Study on the interaction between nucleic acids and cationic surfactants

The interactions of nucleic acids and cationic surfactants (cetylpyridine bromide (CPB) and cetyltrimethylammonium bromide (CTMAB)) in aqueous solution have been studied using the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and NMR assignment measurement. It is considered that CPB or CTMAB can assemble on the surface of nucleic acid via electrostatic and hydrophobic forces, which results in the formation of large associate of nucleic acid-cationic surfactant and RLS enhancement of nucleic acid. Besides these forces, the pi-pi stacking force between CPB and nucleic acid also exists in the associate. In comparison with CTMAB, CPB has larger enhancement on RLS of nucleic acid, which is attributed to that the enhancement of the former is only due to the absorption of the bases of nucleic acid, while the enhancement of the latter is own to the synergetic resonance caused by the absorption of both bases of nucleic acid and the pyridyl in CPB. These results have important implication for understanding the influence of surfactants on nucleic acid functionality in life science.     

Analysis and purification of peptide nucleic acids by denaturing PAGE

A flexible and convenient protocol for the analysis and purification of peptide nucleic acid (PNA) oligomers and PNA-peptide chimeras by denaturing PAGE is described. Vertical slab gel electrophoresis, 26% in polyacrylamide and 8 M urea at pH 3, was suitable for analysis of oligomers ranging in size from tetramers (4-mers) to tetradodecamers (24-mers). Single-base resolution of oligomers was achieved and separations are generally superior to those given by standard RP-HPLC techniques. The separation of a related series of PNA oligomers showed the distance migrated was linearly dependent on the logarithm of the molecular weight. The migration of oligomers through the gel is dependent on the number of basic functional groups present, such as amino groups, and the A and C content of the oligomer. PNAs are amenable to detection by UV-shadowing technique illuminated at 260 nm or Coomassie blue staining, both with similar, sub-microgram per band detection limits.     

Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development

Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.     

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.     

A molecular peptide beacon for the ratiometric sensing of nucleic acids

A pyrene-functionalized cationic oligopeptide 1 efficiently binds to double-stranded DNA, as shown by different spectrophotochemical studies. Upon binding, the conformation of 1 changes from a folded to an extended form, which leads to a distinct change in the fluorescence properties. Thus, 1 functions as a molecular peptide beacon, and as it is easily taken up by cells, 1 can also be used for imaging of nucleic acids within cells.     

Amino acids and peptides—IV: Novel peptide bond formation catalysed by metal ions—II Formation of optically active peptide esters

The novel peptide bond formation previously reported in the reaction of glycine ester with Cu(II) ion in an anhydrous solvent, was examined using several kinds of optically active amino acid esters. Various reaction conditions were studied in detail for Phe-OEt. From Phe-OEt, Ala-OMe, Leu-OMe, Trp-OMe, Ser-OMe, and Met-OMe, the expected dipeptide esters with the same amino acid components were obtained without racemization. Asp(OEt)-OEt, and Glu(OMe)-OMe gave only optically active α-dipeptide esters. No formation of dipeptide esters was observed with Ile-OMe, Cys-OMe, His-OMe, and Pro-OEt. However, Lys-OMe, and Orn-OMe afforded optically active lactam derivatives. Some explanations of these abnormal observations have been given.Attempts to prepare di- and tri-peptide esters carrying different kinds of amino acid units were also studied.     

A DNA-mediated crosslinking strategy to enhance cellular delivery and sensor performance of protein spherical nucleic acids

Intracellular delivery of enzymes is essential for protein-based diagnostic and therapeutic applications. Protein-spherical nucleic acids (ProSNAs) defined by protein core and dense shell of oligonucleotides have been demonstrated as a promising vehicle-free enzyme delivery platform. In this work, we reported a crosslinking strategy to vastly improve both delivery efficiency and intracellular sensor performance of ProSNA. By assembling individual ProSNA with lactate oxidase (LOX) core into a nanoscale particle, termed as crosslinked SNA (X-SNA), the enzyme delivery efficiency increased up to 5–6 times higher. The LOX X-SNA was later demonstrated as a ratiometric probe for quantitative detection of lactate in living cells. More importantly, X-SNA probe showed significantly improved sensor performance with signal-to-noise ratio 4 times as high as ProSNA when detecting intracellular lactate.

