近藤 次郎

Abstract In this study, we report the synthesis of 2′-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5–11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2′-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2′-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2′-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.

Abstract The combination of mass spectrometry and single crystal X‐ray diffraction of HPLC‐purified DNA‐stabilized silver nanoclusters (DNA‐AgNCs) is a powerful tool to determine the charge and structure of the encapsulated AgNC. Such information is not only relevant to design new DNA‐AgNCs with tailored properties, but it is also important for bio‐conjugation experiments and is essential for electronic structure calculations. Here, the efforts to determine the structure of a HPLC‐purified green emissive DNA‐AgNC are presented. Unfortunately, the original DNA‐AgNC, known to have four valence electrons, could not be crystallized. By modifying the stabilizing DNA sequence, while maintaining the original spectroscopic properties, several mutants could be successfully crystallized, and for one of them, single crystal X‐ray diffraction data provided insight into the silver positions. While the DNA conformation is not resolved, the described approach provides valuable insight into the class of green and dual emissive DNA‐AgNCs with four valence electrons. These results constitute a roadmap on how to improve crystallization and crystal quality for X‐ray diffraction measurements.

Numerous applications of metal-mediated base pairs (metallo-base-pairs) to nucleic acid based nanodevices and genetic code expansion have been extensively studied. Many of these metallo-base-pairs are formed in DNA and RNA duplexes containing Watson-Crick base pairs. Recently, a crystal structure of a metal-DNA nanowire with an uninterrupted one-dimensional silver array was reported. We now report the crystal structure of a novel DNA helical wire containing HgII -mediated T:T and T:G base pairs and water-mediated C:C base pairs. The Hg-DNA wire does not contain any Watson-Crick base pairs. Crystals of the Hg-DNA wire, which is the first DNA wire structure driven by HgII ions, were obtained by mixing the short oligonucleotide d(TTTGC) and HgII ions. This study demonstrates the potential of metallo-DNA to form various structural components that can be used for functional nanodevices.

The aminoglycoside resistance conferred by an N1-methylation of A1408 in 16S rRNA by a novel plasmid-mediated methyltransferase NpmA can be a future health threat. In the present study, we have determined crystal structures of the bacterial ribosomal decoding A site with an A1408m(1)A antibiotic-resistance mutation both in the presence and absence of aminoglycosides. G418 and paromomycin both possessing a 6 '-OH group specifically bind to the mutant A site and disturb its function as a molecular switch in the decoding process. On the other hand, binding of gentamicin with a 6 '-NH3+ group to the mutant A site could not be observed in the present crystal structure. These observations agree with the minimum inhibitory concentration of aminoglycosides against Escherichia coli. In addition, one of our crystal structures suggests a possible conformational change of A1408 during the N1-methylation reaction by NpmA. The structural information obtained explains how bacteria acquire resistance against aminoglycosides along with a minimum of fitness cost by the N1-methylation of A1408 and provides novel information for designing the next-generation aminoglycoside.

We have determined a crystal structure of an RNA duplex containing a novel metal-binding motif. The motif is composed of two sheared GOA base pairs, two unpaired A residues and four phosphate groups in close proximity. Four A residues make an A-A-A-A stacking column at the minor groove side and two G bases are highly inclined, thereby forming the pocket-shaped motif at the major groove side. In the present structure, a hydrated Sr2+ ion exists in the pocket and binds to the O6 and N7 atoms of the two G bases and four phosphate groups. According to the previously-reported metal-binding properties to RNA molecules, many of divalent cations, such as Mg2+ Mn2+ Co2+ Zn2+ Ba2+ Pb2+ and Cd2+, may bind to the motif. This metal-binding motif can be used as a modular building block that allows for precise positioning of a single metal ion in functional nucleic acid molecules.

Herein, we determined a high-resolution crystal structure of a B-form DNA duplex containing consecutive dinuclear metal ion-mediated base pairs, namely, 4-thiothymine-2Ag(I)-4-thiothymine (S-2Ag(I)-S), and four Ag(I) ions form a rectangular network and the distances between the Ag(I) ions are 2.8-3.2 angstrom, which may indicate the existence of metallophilic attractions.

The double-helix structure of DNA, in which complementary strands reversibly hybridize to each other, not only explains how genetic information is stored and replicated, but also has proved very attractive for the development of nanomaterials. The discovery of metal-mediated base pairs has prompted the generation of short metal-DNA hybrid duplexes by a bottom-up approach. Here we describe a metallo-DNA nanowire-whose structure was solved by high-resolution X-ray crystallography -that consists of dodecamer duplexes held together by four different metal-mediated base pairs (the previously observed C-Ag-C, as well as G-Ag-G, G-Ag-C and T-Ag-T) and linked to each other through G overhangs involved in interduplex G-Ag-G. The resulting hybrid nanowires are 2 nm wide with a length of the order of micrometres to millimetres, and hold the silver ions in uninterrupted one-dimensional arrays along the DNA helical axis. The hybrid nanowires are further assembled into three-dimensional lattices by interactions between adenine residues, fully bulged out of the double helix.

