Chloride Ligands on DNA-Stabilized Silver Nanoclusters.

DNA-stabilized silver nanoclusters (AgN-DNAs) are known to have one or two DNA oligomer ligands per nanocluster. Here, we present the first evidence that AgN-DNA species can possess additional chloride ligands that lead to increased stability in biologically relevant concentrations of chloride. Mass spectrometry of five chromatographically isolated near-infrared (NIR)-emissive AgN-DNA species with previously reported X-ray crystal structures determines their molecular formulas to be (DNA)2[Ag16Cl2]8+. Chloride ligands can be exchanged for bromides, which red-shift the optical spectra of these emitters. Density functional theory (DFT) calculations of the 6-electron nanocluster show that the two newly identified chloride ligands were previously assigned as low-occupancy silvers by X-ray crystallography. DFT also confirms the stability of chloride in the crystallographic structure, yields qualitative agreement between computed and measured UV-vis absorption spectra, and provides interpretation of the 35Cl-nuclear magnetic resonance spectrum of (DNA)2[Ag16Cl2]8+. A reanalysis of the X-ray crystal structure confirms that the two previously assigned low-occupancy silvers are, in fact, chlorides, yielding (DNA)2[Ag16Cl2]8+. Using the unusual stability of (DNA)2[Ag16Cl2]8+ in biologically relevant saline solutions as a possible indicator of other chloride-containing AgN-DNAs, we identified an additional AgN-DNA with a chloride ligand by high-throughput screening. Inclusion of chlorides on AgN-DNAs presents a promising new route to expand the diversity of AgN-DNA structure-property relationships and to imbue these emitters with favorable stability for biophotonics applications.

Synthesis and properties of fully-modified 4'-selenoRNA, an endonuclease-resistant RNA analog.

A large number of chemically modified oligonucleotides (ONs) have been developed for RNA-based technologies. In most modified RNAs, the characteristic 2'-hydroxyl (2'-OH) groups are removed to enhance both nuclease resistance and hybridization ability. However, the importance of the 2'-OH group in RNA structure and function is well known. Here, we report the synthesis and properties of 4'-selenoRNA in which all four nucleoside units retain the 2'-OH groups but contain a selenium atom instead of an oxygen atom at the 4'-position of the furanose ring. 4'-SelenoRNA has enhanced ability to form duplexes with RNA, and high endonuclease resistance despite the presence of the 2'-OH groups. X-ray crystallography analysis showed that the 4'-selenoRNA duplex adopts an A-conformation, similar to natural RNA, although one 4'-selenocytidine residue has unusual South-type sugar puckering. Furthermore, preliminary studies using 4'-seleno-modified siRNAs suggest that 4'-selenoRNA may be applicable to RNA interference technology. Collectively, our results raise the possibility of a new class of modified RNA in which 2'-OH groups do not need to be masked.

A Novel AgI -DNA Rod Comprising a One-Dimensional Array of 11 Silver Ions within a Double Helical Structure.

DNA/RNA duplexes containing metal-ion-mediated base pairs (metallo-base pairs) have potential applications in developing nucleic acid-based nanodevices and genetic code expansion. Many metallo-base pairs are formed within duplexes stabilized by Watson-Crick base pairs. Recently, the crystal structure of an AgI -DNA nanowire with an uninterrupted one-dimensional silver array was determined. Here, we present a new DNA helical wire, the "AgI -DNA rod", containing an uninterrupted array of 11 AgI ions. The AgI -DNA rod consisted of only C-AgI -C, G-AgI -G, G-AgI -5-bromouracil (Br U), and Br U-AgI -Br U metallo base pairs, with no Watson-Crick pairs. The AgI -DNA rods were connected by non-canonical G-G pairs in crystals. Notably, data from our absorbance, circular dichroism, nuclear magnetic resonance, and mass spectrometry analyses suggested that the AgI -DNA rods formed in solution, as well as within crystals.

Conformational adaptation of UNCG loops upon crowding.

If the A-form helix is the major structural motif found in RNA, the loops that cap them constitute the second most important family of motifs. Among those, two are overrepresented, GNRA and UNCG tetraloops. Recent surveys of RNA structures deposited in the PDB show that GNRA and UNCG tetraloops can adopt tertiary folds that are very different from their canonical conformations, characterized by the presence of a U-turn of a Z-turn, respectively. Crystallographic data from both a lariat-capping (LC) ribozyme and a group II intron ribozyme reveal that a given UUCG tetraloop can adopt a distinct fold depending on its structural environment. Specifically, when the crystal packing applies relaxed constraints on the loop, the canonical Z-turn conformation is observed. In contrast, a highly packed environment induces "squashing" of the tetraloop by distorting its sugar-phosphate backbone in a specific way that expels the first and fourth nucleobases out of the loop, and falls in van der Waals distance of the last base pair of the helix, taking the place of the pair formed between the first and fourth residues in Z-turn loops. The biological relevance of our observations is supported by the presence of similarly deformed loops in the highly packed environment of the ribosome and in a complex between a dsRNA and a RNase III. The finding that Z-turn loops change conformation under higher molecular packing suggests that, in addition to their demonstrated role in stabilizing RNA folding, they may contribute to the three-dimensional structure of RNA by mediating tertiary interactions with distal residues.

