Locked and unlocked nucleosides in functional nucleic acids

Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes.     

Rapid,single-step nucleic acid detection

A rapid detection method for nucleic acid based on bioluminescence resonance energy transfer (BRET) from the luminescence donor Renilla luciferase to an acceptor quantum dot upon oligonucleotide probe hybridization has been developed. Utilizing a competitive assay, we detected the target nucleic acid by correlating the BRET signal with the amount of target present in the sample. This method allows for the detection of as little as 4 pmol (20 nM) of nucleic acid in a single-step, homogeneous format both in vitro in a buffer matrix as well as in a cellular matrix. Using this method, one may perform nucleic acid detection in as little as 30 min, showing much improvement over time-consuming blotting methods and solid-phase methods which require multiple wash steps to remove unbound probe. This is the first report on the use of quantum dots as a BRET acceptor in the development of a nucleic acid hybridization assay. An erratum to this article can be found at     

Synthesis of two mirror image 4-helix junctions derived from glycerol nucleic acid

Structural DNA nanotechnology relies on Watson-Crick base pairing rules to assemble DNA motifs into diverse arrangements of geometric shapes and patterns. While substantial effort has been devoted to expanding the programmability of natural DNA, considerably less attention has been given to the development of nucleic acid structures based on non-natural DNA polymers. Here we describe the use of glycerol nucleic acid (GNA), a simple polymer based on an acyclic repeating unit, as an alternative genetic material for assembling nucleic acid nanostructures independent of RNA or DNA recognition. We synthesized two 4-helix junctions based entirely on GNA self-pairing and showed that GNA provides easy access to highly stable nanostructures with left- and right-handed helical configurations.     

Practical physical aspects of interfacial nucleic acid oligomer hybridisation for biosensor design

A significant amount of research concerning rapid, selective biomolecular analysis has focused on development of analytical methods that make use of nucleic acid hybridisation as the basis for selective recognition. The development of biosensors based on nucleic acid hybridisation requires consideration of the thermodynamics of hybrid formation at a solid interface, because the relative thermodynamic stability can dictate the selectivity of hybridisation. Careful control of hybridisation conditions such as the density of oligonucleotides, as well as the temperature, pH, and ionic strength, may therefore enhance the selectivity, sensitivity and speed of a nucleic acid hybridisation assay that is located at an interface.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.     

Selected aptamer specially combing 5-8F cells based on automatic screening instrument

Since the concept of aptamer emerged, many scientists have launched a rich field of research around it. However, few nucleic acids aptamer which use cell as target can be put into practical applications. We believe that a great deal of this lies in the complexity and irreproducibility of aptamer screening experiments themselves. The complexity is due to the cumbersome processes and the technical requirements for laboratory personnel, whereas irreproducibility arises from the fact that the starting point of such screens is nucleic acid libraries with random fragments, and that different libraries directly determine the differences or even the success or failure of screening results. The complexity and irreproducibility mentioned above, in turn, lead to the inability of this experiment to unfold on a large scale, which naturally cannot lead to excellent results for practical applications. In response to this problem, our group has developed an instrument for automated screening of tumor cell nucleic acid aptamers and characterized the properties of nucleic acid aptamers obtained using this instrument in a comprehensive manner.     

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.     

Detection of miRNA in Live Cells by Using Templated RuII‐Catalyzed Unmasking of a Fluorophore

Reactions templated by cellular nucleic acids are attractive for nucleic acid sensing or responsive systems. Herein we report the use of a photocatalyzed reductive cleavage of an immolative linker to unmask a rhodamine fluorophore, and its application to miRNA imaging. The reaction was found to proceed with a very high turnover (>4000) and provided reliable detection down to 5 pM of template by using γ‐serine‐modified peptide nucleic acid (PNA) probes. The reaction was used for the selective detection of miR‐21 in BT474 cells and miR‐31 in HeLa cells following irradiation for 30 min. The probes were introduced by using reversible permeation with streptolysin‐O (SLO) or a transfection technique.     

