Functional Xeno Nucleic Acids for Biomedical Application

Functional nucleic acids(FNAs) refer to a type of oligonucleotides with functions over the traditional genetic roles of nucleic acids, which have been widely applied in screening, sensing and imaging fields. However, the potential application of FNAs in biomedical field is still restricted by the unsatisfactory stability, biocompatibility, biodistribution and immunity of natural nucleic acids(DNA/RNA). Xeno nucleic acids(XNAs) are a kind of nucleic acid analogues with chemically modified sugar groups that possess improved biological properties, including improved biological stability, increased binding affinity, reduced immune responses, and enhanced cell penetration or tissue specificity. In the last two decades, scientists have made great progress in the research of functional xeno nucleic acids, which makes it an emerging attractive biomedical application material. In this review, we summarized the design of functional xeno nucleic acids and their applications in the biomedical field.     

Recent advances in optical-based and force-based single nucleic acid imaging

The capability to image, as well as control and manipulate single molecules such as nucleic acids(DNA or RNA) can greatly enrich our knowledge of the roles of individual biomolecules in cellular processes and their behavior in native environments. Here we summarize the recent advances of single nucleic acid imaging based on optical observation and force manipulation. We start by discussing the superiority of single molecule image, the central roles nucleic acids play in biosystems, and the significance of single molecule image towards nucleic acids. We then list a series of representative examples in brief to illustrate how nucleic acid of various morphologies has been imaged from different aspects, and what can be learned from such characterizations. Finally,concluding remarks on parts of which should be improved and outlook are outlined.     

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。     

A High-Sensitivity Assay of Nucleic Acids with Tetraphenyl Porphyrin Cobalt Chlorine by Resonance Light Scattering Technique

Abstract

A novel probe, tetraphenyl porphyrin cobalt chlorine (CoTPPCl), is first applied to determine nucleic acids at the nanogram level based on the measurement of resonance light scattering (RLS) signals, which result from the interaction of CoTPPCl with nucleic acids. Under pH 6.37 conditions, the reaction between CoTPPCl and nucleic acid enhances the weak resonance light scattering (RLS) signal of CoTPPCl, and the enhanced light scattering intensity is proportional to the concentration of nucleic acid. The method is sensitive (3.45 ng/mL for ctDNA), simple (one step and a common fluorimeter), and tolerant of the metal ions and other coexistent substances. The mode of the combination between CoTPPCl and nucleic acids and the reasons for RLS enhancement are clearly clarified. Synthetic samples were determined with satisfactory results.     

Recent highlights in modified oligonucleotide chemistry

The synthesis of modified nucleic acids has been the subject of much study ever since the structure of DNA was elucidated by Watson and Crick at Cambridge and Wilkins and Franklin at King's College over half a century ago. This review describes recent developments in the synthesis and application of these artificial nucleic acids, predominantly the phosphoramidites which allow for easy inclusion into oligonucleotides, and is divided into three separate sections. Firstly, modifications to the base portion will be discussed followed secondly by modifications to the sugar portion. Finally, changes in the type of nucleic acid linker will be discussed in the third section. Peptide Nucleic Acids (PNAs) are not discussed in this review as they represent a separate and large area of nucleic acid mimics.     

Analytical methods for locating modifications in nucleic acids

We reviewed and summarized the established methods and the breakthrough of the techniques for locating modifications in nucleic acids. In addition, we discussed the principles, applications, advantages and drawbacks of these methods.     

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.     

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.     

Synthesis of New Chiral Building Blocks for Novel Peptide Nucleic Acids

IntroductionAbouttenyearsago ,PNA ,astructuralmimicofDNAinwhichthesugar phosphatebackboneisreplacedbyN (2 aminoethyl)glycine (aeg)linkageemergedasapotentialanti sensetherapeuticagent.1PNAhassomeadvantages:(1)itisstabletocellularnucleasesandproteases,(2 )ithybridizeswithcomplementaryDNAorRNA (cDNA/RNA)sequenceswithhighaffinity ,(3)ithaslownon specificinteractionwithcellularcontentsand (4 )itiseasilysynthesizedbyadoptionofsolidphasepeptidesynthesischemistry .However,thema jorlimitationo…     