A structural basis for the antibiotic resistance conferred by an N1-methylation of A1408 in 16S rRNA.

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 A1408m1A 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.

Structural Bases for the Fitness Cost of the Antibiotic-Resistance and Lethal Mutations at Position 1408 of 16S rRNA.

To understand a structural basis for the fitness cost of the A1408G antibiotic-resistance mutation in the ribosomal A-site RNA, we have determined crystal structures of its A1408C and A1408U lethal mutants, and made comparison with previously solved structures of the wild type and the antibiotic-resistant mutant. The A-site RNA containing an asymmetric internal loop functions as a molecular switch to discriminate a single cognate tRNA from several near-cognate tRNAs by its conformational ON/OFF switching. Overall structures of the "off" states of the A1408C/U lethal mutants are very similar to those of the wild type and the A1408G antibiotic-resistant mutant. However, significant differences are found in local base stacking interactions including the functionally important A1492 and A1493 residues. In the wild type and the A1408G antibiotic-resistant mutant "off" states, both adenines are exposed to the solvent region. On the other hand, one of the corresponding adenines of the lethal A1408C/U mutants stay deeply inside their A-site helices by forming a purine-pyrimidine AoC or A-U base pair and is sandwiched between the upper and lower bases. Therefore, the ON/OFF switching might unfavorably occur in the lethal mutants compared to the wild type and the A1408G antibiotic-resistant mutant. It is probable that bacteria manage to acquire antibiotic resistance without losing the function of the A-site molecular switch by mutating the position 1408 only from A to G, but not to pyrimidine base C or U.

Crystal structure of a NIR-Emitting DNA-Stabilized Ag16 Nanocluster.

DNA has been used as a scaffold to stabilize small, atomically monodisperse silver nanoclusters, which have attracted attention due to their intriguing photophysical properties. Herein, we describe the X-ray crystal structure of a DNA-encapsulated, near-infrared emitting Ag16 nanocluster (DNA-Ag16 NC). The asymmetric unit of the crystal contains two DNA-Ag16 NCs and the crystal packing between the DNA-Ag16 NCs is promoted by several interactions, such as two silver-mediated base pairs between 3'-terminal adenines, two phosphate-Ca2+ -phosphate interactions, and π-stacking between two neighboring thymines. Each Ag16 NC is confined by two DNA decamers that take on a horse-shoe-like conformation and is almost fully shielded from the solvent environment. This structural insight will aid in the determination of the structure/photophysical property relationship for this class of emitters and opens up new research opportunities in fluorescence imaging and sensing using noble-metal clusters.

A Novel DNA Helical Wire Containing HgII -Mediated T:T and T:G Pairs.

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.

A PNPase dependent CRISPR System in Listeria.

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".

Structure Determination of an Ag(I) -Mediated Cytosine-Cytosine Base Pair within DNA Duplex in Solution with (1) H/(15) N/(109) Ag NMR Spectroscopy.

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 (15) N-(109) Ag J-coupling ((1) J((15) N,(109) Ag): 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±3.0°). The (109) Ag NMR chemical shift of C-Ag(I) -C was recorded for cytidine/Ag(I) complex (δ((109) Ag): 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.

The structure of metallo-DNA with consecutive thymine-HgII-thymine base pairs explains positive entropy for the metallo base pair formation.

We have determined the three-dimensional (3D) structure of DNA duplex that includes tandem Hg(II)-mediated T-T base pairs (thymine-Hg(II)-thymine, T-Hg(II)-T) with NMR spectroscopy in solution. This is the first 3D structure of metallo-DNA (covalently metallated DNA) composed exclusively of 'NATURAL' bases. The T-Hg(II)-T base pairs whose chemical structure was determined with the (15)N NMR spectroscopy were well accommodated in a B-form double helix, mimicking normal Watson-Crick base pairs. The Hg atoms aligned along DNA helical axis were shielded from the bulk water. The complete dehydration of Hg atoms inside DNA explained the positive reaction entropy (ΔS) for the T-Hg(II)-T base pair formation. The positive ΔS value arises owing to the Hg(II) dehydration, which was approved with the 3D structure. The 3D structure explained extraordinary affinity of thymine towards Hg(II) and revealed arrangement of T-Hg(II)-T base pairs in metallo-DNA.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania.

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.