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.     

Aptamers: molecular tools for analytical applications

Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.     

Nucleic Acid Nanoprobes for Biosensor Development in Complex Matrices

Nucleic acid probes in living organisms play an essential role in therapeutics and diagnosis.Through the imaging and sensing of nucleic acid probes in complex biological matrices,a variety of diseases-related biological process,pathogenic process,or pharmacological responses to a therapeutic intervention have been discovered.However,a critical challenge of nucleic acid probes applied in complex matrices lies in enhancing the stability of nucleic acid probes,especially when it suffers from nuclease degradation and protein adsorption.In order to enhance the application of nucleic acid nanoprobes in complex matrices,great efforts have been devoted to improving the stability of probes operated in complex media,including construction of nucleic acid nanoprobes with nuclease resistance and protein adsorption resistance,sample pretreatment,anti-biofouling and signal correction.In this review,we aim to summarize recent advances in the stability of nucleic acid nanoprobes in complex matrices,including the methods of enhancing the stability of probes or signals,and the application of nucleic acid nanoprobes for disease diagnosis.     

A helical amplification system composed of artificial nucleic acids

Herein we report an amplification system of helical excess triggered by nucleic acid hybridization for the first time. It is usually impossible to prepare achiral nanostructures composed of nucleic acids because of their intrinsic chirality. We used serinol nucleic acid (SNA) oligomers for the preparation of achiral nanowires because SNA oligomers with symmetrical sequences are achiral. Nanowire formation was confirmed by atomic force microscopy and size exclusion chromatography. When a chiral nucleic acid with a sequence complementary to SNA was added to the nanostructure, helicity was induced and a strong circular dichroism signal was observed. The SNA nanowire could amplify the helicity of chiral nucleic acids through nucleobase stacks. The SNA nanostructures have potential for use as platforms to detect chiral biomolecules under aqueous conditions because SNA can be readily functionalized and is water-soluble.

Herein we report an amplification system of helical excess triggered by nucleic acid hybridization for the first time.     

Enhancement of anion-exchange chromatography of DNA using compaction agents

The use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. The possibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing their radius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharose anion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mM spermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorption of both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only the binding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids.     

Aptasensors – the future of biosensing?

Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. Aptamers, first reported in 1990, are attracting interest in the areas of therapeutics and diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors. The properties of aptamers, their applicability to biosensor technology, current research and future prospects are addressed in this short review.     

基于化学衍生-质谱技术的生物与临床样本中核酸修饰分析

除了经典碱基外,核酸(DNA和RNA)中还包含许多化学修饰。迄今为止,已经在核酸中鉴定了超过150多种化学修饰。这些化学修饰不会改变核酸的序列,但会改变它们的结构和生化特性,最终调节基因的时空表达。阐明这些修饰的功能可以促进对生命体生理调控机制的深入认识和理解。然而,核酸修饰在体内的丰度通常很低。因此,高灵敏和特异的检测方法对破译这些修饰的功能至关重要。化学衍生与质谱技术相结合对内源性低丰度核酸修饰展现出很好的分析能力。在过去几年中,研究者建立了多种基于化学衍生-质谱分析的分析方法,用于灵敏、高效地分析核酸修饰。该文总结了通过化学衍生-质谱分析方法来破译核酸修饰的最新进展,希望能促进未来对核酸修饰功能的深入研究。     

尿中烷化核酸碱基的研究进展

本文论述了目前关于尿中烷化核酸碱基的重要研究进展,其中包括烷墓核酸碱参的生成机理,尿中烷化核酸碱荃的分析方法,人类暴露于烷基致癌物所产生的烷基核酸碱墓的水平。同时,还评述了烷化核酸碱莎,特别是3一甲墓腺嗦吟作为生物指标的可能性及其应用前景。     

Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples. In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS. Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules. Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals. Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation. This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections.     

Bridging the gap between proteins and nucleic acids: a metal-independent RNAseA mimic with two protein-like functionalities.

Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.