By crosslinking protein spherical nucleic acid (SNA) into a supramolecular architecture X-SNA, the intracellular enzyme delivery efficiency was significantly enhanced, showing 3–4 times higher signal-to-noise ratio in detecting intracellular lactate.     

A method to enhance a1 ions and application for peptide sequencing and protein identification

A simple and effective method was developed for peptide sequencing and protein identification through the determination of its N-terminal residue. The method of N-terminal carbamidomethylation with iodoacetamide could specifically and remarkably enhance the intensity of a₁ ions in the tandem mass spectra of the peptide derivatives without significantly altering their fragmentation pattern, thus allowing determination of their N-terminal residues. The effectiveness and specificity of the method was demonstrated by confirming and extending sequence interpretation of several model peptides and proteins. The developed method was then applied in the LC-MS/MS analysis of the tryptic digests of myoglobin and a whole protein extract from rat heart tissues. The results from database searches were well validated with the enhancement of a₁ ions in tandem mass spectra and the specificity of protein identification was obtained when the information of N-terminal residues was included in the database search.     

Synthesis and properties of peptide nucleic acids containing a psoralen unit

We prepared the psoralen PNA unit from 8-methoxypsoralen and synthesized various PNAs containing psoralen by a typical (t)()Boc method. PNAs containing psoralen (P-PNA) at strand end formed a stable duplex with complementary DNA. The hybridization of P-PNA with complementary DNA resulted in a considerable decrease of the psoralen fluorescence.     

Cellular delivery of peptide nucleic acids and inhibition of human telomerase.

BACKGROUND: Human telomerase has an essential RNA component and is an ideal target for developing rules correlating oligonucleotide chemistry with disruption of biological function. Similarly, peptide nucleic acids (PNAs), DNA analogs that bind complementary sequences with high affinity, are outstanding candidates for inducing phenotypic changes through hybridization. RESULTS: We identify PNAs directed to nontemplate regions of the telomerase RNA that can overcome RNA secondary structure and inhibit telomerase by intercepting the RNA component prior to holoenzyme assembly. Relative potencies of inhibition delineate putative structural domains. We describe a novel protocol for introducing PNAs into eukaryotic cells and report efficient inhibition of cellular telomerase by PNAs. CONCLUSIONS: PNAs directed to nontemplate regions are a new class of telomerase inhibitor and may contribute to the development of novel antiproliferative agents. The dependence of inhibition by nontemplate-directed PNAs on target sequence suggests that PNAs have great potential for mapping nucleic acid structure and predictably regulating biological processes. Our simple method for introducing PNAs into cells will not only be useful for probing the complex biology surrounding telomere length maintenance but can be broadly applied for controlling gene expression and functional genomics.     

Amino acids and peptides—V: Novel peptide bond formation catalyzed by metal ions—III Elucidation of the formation mechanism

Elucidation of the mechanism for peptide bond formation observed when an amino acid ester is treated with anhyd CuCl₂ in an anhyd alcoholic solvent, was attempted using results of IR and visible spectra measurements of the amino acid ester-CuCl₂ complex and accumulated experimental data.This novel reaction proceeds through a mechanism in which the amino anion produced by elimination of the proton from the Cu(II)-coordinating amino group, attacks the non-activated ester CO group of the amino acid ester which shares a common Cu(II) ion. It differs completely from the peptide formation reaction featuring a Co(III)-amino acid ester complex.     

An experimental and theoretical study of the gas-phase decomposition of monoprotonated peptide nucleic acids

Peptide nucleic acids (PNAs) are DNA/RNA mimics which have recently generated considerable interest due to their potential use as antisense and antigene therapeutics and as diagnostic and molecular biology tools. These synthetic biomolecules were designed with improved properties over corresponding oligonucleotides such as greater binding affinity to complementary nucleic acids, enhanced cellular uptake, and greater stability in biological systems. Because of the stability and unique structure of PNAs, traditional sequence confirmation methods are not effective. Alternatively, electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry shows great potential as a tool for the characterization and structural elucidation of these oligonucleotide analogs. Extensive gas-phase fragmentation studies of a mixed nucleobase 4-mer (AACT) and a mixed nucleobase 4-mer with an acetylated N-terminus (N-acetylated AACT) have been performed. Gas-phase collision-induced dissociation of PNAs resulted in water loss, cleavage of the methylene carbonyl linker containing a nucleobase, cleavage of the peptide bond, and the loss of nucleobases. These studies show that the fragmentation behavior of PNAs resembles that of both peptides and oligonucleotides. Molecular mechanics (MM+), semiempirical (AM1), and ab initio (STO-3G) calculations were used to investigate the site of protonation and determine potential low energy conformations. Computational methods were also employed to study prospective intramolecular interactions and provide insight into potential fragmentation mechanisms.     

Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: role of water activity

Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for immobilized metal-chelate affinity adsorbents. Addition of 20% (v/v) of solutes such as ethanol, methanol, isopropanol, n-propanol, and dimethyl sulfoxide enhances the initial affinity of binding of total yeast RNA by 4.4-, 3.8-, 3.7-, 3.0-, and 2.8-fold, respectively for Cu(II)-iminodiacetic acid (IDA) agarose adsorbent, and the weaker adsorption by Cu(II)-nitrilotriacetic acid (NTA) agarose was even more strongly enhanced. The adsorption affinities of the smaller oligodeoxynucleotide molecules A20, G20, C20 and T20 also increase with the addition of ethanol, suggesting that the effect is not significantly mediated by conformational changes. Binding enhancement generally correlates with reduction of water activity by the various solutes, as predicted by several models of solution thermodynamics, consistent with an entropic contribution by displacement of waters from the metal-chelate. Interestingly, the enhancement was not seen with the proteins bovine serum albumin and lysozyme.     

Lanthanide ions as luminescent chromophores for the liquid chromatographic detection of polynucleotides and nucleic acids

Europium(III) and terbium(III) can be used as luminescent chromophores for the liquid chromatographic detection of certain nucleotides and nucleic acids. The method is dependent upon an energy transfer from the nucleic acid to the lanthanide ion. Of the base moieties, only xanthine, guanine, and thiouridine have appropriate excited state energy levels for efficient energy transfer. The lanthanide ion can be added in a pre- or post-column mode. The applicability of the method was demonstrated for the detection of homologous polynucleotides such as poly X and poly G. The method was also used to detect transfer RNA from Escherichia coli.     

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Direct determination of pKa values of cationic acids conjugated to heterocyclic amine N-oxides in polar aprotic and amphiprotic solvents

The applicability of the direct method of pK_a determination in the case of protonated heterocyclic amine N-oxides in a series of polar non-aqueous (aprotic and amphiprotic) solvents has been tested. The method is based on the pH determination of the non-aqueous solution of complex salt (the semiperchlorate in this case) formed by the N-oxides studied. The direct method not only provides for quick (one data point per each pK_a determined), but also relatively accurate estimates of acidic dissociation constants. It has been experimentally shown on the example of substituted pyridine N-oxides that this method is precise enough in all studied non-aqueous solvents when applied to compounds of not too low basicity (the pK_a being of the order of 5 or higher). To prove this, the pK_a values of protonated monocyclic N-oxides obtained by the direct method have been compared with those resulting from the potentiometric titration curve. The agreement between the results found by using both methods is very good in most cases, the differences being within standard deviations. Based upon this observation it can be inferred that the pK_a values of protonated bicyclic N-oxides in solvents studied determined by using the direct method can be also considered reliable, especially in the case of polar aprotic solvents.     

Side chain homologation of alanyl peptide nucleic acids: pairing selectivity and stacking

Alanyl peptide nucleic acids (alanyl-PNAs) are oligomers based on a regular peptide backbone with alternating configuration of the amino acids. All side chains are modified by covalently linked nucleobases. Alanyl-PNAs form very rigid, well defined, and linear double strands based on hydrogen bonding of complementary strands, stacking, and solvation. Side chain homology was examined by comparing a methylene linker (alanyl-PNA) with an ethylene linker (homoalanyl-PNA), a trimethylene linker (norvalyl-PNA), and PNA sequences with mixed linker length between nucleobase and backbone. Side chain homology in combination with a linear double strand topology turned out to be valuable in order to selectively manipulate pairing selectivity (pairing mode) and base pair stacking.     

Preparation of trimethylsilyl derivatives of amino acids and peptides for peptide synthesis

Russian Chemical Bulletin -