Nucleic acid based molecular devices

In biology, nucleic acids are carriers of molecular information: DNA's base sequence stores and imparts genetic instructions, while RNA's sequence plays the role of a messenger and a regulator of gene expression. As biopolymers, nucleic acids also have exciting physicochemical properties, which can be rationally influenced by the base sequence in myriad ways. Consequently, in recent years nucleic acids have also become important building blocks for bottom-up nanotechnology: as molecules for the self-assembly of molecular nanostructures and also as a material for building machinelike nanodevices. In this Review we will cover the most important developments in this growing field of nucleic acid nanodevices. We also provide an overview of the biochemical and biophysical background of this field and the major "historical" influences that shaped its development. Particular emphasis is laid on DNA molecular motors, molecular robotics, molecular information processing, and applications of nucleic acid nanodevices in biology.     

MVF Sensor Enables Analysis of Nucleic Acids with Stable Secondary Structures

One major challenge in nucleic acids analysis by hybridization probes is a compromise between the probe's tight binding and sequence‐selective recognition of nucleic acid targets folded into stable secondary structures. We have been developing a four‐way junction (4WJ)‐based sensor that consists of a universal stem‐loop (USL) probe immobilized on an electrode surface and two adaptor strands (M and F). The sensor was shown to be highly selective towards single base mismatches at room temperature, able to detect multiple targets using the same USL probe, and have improved ability to detect folded nucleic acids. However, some nucleic acid targets, including natural RNA, are folded into very stable secondary and tertiary structures, which may represent a challenge even for the 4WJ sensors. This work describes a new sensor, named MVF since it uses three probe stands M, V and F, which further improves the performance of 4WJ sensors with folded targets. The MVF sensor interrogating a 16S rRNA NASBA amplicon with calculated folding energy of ?32.82 kcal/mol has demonstrated 2.5‐fold improvement in a signal‐to‐background ratio in comparison with a 4WJ sensor lacking strand V. The proposed design can be used as a general strategy in the analysis of folded nucleic acids including natural RNA.     

Interactions of Safranin T with Nucleic Acids

The interactions of Safranin T (ST) with several nucleic acids have been investigated by electrochemical, UV‐visible and CD spectroscopic techniques. The form of the nucleic acid‐ST complexes is sensitive to the ratio of the two species. Two electrochemically inactive complexes such as, nucleic acid‐ST and nucleic acid‐2ST, were formed while ST interacts with nucleic acids. Two processes were obtained from spectral experiments: (1) at the high value of R (R is defined as the ratio of the total concentration of ST to that of nucleic acid), ST is groove‐binding with stacking, (2) at the low value of R, ST is groove‐binding without stacking. Intrinsic binding constants were obtained by spectral methods. The experiments also show that electrostatic binding plays an important role in the interaction of ST with nucleic acids.     

The binding of single-stranded DNA and PNA to single-walled carbon nanotubes probed by flow linear dichroism

The binding of single-stranded DNAs and a neutral DNA analogue (peptide nucleic acid, PNA) to single-walled carbon nanotubes in solution phase has been probed by absorbance spectroscopy and linear dichroism. The nanotubes are solubilised by aqueous sodium dodecyl sulfate, in which the nucleic acids also dissolve. The linear dichroism (LD) of the nanotubes, when subtracted from that due to the nanotubes/nucleic acid samples, gives the LD of the bound nucleic acid. The binding of the single-stranded DNA to the single-walled nanotubes is quite different from that previously observed for double-stranded DNA. It is likely that the nucleic acid bases lie flat on the nanotube surface with the backbone wrapping round the nanotube at an oblique angle in the region of 45 degrees . The net effect is like beads on a string. The base orientation with the single-stranded PNA is inverted with respect to that of the single-stranded DNA, as shown by their oppositely signed LD signals.     

