.     

Site-selective screening by NMR spectroscopy with labeled amino acid pairs

A new method for site-selective screening by NMR is presented. The core of the new method is the dual amino acid sequence specific labeling technique. Amino acid X is labeled with (13)C and amino acid Y is labeled with (15)N. Provided only one XY pair occurs in the amino acid sequence, only one signal in the 1D carbonyl (13)C spectrum will display a splitting due to the (1)J(C'N) coupling. Using this labeling strategy it is possible to screen selectively for binding to a selected epitope without the need for sequence specific assignments. An HNCO spectrum (1D or 2D) can be used either directly as a screening experiment or indirectly to identify what signals to monitor in a 2D (1)H-(15)N correlation spectrum. Chemical shift perturbations upon addition of a potential ligand are easily detected even for large proteins due to the reduced spectral complexity resulting from the use of a selectively labeled sample. The new technique is demonstrated on the human adipocyte fatty acid binding protein FABP-4. Due to the reduced spectral complexity, the method should be applicable to larger proteins than are conventional methods.     

Site-selective photochemistry in anthracene crystals

Site-selective heterodimer formation in anthracene crystals containing a small amount of 2-hydroxyanthracene (2OHA) has been investigated. While direct irradiation at low temperatures of the absorption origins of the two 2OHA sites (O₈ and O₉) in anthracene did not lead to dimer formation, one of these sites (O₉) is photoactive when irradiated at room temperature. From an analysis of the O₈ and O₉ fluorescence excitation spectra we infer an involvement of the X-traps in heterodimer formation. Furthermore it seems likely that an intermediate, perhaps an excimer is involved in the process.     

Site-selective patterning using surfactant-based resists

Patterned SAMs of alkanethiols on gold or silver are explored as resists for electrodeposition and exhibit surprisingly rich behavior depending on the overpotential and the length of the alkane chain. At small overpotentials, SAMs are positive resists with deposition only in the surfactant-free regions. At larger overpotentials, SAMs are negative resists with preferential deposition in the SAM-modified regions. Tunable surfactant-based resists are potentially versatile tools to dictate the deposition of materials and are demonstrated as a means of creating complex, three-dimensional structures.     

Site-selective catalysis: toward a regiodivergent resolution of 1,2-diols

This paper demonstrates that the secondary hydroxyl can be functionalized in preference to the primary hydroxyl of a 1,2-diol. The site selectivity is achieved by using an enantioselective organic catalyst that is able to bond to the diol reversibly and covalently. The reaction has been parlayed into a divergent kinetic resolution on a racemic mixture, providing access to highly enantioenriched secondary-protected 1,2-diols in a single synthetic step.     

Site-selective metal-coordination-based patterning of silane monolayers

Herein, we report a method to pattern a derivatized nifedipine silane monolayer that aromatizes under UV irradiation. Using a functionalized SCS-Pd(II) pincer complex, we demonstrate that a strong metal-coordination complex between the aromatized nifedipine derivative and the pincer complex is formed. This methodology integrates SAM photolithography (top-down) and molecular recognition directed self-assembly (bottom-up) strategies to create simple and rapid synthesizable functional patterned surfaces.     

Novel biomaterials derived from deoxyribozyme and NAPzyme

We report the potential of a small Ca²⁺-dependent deoxyribozyme as a novel biomaterial to distinguish RNA foldings. It is found that an immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The RNA cleavage reaction is also detected using the deoxyribozyme SPR sensor chip. Furthermore, we develop a novel NAPzyme (nucleic acid peptide deoxyribozyme) with its RNA cleavage function in the absence of divalent metal ions.     

Site-selective surface modification using enzymatic soft lithography

Surface modification with functional polymers or molecules offers great promise for the development of smart materials and applications. Here, we describe a versatile and easy-to-use method of site-selective surface modification based on the ease of microcontact printing and the exquisite selectivity of enzymatic degradation. A micropatterned poly-L-lysine (PLL) layer on solid substrates was prepared by enzymatic degradation using trypsin enzyme immobilized on a prestructured poly(dimethlylsiloxane) (PDMS) stamp. After the enzymatic degradation of PLL and the removal of the degradation products, very well defined patterning was revealed over a large scale by fluorescence microscopy and atomic force microscopy (AFM). We investigate the advantage of our method by comparison with traditional microcontact printing and found that lateral diffusion was reduced, yielding a more accurate reproduction of the master. We also demonstrate that the stamp can be reused without reinking. The patterned surface was used for site-selective modification. The strategy was applied to two applications: the first is dedicated to the creation of amino-silane patterned surfaces, and the second illustrates the possibility of patterning polyelectrolyte multilayered thin films.