The structure of an Ag-I-mediated cytosine-cytosine base pair, C-Ag-I-C, was determined with NMR spectroscopy in solution. The observation of 1-bond N-15-Ag-109 J-coupling ((1)J(N-15, Ag-109): 83 and 84 Hz) recorded within the C-Ag-I-C base pair evidenced the N3-Ag-I-N3 linkage in C-Ag-I-C. The triplet resonances of the N4 atoms in C-Ag-I-C demonstrated that each exocyclic N4 atom exists as an amino group (-NH2), and any isomerization and/or N4-Ag-I bonding can be excluded. The 3D structure of Ag-I-DNA complex determined with NOEs was classified as a B-form conformation with a notable propeller twist of C-Ag-I-C -18.3 +/- 3.08). The Ag-109 NMR chemical shift of C-Ag-I-C was recorded for cytidine/Ag-I complex (delta(Ag-109): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C-Ag-I-C base pair.

It has been confirmed by our previous studies that a 2',4'-BNA(NC)[N-Me]-modified antisense gapmer displays high affinity and selectivity to the target RNA strand, promising mRNA inhibitory activity and excellent nuclease resistance. Herein, we have obtained a crystal structure that provides insights into these excellent antisense properties.

The effect of microwave heating (MW) on the activity of a well-known enzyme (catalase) was elucidated by examining the catalase-assisted decomposition of hydrogen peroxide (H2O2 at various heating times (0 to 12 min)). For comparison, conventional water bath heating (WB) was also examined under identical temperature conditions. Microwave radiation had a positive effect on the activity of catalase only over a very short time (less than 3 min), presumably because of the possible disruption of the catalase structural integrity under microwave irradiation at longer times (a negative influence) as evidenced by Gel Permeation Chromatographic (GPC) and MALDI Time-Of-Flight-Mass-Spectrometric (MALDI-TOFMS) analyses. The effect of temperature on the catalase activity was also probed at 39, 37, and 25 degrees C. Results indicate that utilizing a hybrid heating approach with conventional heating (water bath) coupled to microwaves was more effective provided microwave irradiation was carried out for a short time (also less than 3 min). Moreover, it is demonstrated that microwave heating in degrading hydrogen peroxide was most effective when the enzymatic reaction was carried out at a lower temperature, particularly at 25 degrees C.

Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing (THgT)-T-II base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases (CAgC)-C-I formed in an RNA duplex. One Ag-I occupies the center between two cytosines and forms a (CAgC)-C-I base pair through N3Ag(I)N3 linear coordination. The (CAgC)-C-I base pair formation does not disturb the standard A-form conformation of RNA. Since the (CAgC)-C-I base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices.

Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T-HgII-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-AgI-C formed in an RNA duplex. One AgI occupies the center between two cytosines and forms a C-AgI-C base pair through N3-AgI-N3 linear coordination. The C-AgI-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-AgI-C base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices. The high-resolution structures of C-AgI-C base pairs in A-form RNA duplexes have been solved. Structural information on the present metallo-base pair together with the previously reported T-HgII-T base pair widely open the possibility of structure-based design of nucleic-acid-based nanodevices containing the natural metallo-base pairs.

Recently, metal-mediated base-pairs (metallo-base-pairs) have been studied extensively with the aim of exploring novel base-pairs; their structures, physicochemical properties, and applications have been studied. This trend has become more evident after the discovery of Hg-II-mediated thymine-thymine (T-Hg-II-T) and Ag-I-mediated cytosine-cytosine (C-Ag-I-C) base-pairs. In this article, we focus on the basic science and applications of these metallo-base-pairs, which are composed of natural bases.

Aminoglycoside antibiotics are pseudosaccharides decorated with ammonium groups that are critical for their potent broad-spectrum antibacterial activity. Despite over three decades of speculation whether or not modulation of pK&lt inf&gt a&lt /inf&gt is a viable strategy to curtail aminoglycoside kidney toxicity, there is a lack of methods to systematically probe amine-RNA interactions and resultant cytotoxicity trends. This study reports the first series of potent aminoglycoside antibiotics harboring fl uorinated N1-hydroxyaminobutyryl acyl (HABA) appendages for which fluorine-RNA contacts are revealed through an X-ray cocrystal structure within the RNA A-site. Cytotoxicity in kidney-derived cells was significantly reduced for the derivative featuring our novel β,β-difluoro-HABA group, which masks one net charge by lowering the pK&lt inf&gt a&lt /inf&gt without compromising antibacterial potency. This novel side-chain assists in evasion of aminoglycoside-modifying enzymes, and it can be easily transferred to impart these properties onto any number of novel analogs.

