Synthesis of a novel nucleotide unit containing cytosine analog bearing phosphodiester function and formation of silver(I)-mediated DNA duplex.

Novel modified cytosine analogs bearing phosphodiester/thiophosphodiester functionality were synthesized. The interactions between different metal ions and modified cytosine-cytosine base-pairs in DNA duplexes were investigated by UV-melting experiments. The thiophosphodiester modification binds to the Ag(I) ions strongly compared to the phosphodiester counterpart as examined in ESI-MS spectra as well.

Dye-Conjugated Spinach RNA by Genetic Alphabet Expansion.

Light-emitting systems using an RNA aptamer-dye pair, such as Spinach RNA, are an attractive method for imaging and tracing RNA expression in vitro and in vivo. We present an alternative Spinach method by genetic alphabet expansion using an unnatural base pair system, in which a dye-conjugated unnatural base substrate is site-specifically incorporated at a specific position in Spinach RNA by transcription involving the third base pair. The incorporation position was predicted by molecular dynamics simulations. This dye-conjugated Spinach RNA increased the thermal stability of the fluorescence, the robustness against ion sensitivity, and the resistance against photobleaching. Furthermore, we applied our method to Baby Spinach, a shorter version of Spinach, for dye conjugation toward the visible detection of transcripts. This is the first demonstration of an alternative RNA imaging method for a detection system using genetic alphabet expansion.

Cognate base-pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase.

We present cognate base pair selectivity in template-dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds-Pa. T4 DNA ligase efficiently recognizes the Ds-Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base-pair selectivity, relative to the hydrogen-bond interactions between pairing bases. The efficient ligation using Ds-Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi-synthetic organisms containing UBPs.

Synthesis and Characterization of Cell-Permeable Oligonucleotides Bearing Reduction-Activated Protecting Groups on the Internucleotide Linkages.

Cell-permeable oligodeoxyribonucleotides (ODNs) bearing reduction-activated protecting groups were synthesized as oligonucleotide pro-drugs. Although these oligonucleotides were amenable to solid-phase DNA synthesis and purification, the protecting group on their phosphodiester moiety could be readily cleaved by nitroreductase and NADH. Moreover, these compounds exhibited good nuclease resistance against 3'-exonuclease and endonuclease and good stability in human serum. Fluorescein-labeled ODNs modified with reduction-activated protecting groups showed better cellular uptake compared with that of naked ODNs.

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.

High Fidelity, Efficiency and Functionalization of Ds-Px Unnatural Base Pairs in PCR Amplification for a Genetic Alphabet Expansion System.

Genetic alphabet expansion of DNA using an artificial extra base pair (unnatural base pair) could augment nucleic acid and protein functionalities by increasing their components. We previously developed an unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px), which exhibits high fidelity as a third base pair in PCR amplification. Here, the fidelity and efficiency of Ds-Px pairing using modified Px bases with functional groups, such as diol, azide, ethynyl and biotin, were evaluated by an improved method with optimized PCR conditions. The results revealed that all of the base pairs between Ds and either one of the modified Px bases functioned with high amplification efficiency (0.76-0.81), high selectivity (≥99.96% per doubling), and less sequence dependency, in PCR using 3'-exonuclease-proficient Deep Vent DNA polymerase. We also demonstrated that the azide-Px in PCR-amplified DNA was efficiently modified with any functional groups by copper-free click reaction. This genetic alphabet expansion system could endow nucleic acids with a wide variety of increased functionalities by the site-specific incorporation of modified Px bases at desired positions in DNA.

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.

Development of a photolabile protecting group for phosphodiesters in oligonucleotides.

A photolabile protecting group, consisting of an o-nitrobenzyl group and a 3-(2'-hydroxy-3',6'-dimethylphenyl)-2,2-dimethylpropyl moiety, was developed for phosphodiesters in oligodeoxyribonucleotides. Deprotection was triggered by photoirradiation and subsequent spontaneous cyclization to release the naked oligonucleotide.

Parallel synthesis of cell-penetrating peptide conjugates of PMO toward exon skipping enhancement in Duchenne muscular dystrophy.

We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening.

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.

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.

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.

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.

Metal ion-binding properties of DNA duplexes containing thiopyrimidine base pairs.

Thiopyrimidine pairs in DNA duplexes were unexpectedly largely stabilized by complexation with two equivalents of Ag(I) ions and their binding properties were evaluated. The metal ion-binding properties of the thiopyrimidine base pairs differed significantly from those of unpaired bases.

