Rie Kawai

An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA

Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, γ-amidotriphosphates, as substrates of 3′ to 5′ exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules.

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.

A facile synthesis of cyclic bis(3'→5')diguanylic acid

Abstract This paper describes a new method for synthesizing biologically important cyclic bis(3′→5′)diguanylic acid (cGpGp) in a higher yield than that of the existing synthetic method. In the new synthesis, the following two means, in place of those used in the existing synthesis are employed as main strategies to cause the increase in product yield. One of these distinctive strategies in the new synthesis is that the phosphoramidite method is used for the preparation of a key synthetic intermediate of a linear guanylyl(3′→5′)guanylic acid derivative. This method allowed higher-yield formation of the intermediate than that by the triester method used in the existing synthesis. The second distinctive strategy used in the new synthesis is that allyloxycarbonyl and allyl groups are used for the protection of two guanine bases and two internucleotide bonds, respectively. These four allylic protectors can be removed all at once by the organopalladium-catalyzed reaction under neutral conditions. Thus, deprotection of the protected cGpGp precursor was achieved in the present synthesis in a shorter step and under milder conditions than the deprotection achieved in the existing synthesis, which uses diphenylacetyl and o-chlorophenyl groups as protectors for two guanine bases and two internucleotide bonds, respectively, whose full removal requires two different procedures including rather harsh basic treatment. As a result, technical loss and decomposition of the target product in the new synthesis is remarkably reduced.

Crystal Structure of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete chrysosporium

Glycoside hydrolase family 55 consists of β-1,3-glucanases mainly from filamentous fungi. A β-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes β-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from β-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two β-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two β-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various β-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two β-helical domains, demonstrating an unexpected variety of carbohydrate binding modes.

Site-specific incorporation of functional components into RNA by an unnatural base pair transcription system.

Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

X-Ray Crystal Structures of Phanerochaete Chrysosporium Laminarinase 16A in Complex with Products from Lichenin and Laminarin Hydrolysis

The 1,3(4)‐β‐d‐glucanases of glycoside hydrolase family 16 provide useful examples of versatile yet specific protein–carbohydrate interactions. In the present study, we report the X‐ray structures of the 1,3(4)‐β‐d‐glucanase Phanerochaete chrysosporium Laminarinase 16A in complex with β‐glucan products from laminarin (1.6 Å) and lichenin (1.1 Å) hydrolysis. The G6G3G3G glucan, in complex with the enzyme, showed a β‐1,6 branch in the acceptor site. The G4G3G ligand–protein complex showed that there was no room for a β‐1,6 branch in the −1 or −2 subsites; furthermore, the distorted residue in the −1 subsite and the glucose in the −2 subsite required a β‐1,3 bond between them. These are the first X‐ray crystal structures of any 1,3(4)‐β‐d‐glucanase in complex with glucan products. They provide details of both substrate and product binding in support of earlier enzymatic evidence.

Synthesis of cyclic β-glucan using Laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-beta-D-glucanase nucleophile mutant E115S of Phanerochaete chrysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and nonreducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with alpha-laminariheptaosyl fluoride (alphaL7F) revealed how alphaL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with alphaL7F the major product was a cyclic beta-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely beta-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic beta-glucan.

Effect of molecular sieves in the liquid-phase synthesis of nucleotides via the phosphoramidite method

Abstract It is demonstrated that the reaction of a nucleoside phosphoramidite and a nucleoside aided by a suitable promoter in the presence of molecular sieves 3A or 4A in a liquid phase is efficiently performed by the use of stoichiometric amounts of the reactants to give the desired coupling product in an excellent yield.

Site-specific incorporation of functional components into RNA by transcription using unnatural base pair systems

The creation of an extra, unnatural base pair that functions in replication, transcription, and translation, would provide a new system for the expansion of the genetic alphabet. In transcription, an unnatural base pair system could be used for making new RNA molecules containing functional components of interest at specific positions. We have developed several unnatural base pairs that can function in replication and transcription. Among them, the 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) pair and the Ds and pyrrole-2-carbaldehyde (Pa) pair exhibit high selectivity and efficiency in PCR amplification and T7 transcription, respectively. We performed PCR amplification involving the Ds-Px pair for DNA template preparation and T7 transcription involving the Ds-Pa pair with several modified Pa substrates for the incorporation of functional components, such as biotin- and fluorescein-linked Pa nucleotides, into RNA. This Ds-Px and Ds-Pa pair system could provide a powerful tool for the site-specific fluorescent labeling and immobilization of RNA molecules, as a detection system for RNA and RNA-protein interactions.

X-ray crystallographic native sulfur SAD structure determination of laminarinase Lam16A from Phanerochaete chrysosporium.

Laminarinase Lam16A from Phanerochaete chrysosporium was recombinantly expressed in Pichia pastoris, crystallized and the structure was solved at 1.34 A resolution using native sulfur SAD X-ray crystallography. It is the first structure of a non-specific 1,3(4)-beta-D-glucanase from glycoside hydrolase family 16 (GH16). P. chrysosporium is a wood-degrading basidiomycete fungus and Lam16A is the predominant extracellular protein expressed when laminarin is used as the sole carbon source. The protein folds into a curved beta-sandwich homologous to those of other known GH16 enzyme structures (especially kappa-carrageenase from Pseudoalteromonas carrageenovora and beta-agarase from Zobelia galactanivorans). A notable likeness is also evident with the related glycoside hydrolase family 7 (GH7) enzymes. A mammalian lectin, p58/ERGIC, as well as polysaccharide lyase (PL7) enzymes also showed significant similarity to Lam16A. The enzyme has two potential N-glycosylation sites. One such site, at Asn43, displayed a branched heptasaccharide sufficiently stabilized to be interpreted from the X-ray diffraction data. The other N-glycosylation motif was found close to the catalytic centre and is evidently not glycosylated.