Oxovanadium complex as potential nucleic acid binder

DNA binding study of a vanadium(V) complex, Oxo-chloro-bis-N-phenylbenzohydroxamto-vanadium(V), derived from N-phenylbenzohydroxamic acid(PBHA) form a violet color complex with vanadium (V) in presence of hydrochloric acid is performed using absorption, fluorescence and viscometric techniques. The binding parameters of the PBHA-V(V) complex using calf thymus DNA (ct-DNA) and torula yeast RNA (t-RNA) have been determined. The complex shows the ability of cooperatively minor groove binding with ct-DNA as indicated by remarkable hyperchromicity and a blue shift of the absorption spectra. Quenching of metal complex calculation was carried out with Stern-Volmer equation and K_sv was found to be 2.32 ± 0.18 × 10⁴ M^?1, while in the case of t-RNA, enhancement is observed and that means the compound was not able to displace the Ethidium Bromide(EB)-t-RNA complex. Molecular docking was also applied to predict the mode of interaction of the hydroxamic acid with ct-DNA and t-RNA. DNA binding results of the complex are compared with those of the parent ligand.     

Polarizable model potential function for nucleic acid bases

A polarizable model potential (PMP) function for adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) is developed on the basis of ab initio molecular orbital calculations at the MP2/6-31+G* level. The PMP function consists of Coulomb, van der Waals, and polarization terms. The permanent atomic charges of the Coulomb term are determined by using electrostatic potential (ESP) optimization. The multicenter polarizabilities of the polarization term are determined by using polarized one-electron potential (POP) optimization in which the electron density changes induced by a test charge are target. Isotropic and anisotropic polarizabilities are adopted as the multicenter polarizabilities. In the PMP calculations using the optimized parameters, the interaction energies of Watson-Crick type A-T and C-G base pairs were -15.6 and -29.4 kcal/mol, respectively. The interaction energy of Hoogsteen type A-T base pair was -17.8 kcal/mol. These results reproduce well the quantum chemistry calculations at the MP2/6-311++G(3df,2pd) level within the differences of 0.6 kcal/mol. The stacking energies of A-T and C-G were -9.7 and -10.9 kcal/mol. These reproduce well the calculation results at the MP2/6-311++G (2d,2p) level within the differences of 1.3 kcal/mol. The potential energy surfaces of the system in which a sodium ion or a chloride ion is adjacent to the nucleic acid base are calculated. The interaction energies of the PMP function reproduced well the calculation results at the MP2/6-31+G* or MP2/6-311++G(2d,2p) level. The reason why the PMP function reproduces well the high-level quantum mechanical interaction energies is addressed from the viewpoint of each energy terms.     

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.     

Reaction of nucleobases with α-acetylenic esters,potentially useful for chemical modification of nucleic acids

The NH₂ group and the adjacent ring nitrogen of adenosine and cytidine react with α-acetylenic esters by addition across the triple bond and formation of a lactam with the ester group.     

Electrochemical analysis of nucleic acids as potential cancer biomarkers

In recent years, a huge progress has been made regarding the development of electrochemical (EC) assays for detection of nucleic acids — DNA or RNA — as potential cancer biomarkers. Various ingenious strategies for determination of DNA methylation of gene promoters, circulating tumor DNAs, viral nucleic acids, or short noncoding microRNAs were presented, many of them showing remarkable sensitivities. However, a majority of these assays were not applied into clinical samples from patients, which is crucial should the electrochemistry compete with conventional, routinely used techniques. In this review, we critically evaluate strengths and weaknesses of EC assays that recognized this necessity and successfully determined endogenous DNA or RNA in patient samples with various forms of tumors.     

