Importance of isothermal titration calorimetry for the detection of the direct binding of metal ions to mismatched base pairs in duplex DNA.

Metal ion-nucleic acid interactions contribute substantially to the structure and biological activity of nucleic acids and have a wide range of potential applications in nanotechnology. In this study, we examined the interactions between metal ions and mismatched base pairs in duplex DNA to reveal the underlying molecular mechanism. UV melting analyses showed that the melting temperature (Tm) of a 21-base pair duplex DNA with each of the C-A, C-C and C-T mismatched base pairs increased upon the addition of Ag+. However, isothermal titration calorimetry (ITC) demonstrated that Ag+ only bound to the C-C mismatched base pair of the duplex DNA to form C-Ag-C bonds, without binding to the C-A and C-T mismatches. These results showed that Tm increased even when metal ions did not bind to the mismatched base pairs of the duplex DNA. Although the increase in Tm upon the addition of the metal ions is often used to detect metal ion binding to mismatched base pairs of duplex DNA, these results indicated that UV melting analyses are unable to detect the direct binding of metal ions to the mismatched base pairs. Because ITC analyses directly detect the heat derived from metal ion binding to mismatched base pairs of duplex DNA, we concluded that this may be an effective detection approach.

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.

Replication-stress-associated DSBs induced by ionizing radiation risk genomic destabilization and associated clonal evolution.

Exposure to ionizing radiation is associated with cancer risk. Although multiple types of DNA damage are caused by radiation, it remains unknown how this damage is associated with cancer risk. Here, we show that after repair of double-strand breaks (DSBs) directly caused by radiation (dir-DSBs), irradiated cells enter a state at higher risk of genomic destabilization due to accumulation of replication-stress-associated DSBs (rs-DSBs), ultimately resulting in clonal evolution of cells with abrogated defense systems. These effects were observed over broad ranges of radiation doses (0.25-2 Gy) and dose rates (1.39-909 mGy/min), but not upon high-dose irradiation, which caused permanent cell-cycle arrest. The resultant genomic destabilization also increased the risk of induction of single-nucleotide variants (SNVs), including radiation-associated SNVs, as well as structural alterations in chromosomes. Thus, the radiation-associated risk can be attributed to rs-DSB accumulation and resultant genomic destabilization.

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

A G-quadruplex-forming RNA aptamer binds to the MTG8 TAFH domain and dissociates the leukemic AML1-MTG8 fusion protein from DNA.

MTG8 (RUNX1T1) is a fusion partner of AML1 (RUNX1) in the leukemic chromosome translocation t(8;21). The AML1-MTG8 fusion gene encodes a chimeric transcription factor. One of the highly conserved domains of MTG8 is TAFH which possesses homology with human TAF4 [TATA-box binding protein-associated factor]. To obtain specific inhibitors of the AML1-MTG8 fusion protein, we isolated RNA aptamers against the MTG8 TAFH domain using systematic evolution of ligands by exponential enrichment. All TAF aptamers contained guanine-rich sequences. Analyses of a TAF aptamer by NMR, CD, and mutagenesis revealed that it forms a parallel G-quadruplex structure in the presence of K+ . Furthermore, the aptamer could bind to the AML1-MTG8 fusion protein and dissociate the AML1-MTG8/DNA complex, suggesting that it can inhibit the dominant negative effects of AML1-MTG8 against normal AML1 function and serve as a potential therapeutic agent for leukemia.

Resveratrol and its Related Polyphenols Contribute to the Maintenance of Genome Stability.

Genomic destabilisation is associated with the induction of mutations, including those in cancer-driver genes, and subsequent clonal evolution of cells with abrogated defence systems. Such mutations are not induced when genome stability is maintained; however, the mechanisms involved in genome stability maintenance remain elusive. Here, resveratrol (and related polyphenols) is shown to enhance genome stability in mouse embryonic fibroblasts, ultimately protecting the cells against the induction of mutations in the ARF/p53 pathway. Replication stress-associated DNA double-strand breaks (DSBs) that accumulated with genomic destabilisation were effectively reduced by resveratrol treatment. In addition, resveratrol transiently stabilised the expression of histone H2AX, which is involved in DSB repair. Similar effects on the maintenance of genome stability were observed for related polyphenols. Accordingly, we propose that polyphenol consumption can contribute to the suppression of cancers that develop with genomic instability, as well as lifespan extension.

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases.

Spectroscopic characterization of AgI -ion-mediated C-AgI -A and C-AgI -T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by AgI ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of AgI ions is in the order C-A≈C-T>C-C, the stabilizing effect of AgI ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to AgI -mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.

Targeting miR-223 in neutrophils enhances the clearance of Staphylococcus aureus in infected wounds.

Argonaute 2 bound mature microRNA (Ago2-miRNA) complexes are key regulators of the wound inflammatory response and function in the translational processing of target mRNAs. In this study, we identified four wound inflammation-related Ago2-miRNAs (miR-139-5p, miR-142-3p, miR-142-5p, and miR-223) and show that miR-223 is critical for infection control. miR-223Y/- mice exhibited delayed sterile healing with prolonged neutrophil activation and interleukin-6 expression, and markedly improved repair of Staphylococcus aureus-infected wounds. We also showed that the expression of miR-223 was regulated by CCAAT/enhancer binding protein alpha in human neutrophils after exposure to S. aureus peptides. Treatment with miR-223Y/--derived neutrophils, or miR-223 antisense oligodeoxynucleotides in S. aureus-infected wild-type wounds markedly improved the healing of these otherwise chronic, slow healing wounds. This study reveals how miR-223 regulates the bactericidal capacity of neutrophils at wound sites and indicates that targeting miR-223 might be of therapeutic benefit for infected wounds in the clinic.