Determination of nucleic acids using fluorescence probe sensitized by microemulsion

Because the fluorescence of azur A can be quenched by adding nucleic acid, a sensitive fluorometric method for determination of nucleic acids at nanogram levels was established. Using optimal conditions, the calibration curves were linear in the range of 0-6.0 microg/mL for calf thymus deoxyribonucleic acid (ct DNA) and 0-7.0 microg/mL for herring sperm DNA (hs DNA). The limits of determination were 3.5 and 3.8 ng/mL, respectively, which shows the high sensitivity of this method. Triton X-100 microemulsion was applied as a sensitive media to enhance the sensitivity. The binding mode concerning the interactions of azur A with nucleic acids was also studied and the association constant with different binding numbers was obtained. The method has been applied to the determination of nucleic acid in both synthetic and real samples, such as cauliflower and pork liver, with satisfactory results.     

Interaction of morin-cetyltrimethylammonium bromide with nucleic acids and determination of nucleic acids at nanograms per milliliter levels based on the enhancement of preresonance light scattering

A new preresonance light scattering (PRLS) assay of nucleic acids is presented. At pH 7.30, the weak PRLS of morin-cetyltrimethylammonium bromide (CTMAB) can be greatly enhanced by the addition of nucleic acids, owing to the interaction between the nucleic acid and morin-CTMAB. After the addition of morin and CTMAB to DNA, the zeta potential of DNA decreases and changes from negative to positive, which is due to the formation of an associate, the aggregation of morin on nucleic acids and the electric neutralization between DNA and the cationic surfactant CTMAB. Mechanism studies showed that the enhanced PRLS comes from the aggregation of morin in the presence of nucleic acids and CTMAB. The enhanced intensity of PRLS is in proportion to the concentration of nucleic acids in the range 7.5 x 10(-9)-1.0 x 10(-5) g ml(-1) for calf thymus DNA, 7.5 x 10(-9)-1.0 x 10(-6) g ml(-1) for salmon sperm DNA and 1.0 x 10(-8)-1.0 x 10(-6) g ml(-1) for yeast RNA. The detection limits are 3.4, 6.2 and 4.1 ng ml(-1) for calf thymus DNA, salmon sperm DNA and yeast RNA, respectively. Synthetic samples were analyzed satisfactorily.     

Invite Article: The Liquid-Crystalline Phases of Double-Stranded Nucleic Acids in Vitro and in Vivo

This article describes the state of and progress in experimental studies of liquid crystals of naturally occurring nucleic acids and synthetic polynucleotides. The areas considered in this review include: (i) the liquid-crystalline phase of nucleic acids in aqueous salt solutions, (ii) the liquid-crystalline phase of nucleic acids in aqueous polymer solutions, (iii) the liquid-crystalline phase of nucleic acids in living systems. Some unsolved problems which are of interest from both a physicochemical and a biological point of view are discussed.     

PNA-based probe for quantitative chemiluminescent in situ hybridisation imaging of cellular parvovirus B19 replication kinetics

To allow the ultrasensitive localization and the quantitative detection of parvovirus B19 nucleic acids in single infected cells at various times post-infection, a peptide nucleic acid (PNA)-based in situ hybridisation (ISH) assay with chemiluminescent detection has been developed. The assay is based on the use of a biotin-labelled PNA probe detected by a streptavidin-linked alkaline phosphatase and a chemiluminescent dioxetane phosphate derivative substrate. The luminescent signal was quantified and imaged with an ultrasensitive nitrogen-cooled CCD camera connected to an epifluorescence microscope. The assay was used to analyze the parvovirus B19 infection process in cell cultures and to quantify the amount of viral nucleic acids at different times after infection.The chemiluminescent ISH-PNA assay is characterized by high resolution providing a sharp localization of B19 nucleic acids within single cells, with higher sensitivity with respect to conventional colorimetric ISH detection. Thanks to the high detectability and wide linear range of chemiluminescence detection, an objective evaluation of the percentage of infected cells, which reached its maximum at 24 h after infection, following a B19 virus infectious cycle could be accurately evaluated. Chemiluminescence detection also allowed the quantitative analysis of viral nucleic acids at the single-cell level, showing a continuous increase of the content of viral nucleic acids in infected cells with time after infection.The developed chemiluminescent ISH-PNA assay could thus represent a potent tool for the assessment of viral infections and for the quantitative evaluation of the virus nucleic acid load of infected cells in virus studies and diagnostics.