Raman spectroscopic detection of the T-Hg II-T base pair and the ionic characteristics of mercury.

Developing applications for metal-mediated base pairs (metallo-base-pair) has recently become a high-priority area in nucleic acid research, and physicochemical analyses are important for designing and fine-tuning molecular devices using metallo-base-pairs. In this study, we characterized the Hg(II)-mediated T-T (T-Hg(II)-T) base pair by Raman spectroscopy, which revealed the unique physical and chemical properties of Hg(II). A characteristic Raman marker band at 1586 cm(-1) was observed and assigned to the C4=O4 stretching mode. We confirmed the assignment by the isotopic shift ((18)O-labeling at O4) and density functional theory (DFT) calculations. The unusually low wavenumber of the C4=O4 stretching suggested that the bond order of the C4=O4 bond reduced from its canonical value. This reduction of the bond order can be explained if the enolate-like structure (N3=C4-O4(-)) is involved as a resonance contributor in the thymine ring of the T-Hg(II)-T pair. This resonance includes the N-Hg(II)-bonded state (Hg(II)-N3-C4=O4) and the N-Hg(II)-dissociated state (Hg(II+) N3=C4-O4(-)), and the latter contributor reduced the bond order of N-Hg(II). Consequently, the Hg(II) nucleus in the T-Hg(II)-T pair exhibited a cationic character. Natural bond orbital (NBO) analysis supports the interpretations of the Raman experiments.

Binding of metal ions by pyrimidine base pairs in DNA duplexes.

Pyrimidine base pairs in DNA duplexes selectively capture metal ions to form metal ion-mediated base pairs, which can be evaluated by thermal denaturation, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. In this critical review, we discuss the metal ion binding of pyrimidine bases (thymine, cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA duplexes. Thymine-thymine (T-T) and cytosine-cytosine (C-C) base pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the metallo-base pairs, T-Hg(II)-T and C-Ag(I)-C, are formed in DNA duplexes. The metal ion binding properties of the pyrimidine-pyrimidine pairs can be changed by small chemical modifications. The binding selectivity of a metal ion to a 5-fluorouracil-5-fluorouracil pair in a DNA duplex can be switched by changing the pH of the solution. Two silver ions bind to each thiopyrimidine-thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized. Oligonucleotides containing these bases are commercially available and can readily be applied in many scientific fields (86 references).

Synthesis of covalently linked parallel and antiparallel DNA duplexes containing the metal-mediated base pairs T-Hg(II)-T and C-Ag(I)-C.

DNA duplexes fixed in anti-parallel and parallel orientations by introducing covalent linkages have been synthesized and metal ions, Hg(II) and Ag(I), were incorporated into pyrimidine-pyrimidine base pairs.

Metal ion binding of modified pyrimidine-base pairs in DNA duplexes.

We report the synthesis and metal ion-binding properties of DNA duplexes containing 5-substituted uracil ((X)U) pairs, such as 5-bromouracil, 5-fluorouracil and 5-cyanouracil pairs. Thermal denaturation studies of these modified DNA duplexes revealed that the DNA duplexes were stabilized in the presence of Hg(II) ions in acidic and neutral solutions. On the other hand, the duplexes were stabilized in the presence of Hg(II) and Ag(I) ions. These results indicated that (X)U-Hg(II)-(X)U complex was formed in the acidic and neutral solutions, then, in the basic solutions (X)U-Ag(I)-(X)U complex as well as the (X)U-Hg(II)-(X)U complex were formed. ESI-TOF MS analysis also indicated formation of the metal ion-DNA complexes.

Synthesis and thermal denaturation studies of covalently linked DNA triplexes.

We report on the synthesis and thermal stability of small covalently linked DNA triplexes. These modified triplexes were found to contain covalently linked T-T pairs at the edges, and thermal denaturation studies revealed that the covalent linking efficiently stabilized triplex formation.

Metal-ion selectivity of chemically modified uracil pairs in DNA duplexes.

DNA duplexes containing 5-modified uracil pairs (5-bromo, 5-fluoro, and 5-cyanouracil) bind selectivity to metal ions. Their selectivity is sensitive to the pH value of the solution (see picture), as the acidities of the modified uracil bases vary according to the electron-withdrawing properties of the substituents.