Aminoglycoside antibiotics are pseudosaccharides decorated with ammonium groups that are critical for their potent broad-spectrum antibacterial activity. Despite over three decades of speculation whether or not modulation of pK(a) is a viable strategy to curtail aminoglycoside kidney toxicity, there is a lack of methods to systematically probe amine-RNA interactions and resultant cytotoxicity trends. This study reports the first series of potent aminoglycoside antibiotics harboring fluorinated N1-hydroxyaminobutyryl acyl (HABA) appendages for which fluorine-RNA contacts are revealed through an X-ray cocrystal structure within the RNA A-site. Cytotoxicity in kidney-derived cells was significantly reduced for the derivative featuring our novel beta,beta-difluoro-HABA group, which masks one net charge by lowering the pK(a) without compromising antibacterial potency. This novel side-chain assists in evasion of aminoglycoside-modifying enzymes, and it can be easily transferred to impart these properties onto any number of novel analogs.

The metallo DNA duplex containing mercury-mediated T-T base pairs is an attractive biomacromolecular nanomaterial which can be applied to nanodevices such as ion sensors. Reported herein is the first crystal structure of a B-form DNA duplex containing two consecutive T-Hg-II-T base pairs. The Hg-II ion occupies the center between two T residues. The N3-Hg-II bond distance is 2.0 angstrom. The relatively short Hg-II-Hg-II distance (3.3 angstrom) observed in consecutive T-Hg-II-T base pairs suggests that the metallophilic attraction could exist between them and may stabilize the B-form double helix. To support this, the DNA duplex is largely distorted and adopts an unusual nonhelical conformation in the absence of Hg-II. The structure of the metallo DNA duplex itself and the Hg-II-induced structural switching from the nonhelical form to the B-form provide the basis for structure-based design of metal-conjugated nucleic acid nanomaterials.

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology".

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology". © 2014 Sesto et al.

This study reports the synthesis, antibacterial evaluation and nature of fluorine-rRNA contacts revealed by an X-ray co-crystal structure of a series of 4'-deoxy-4'-fluoro B-neomycin analogs. 4'-Deoxyfluorination improves the inhibition profile towards resistant enzymes and renders equally potent antibiotics compared to the parent neomycin B. The 4'-deoxy-4'-fluoro-4'-epi neomycin analogs showed a preferential inhibition over the 4'-deoxy-4'-fluoro neomycin counterpart against the strains of P. aeruginosa carrying a chromosomal APH(3')-IIb enzyme, known to inactivate the parent aminoglycoside. To the best of our knowledge, this is the first example of a neighboring-group aminoglycoside-modifying enzyme evasion by fluorine substitution. A unique F-G1491 stacking was observed in a co-crystal structure of 4'-deoxy-4'-fluoro-4'-epi neomycin with a bacterial ribosomal RNA A-site.

Leishmaniasis, a parasitic disease caused by protozoa of the genus Leishmania, affects millions of people worldwide. Aminoglycosides are mostly known as highly potent, broad-spectrum antibiotics that exert their antibacterial activity by selectively targeting the decoding A site of the bacterial ribosome, leading to aberrant protein synthesis. Recently, some aminoglycosides have been clinically approved and are currently used worldwide for the treatment of leishmaniasis however the molecular details by which aminoglycosides induce their deleterious effect on Leishmaina is still rather obscure. Based on high conservation of the decoding site among all kingdoms, it is assumed that the putative binding site of these agents in Leishmania is the ribosomal A site. However, although recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics, no such data are presently available regarding their binding site in Leishmania. We present crystal structures of two different aminoglycoside molecules bound to a model of the Leishmania ribosomal A site: Geneticin (G418), a potent aminoglycoside for the treatment of leishmaniasis at a 2.65-Å resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome lacking an antileishmanial activity at 1.4-Å resolution. The structural data, coupled with in vitro inhibition measurements on two strains of Leishmania, provide insight as to the source of the difference in inhibitory activity of different Aminoglycosides. The combined structural and physiological data sets the ground for rational design of new, and more specific, aminoglycoside derivatives as potential therapeutic agents against leishmaniasis.