Probing binding preferences of DNA and RNA: backbone chirality of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids to differentiate DNA versus RNA selective binding

Subtle differences in RNA and DNA duplex geometry could be sensed by the changed stereochemistry at 3'-amino function in the 5-atom thioacetamido linker of thioacetamido-linked nucleic acids and iso-thioacetamido-linked nucleic acids modified oligomers. In contrast to the preferred N-type sugar conformations for either 3'- ribo- or xylo amino nucleosides, predominant S-type sugar conformations were found in the dimers. Although the CD spectral differences for the dimer blocks were found to be identical for those found in phosphodiester linked ribo/xylo dimers, the 5-atom thioactamido linker could reverse the RNA binding selectivity to DNA binding selectivity by the change in configuration at the 3'-amino-substituted sugar.     

Synthesis and RNA binding selectivity of oligonucleotides modified with five-atom thioacetamido nucleic acid backbone structures

Convenient chemical synthesis and incorporation of dithymidine and thymidine-cytidine dimer blocks connected with a five-atom amide linker N3'-CO-CH2-S-CH2 into oligonucleotides (ONs) are reported. The UV-Tm experiments for binding affinities of these mixed backbone ONs with complementary DNA and RNA sequences revealed important results such as significantly higher RNA-binding selectivity as compared with complementary DNA. NMR studies of the dimer blocks suggested a marginal increase in the N-type sugar conformations over that of the native DNA.     

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes.

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2-4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68 degrees C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.     

A simple gamma-backbone modification preorganizes peptide nucleic acid into a helical structure

Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA, developed more than a decade ago in which the naturally occurring sugar phosphate backbone has been replaced by the N-(2-aminoethyl) glycine units. Unlike DNA or RNA in the unhybridized state (single strand) which can adopt a helical structure through base-stacking, although highly flexible, PNA does not have a well-defined conformational folding in solution. Herein, we show that a simple backbone modification at the gamma-position of the N-(2-aminoethyl) glycine unit can transform a randomly folded PNA into a helical structure. Spectroscopic studies showed that helical induction occurs in the C- to N-terminal direction and is sterically driven. This finding has important implication not only on the future design of nucleic acid mimics but also on the design of novel materials, where molecular organization and efficient electronic coupling are desired.     

The oligodeoxynucleotide probes for the site-specific modification of RNA

As the knowledge of the biological functions of RNA expands, the demand for research tools to investigate intracellular RNA is increasing. Oligonucleotides can be rationally designed for the target RNA sequence, and therefore, have become a reliable platform for the development of specific molecules for RNA. The chemical modification of RNA has a strong impact on RNA research; the fluorescent labeling of RNA is useful to monitor RNA production, processing, relocation in the cell, interaction with other intracellular components and degradation, etc. Chemical modification may affect the RNA function through a variety of pathways, and therefore, would be potentially useful for biological research, therapeutic approach and artificial manipulation of the RNA function. This tutorial review starts with an introduction of the biological relevance of modified RNA, and focuses on the recent progress of the oligodeoxynucleotide probes for the covalent modifications of RNA. The prospects of this new technology are also discussed.     

Alpha-LNA (locked nucleic acid with alpha-D-configuration): synthesis and selective parallel recognition of RNA

Alpha-LNA is presented as a stereoisomer of LNA (locked nucleic acid) with alpha-D-configuration. Three different approaches towards the thymine alpha-LNA monomer as well as the 5-methylcytosine alpha-LNA monomer are presented. Different alpha-LNA sequences have been synthesised and their hybridisation with complementary DNA and RNA has been evaluated by means of thermal stability experiments and circular dichroism spectroscopy. In a mixed pyrimidine sequence, alpha-LNA displays unprecedented parallel-stranded and selective RNA binding. Furthermore, a remarkable selectivity for hybridisation with RNA over DNA is indicated.     

Nanofibrillated cellulose: surface modification and potential applications

Interest in nanofibrillated cellulose has been increasing exponentially because of its relatively ease of preparation in high yield, high specific surface area, high strength and stiffness, low weight and biodegradability etc. This bio-based nanomaterial has been used mainly in nanocomposites due to its outstanding reinforcing potential. Solvent casting, melt mixing, in situ polymerization and electrospinning are important techniques for the fabrication of nanofibrillated cellulose-based nanocomposites. Due to hydrophilic character along with inherent tendency to form strong network held through hydrogen-bonding, nanofibrillated cellulose cannot uniformly be dispersed in most non-polar polymer matrices. Therefore, surface modification based on polymer grafting, coupling agents, acetylation and cationic modification was used in order to improve compatibility and homogeneous dispersion within polymer matrices. Nanofibrillated cellulose opens the way towards intense and promising research with expanding area of potential applications, including nanocomposite materials, paper and paperboard additive, biomedical applications and as adsorbent.     

Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification

Nucleic acid sequence based amplification (NASBA) is a versatile in vitro nucleic acid amplification method. In this work, RNA amplification and labeling by NASBA and microarray analysis are combined in a one-step process. The NASBA reaction is performed in direct contact with capture probes. These probes are bound to surface-attached hydrogel spots generated at the chip surfaces by using a simple printing and UV irradiation process. Five gene expression and SNP parameters with known relevance in breast cancer diagnostics were chosen to demonstrate that multiplex NASBA-on-microarray analysis is possible. A minimum amount of 10 pg of total RNA was shown to be sufficient for the detection of the reference parameter RPS18, which demonstrates that the detection limit of the microarray-based NASBA assays theoretically allows single-cell assays to be performed.     

Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids

Locked nucleic acids (LNAs) containing one or more 2'-O,4'-C-methylene-linked bicyclic ribonucleoside monomers possess a number of the prerequisites of an effective antisense oligonucleotide, e.g. unprecedented helical thermostability when hybridized with cognate RNA and DNA. To acquire a detailed understanding of the structural features of LNA giving rise to its remarkable properties, we have conducted structural studies by use of NMR spectroscopy and now report high-resolution structures of two LNA:RNA hybrids, the LNA strands being d(5'-CTGAT(L)ATGC-3') and d(5'-CT(L)GAT(L)AT(L)GC-3'), respectively, T(L) denoting a modified LNA monomer with a thymine base, along with the unmodified DNA:RNA hybrid. In the structures, the LNA nucleotides are positioned as to partake in base stacking and Watson-Crick base pairing, and with the inclusion of LNA nucleotides, we observe a progressive change in duplex geometry toward an A-like duplex structure. As such, with the inclusion of three LNA nucleotides, the hybrid adopts an almost canonical A-type duplex geometry, and thus it appears that the number of modifications has reached a saturation level with respect to structural changes, and that further incorporations would furnish only minute changes in the duplex structure. We attempt to rationalize the conformational steering induced by the LNA nucleotides by suggesting that the change in electronic density at the brim of the minor groove, introduced by the LNA modification, is causing an alteration of the pseudorotational profile of the 3'-flanking nucleotide, thus shifting this sugar equilibrium toward N-type conformation.     

Chemical modification of siRNA bases to probe and enhance RNA interference

Considerable attention has focused on the use of alternatives to the native ribose and phosphate backbone of small interfering RNAs for therapeutic applications of the RNA interference pathway. In this synopsis, we highlight the less common chemical modifications, namely, those of the RNA nucleobases. Base modifications have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.     

Backbone modifications of aromatic peptide nucleic acid (APNA ) monomers and their hybridization properties with DNA and RNA

Aromatic peptide nucleic acid (APNA) monomers containing N-(2-aminobenzyl)-glycine, N-(2-aminobenzyl)-(R)- or -(S)-alanine, and N-(2-aminobenzyl)-beta-alanine moieties as part of their backbone were synthesized. These novel analogues were incorporated as a single "point mutation" in PNA hexamers, and their physicochemical properties were investigated by UV thermal denaturation and CD experiments. Destabilization in triplex formation between the PNA-APNA chimeras and complementary DNA or RNA oligomers was observed, as compared to the PNA control. The APNA monomer composed of the N-(2-aminobenzyl)-glycine backbone led to the smallest decrease in the thermal stability of the triplexes formed with DNA and RNA, while maintaining selectivity for base-pairing recognition. Since the PNA-APNA chimeras are more lipophilic than the corresponding PNA homopolymers, these oligomers may also exhibit better cell membrane permeability properties.