Conjugation of two RNA aptamers improves binding affinity to AML1 Runt domain.

To develop a high-affinity aptamer against AML1 Runt domain, two aptamers were conjugated based on their structural information. The newly designed aptamer Apt14 was generated by the conjugation of two RNA aptamers (Apt1 and Apt4) obtained by SELEX against AML1 Runt domain, resulting in improvement in its binding performance. The residues of AML1 Runt domain in contact with Apt14 were predicted in silico and confirmed by mutation and NMR analyses. It was suggested that the conjugated internal loop renders additional contacts and is responsible for the enhancement in the binding affinity. Conjugation of two aptamers that bind to different sites of the target protein is a facile and robust strategy to develop an aptamer with higher performance.

The crystal structure of a 2',4'-BNA(NC)[N-Me]-modified antisense gapmer in complex with the target RNA.

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.

Structures, physicochemical properties, and applications of T-Hg(II)-T, C-Ag(I)-C, and other 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.

Hg2+-trapping beads: Hg2+-specific recognition through thymine-Hg(II)-thymine base pairing.

Mercury pollution poses a severe threat to human health. To remove Hg(2+) from contaminated water, we synthesized Hg(2+)-trapping beads that include oligo-thymidine functionalities that can form thymine-Hg(II)-thymine base pairs on the solid support. The beads can selectively trap Hg(2+) even in the presence of other metal cations. More interestingly, Hg(2+)-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg(2+)-trapping beads can capture Hg(2+) without affecting the mineral balance of water so much. The Hg(2+)-trapping beads presented here show promise for removing Hg(2+) from environmental water.

A solution 17O-NMR approach for observing an oxidized cysteine residue in Cu,Zn-superoxide dismutase.

Solution (17)O-NMR application to biological macromolecules is extremely limited. We describe here (17)O-NMR observation of the (17)O(2)-oxidized cysteine side chain of human Cu,Zn-superoxide dismutase in solution using selective (17)O(2) oxidation. (17)O-NMR with the aid of (17)O-labeling has wide potential to probe the environment and dynamics of oxidizable functionalities in proteins.

Thermodynamic and structural properties of the specific binding between Ag⁺ ion and C:C mismatched base pair in duplex DNA to form C-Ag-C metal-mediated base pair.

Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences.

Structural switching of Cu,Zn-superoxide dismutases at loop VI: insights from the crystal structure of 2-mercaptoethanol-modified enzyme.

Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102-115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys111 in the loop, is readily oxidized and alkylated. We have found that modification of this Cys111 with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly108 to Cys111 in loop VI. One loop VI of the dimer forms a 310-helix (Gly108 to His110) within a unique ß-bridge stabilized by a hydrogen bond between Ser105-NH and His110-CO, while the other forms a ß-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.

Ag(I) ion mediated formation of a C-A mispair by DNA polymerases.

Silver turns up the A-C: In the presence of Ag(I) ions, a DNA polymerase incorporated deoxyadenosine (from dATP) at the site opposite cytosine in the template strand to afford the full-length product (see scheme), meaning that DNA polymerases prefer a C-Ag(I)-A base pair to the more thermodynamically stable C-Ag(I)-C base pair.

2'-O,4'-C-ethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability under physiological condition.

Since pyrimidine motif triplex DNA is unstable at physiological neutral pH, triplex stabilization at physiological neutral pH is important for improvement of its potential to be applied to various methods in vivo, such as repression of gene expression, mapping of genomic DNA and gene-targeted mutagenesis. For this purpose, we studied the thermodynamic and kinetic effects of a chemical modification, 2'-O,4'-C-ethylene bridged nucleic acid (ENA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological neutral pH. Thermodynamic investigations indicated that the modification achieved more than 10-fold increase in the binding constant of the triplex formation. The increased number of the modification in TFO enhanced the increased magnitude of the binding constant. On the basis of the obtained thermodynamic parameters, we suggested that the remarkably increased binding constant by the modification may result from the increased stiffness of TFO in the unbound state. Kinetic studies showed that the considerably decreased dissociation rate constant resulted in the observed increased binding constant by the modification. We conclude that ENA modification of TFO could be a useful chemical modification to promote the triplex formation under physiological neutral condition, and may advance various triplex formation-based methods in vivo.

Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH.

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice.

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2',4'-BNA (also called as locked nucleic acid (LNA)) and 2',4'-BNA(NC) chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2',4'-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2',4'-BNA-AON, 2',4'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

Development of a 2',4'-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo.

Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2',4'-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2',4'-BNA/LNA in its sense strand and with 2'-methoxy (2'-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2',4'-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5'-UG-3' in the sense strand would ameliorate the negative immune response.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.32; published online 18 September 2012.