High-Resolution Crystal Structure of a Silver(I)-RNA Hybrid Duplex Containing Watson-Crick-like C-Silver(I)-C Metallo-Base Pairs.

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-Hg(II)-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-Ag(I)-C formed in an RNA duplex. One Ag(I) occupies the center between two cytosines and forms a C-Ag(I)-C base pair through N3-Ag(I)-N3 linear coordination. The C-Ag(I)-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-Ag(I)-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.

Crystal structure of metallo DNA duplex containing consecutive Watson-Crick-like T-Hg(II)-T base pairs.

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 Å. The relatively short Hg(II)-Hg(II) distance (3.3 Å) 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.

Intra-tumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer.

The dense stroma is one cause of poor efficacy of T cell-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC). Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan-synthetic enzyme responsible for remodeling tumor stroma. Intra-tumoral injection of CHST15 small interfering RNA (siRNA) has been shown to increase the tumor-infiltrating T cells (TILs) in patients with unresectable PDAC. However, the mechanism underlying the enhanced accumulation of TILs is not fully explored. Here, we demonstrate that intra-tumoral injection of CHST15 siRNA locally and remotely diminishes myeloid-derived suppressor cells (MDSCs) and enhances TILs in mice. CHST15 was expressed by tumor cells and MDSCs in both tumor and tumor-draining lymph nodes (TDLNs), and CHST15 siRNA repressed stromal density, neutrophil extracellular traps, and Ly6C/G+ MDSCs in vivo. Remarkably, tumor growth inhibition was only observed in the immunocompetent KPC model, which is associated with enhanced TILs. In vitro, CHST15 siRNA significantly downregulated the levels of CHST15 and indoleamine 2,3-dioxygenase mRNA in CD33+ MDSCs derived from human peripheral blood mononuclear cells. These results suggest a dual role for intra-tumorally injected CHST15 siRNA on modulating the tumor immune microenvironment for T cell entry and remotely diminishing CHST15+ MDSCs, decreasing T cell suppression and expanding T cells in the TDLN, ultimately leading to an enhanced accumulation of TILs.

Structure of an A-form RNA duplex obtained by degradation of 6S RNA in a crystallization droplet.

In the course of a crystallographic study of a 132 nt variant of Aquifex aeolicus 6S RNA, a crystal structure of an A-form RNA duplex containing 12 base pairs was solved at a resolution of 2.6 Å. In fact, the RNA duplex is part of the 6S RNA and was obtained by accidental but precise degradation of the 6S RNA in a crystallization droplet. 6S RNA degradation was confirmed by microscopic observation of crystals and gel electrophoresis of crystallization droplets. The RNA oligomers obtained form regular A-form duplexes containing three GoU wobble-type base pairs, one of which engages in intermolecular contacts through a ribose-zipper motif at the crystal-packing interface.

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

Specific binding of Hg2+ to mismatched base pairs involving 5-hydroxyuracil in duplex DNA.

Metal ion-nucleic acid interactions contribute significantly to nucleic acid structure and biological activity and have potential applications in nanotechnology. Hg2+ specifically binds to the natural T-T mismatched base pair in duplex DNA to form a T-Hg-T base pair. Metal ions may enhance DNA damage induced by DNA-damaging agents, such as oxidative agents. The interactions between metal ions and damaged DNAs, such as mismatched oxidized bases, have not been well characterized. Here, we examined the possibility of Hg2+ binding to an asymmetric mismatched base pair involving thymine and 5-hydroxyuracil (OHdU), an oxidized base produced by the oxidative deamination of cytosine. UV melting analyses showed that only the melting temperature of the single T-OHdU mismatched duplex DNA increased upon Hg2+ addition. CD spectra indicated no significant change in the higher-order structure of the single T-OHdU mismatched duplex DNA upon Hg2+ addition. X-ray crystallographic structure with two consecutive T-OHdU mismatched base pairs and isothermal titration calorimetric analyses with the single T-OHdU mismatched base pair showed that Hg2+ specifically binds to the N3 positions of both T and OHdU in T-OHdU at 1:1 molar ratio, with a 5×105 M-1 binding constant of to form the T-Hg-OHdU base pair. The Hg2+-bound structure and the Hg2+-binding affinity for T-OHdU was similar to those for T-T. This study on T-Hg-OHdU metal-mediated base pair could aid in studying the molecular mechanism of metal ion-mediated DNA damage and their potential applications in nanotechnology.

A structural basis for the antibiotic resistance conferred by an A1408G mutation in 16S†rRNA and for the antiprotozoal activity of aminoglycosides.

Resistance explained: The crystal structures of the ribosomal decoding A site with an A1408G antibiotic-resistance mutation were solved in the presence and absence of the aminoglycoside geneticin (see structure, geneticin carbon framework in yellow). These structures show how bacteria acquire high-level resistance against aminoglycosides by the mutation.