Natural Nucleic Acids Research Articles

Exosomes: Biogenesis, Composition, Functions, and Their Role in Pre-metastatic Niche Formation

Exosomes are a subclass of extracellular vesicles that are released by normal or diseased cells. Exosomes are natural nanoparticles, contain natural cargo, including lipids, proteins, and nucleic acids, and are involved in intercellular communication. Exosomes are detected in the tumor microenvironment and participate in tumorigenesis by regulating angiogenesis, immunity, metastasis, and formation of pre-metastatic niches. This review mainly focuses on exosome biogenesis, composition, their biological and pathological functions, and roles in pre-metastatic niche formation.

Multi-level patterning nucleic acid photolithography

The versatile and tunable self-assembly properties of nucleic acids and engineered nucleic acid constructs make them invaluable in constructing microscale and nanoscale devices, structures and circuits. Increasing the complexity, functionality and ease of assembly of such constructs, as well as interfacing them to the macroscopic world requires a multifaceted and programmable fabrication approach that combines efficient and spatially resolved nucleic acid synthesis with multiple post-synthetic chemical and enzymatic modifications. Here we demonstrate a multi-level photolithographic patterning approach that starts with large-scale in situ surface synthesis of natural, modified or chimeric nucleic acid molecular structures and is followed by chemical and enzymatic nucleic acid modifications and processing. The resulting high-complexity, micrometer-resolution nucleic acid surface patterns include linear and branched structures, multi-color fluorophore labeling and programmable targeted oligonucleotide immobilization and cleavage.

Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics.

The remarkable physicochemical properties of the natural nucleic acids, DNA and RNA, define modern biology at the molecular level and are widely believed to have been central to life's origins. However, their ability to form repositories of information as well as functional structures such as ligands (aptamers) and catalysts (ribozymes/DNAzymes) is not unique. A range of nonnatural alternatives, collectively termed xeno nucleic acids (XNAs), are also capable of supporting genetic information storage and propagation as well as evolution. This gives rise to a new field of "synthetic genetics," which seeks to expand the nucleic acid chemical toolbox for applications in both biotechnology and molecular medicine. In this review, we outline XNA polymerase and reverse transcriptase engineering as a key enabling technology and summarize the application of "synthetic genetics" to the development of aptamers, enzymes, and nanostructures.

Sequence- And Structure-Specific Probing of RNAs by Short Nucleobase-Modified dsRNA-Binding PNAs Incorporating a Fluorescent Light-up Uracil Analog.

RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil (btU). The incorporation of btU into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of btU-modified dbPNAs is sequence- and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1-1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating btU-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.

Original article - Cytoplasmic nucleic acid-based XNAs directly enhance live cardiac cell function by a Ca2+ cycling-independent mechanism via the sarcomere

Nucleic acid - protein interactions are critical for regulating gene activation in the nucleus. In the cytoplasm, however, potential nucleic acid-protein functional interactions are less clear. The emergence of a large and expanding number of non-coding RNAs and DNA fragments raises the possibility that the cytoplasmic nucleic acids may interact with cytoplasmic cellular components to directly alter key biological processes within the cell. We now show that both natural and synthetic nucleic acids, collectively XNAs, when introduced to the cytoplasm of live cell cardiac myocytes, markedly enhance contractile function via a mechanism that is independent of new translation, activation of the TLR-9 pathway or by altered intracellular Ca2+ cycling. Findings show a steep XNA oligo length-dependence, but not sequence dependence or nucleic acid moiety dependence, for cytoplasmic XNAs to hasten myocyte relaxation. XNAs localized to the sarcomere in a striated pattern and bound the cardiac troponin regulatory complex with high affinity in an electrostatic-dependent manner. Mechanistically, XNAs phenocopy PKA-based modified troponin to cause faster relaxation. Collectively, these data support a new role for cytoplasmic nucleic acids in directly modulating live cell cardiac performance and raise the possibility that cytoplasmic nucleic acid - protein interactions may alter functionally relevant pathways in other cell types.

Side chain determinants of biopolymer function during selection and replication.

The chemical functionalities within biopolymers determine their physical properties and biological activities. The relationship between the side-chains available to a biopolymer population and the potential functions of the resulting polymers, however, has proven difficult to study experimentally. Using seven sets of chemically diverse charged, polar, and nonpolar side-chains, we performed cycles of artificial translation, in vitro selections for binding to either PCSK9 or IL-6 protein, and replication on libraries of random side-chain-functionalized nucleic acid polymers. Polymer sequence convergence, bulk population target binding, affinity of individual polymers, and head-to-head competition among post-selection libraries collectively indicate that polymer libraries with nonpolar side-chains outperformed libraries lacking these side-chains. The presence of nonpolar groups, resembling functionality present in proteins but missing from natural nucleic acids, thus may be strong determinants of binding activity. This factor may contribute to the apparent evolutionary advantage of proteins over their nucleic acid precursors for some molecular recognition tasks.

Aptazymes: Expanding the Specificity of Natural Catalytic Nucleic Acids by Application of In Vitro Selected Oligonucleotides.

Aptazymes are synthetic molecules composed of an aptamer domain and a catalytic active nucleic acid unit, which may be a ribozyme or a DNAzyme. In these constructs the aptamer domain serves as a molecular switch that can regulate the catalytic activity of the ribozyme or DNAzyme subunit. This regulation is triggered by binding of the aptamers target molecule, which causes significant structural changes in the aptamer and thus in the entire aptazyme. Therefore, aptazymes function similar to allosteric enzymes, whose catalytic activity is regulated by binding of ligands (effectors) to allosteric sites due to alteration of the three-dimensional structure of the active site of the enzyme. In case of aptazymes, the allosteric site is composed of an aptamer. Aptazymes can be designed for different applications and have already been used in analytical assays as well as for the regulation of gene expression.

Aminoglycoside Functionalization as a Tool for Targeting Nucleic Acids.

Aminoglycoside functionalization as a tool for targeting natural and unnatural nucleic acids holds great promise in their development as diagnostic probes and medicinally relevant compounds. Simple synthetic procedures designed to easily and quickly manipulate amino sugar (neomycin, kanamycin) to more powerful and selective ligands are presented in this chapter. We describe representative procedures for (a) aminoglycoside conjugation and (b) preliminary screening for their nucleic acid binding and selectivity.

Dynamic Single Molecular Rulers: Toward Quantitative Detection of MicroRNA-21 in Living Cells.

Innovative techniques to measure microRNA (miRNA) in vivo could greatly improve the fundamental understanding of complex cellular processes. Herein, we report a novel method for real-time, quantitative miRNA detection inside living cells based on core-satellite plasmon rulers (PRs). This approach allows for the statistical analysis of single hybridization event caused by target miRNA. We investigated hundreds of satellite leaving events and found that the distribution of the time range for one strand displacement event is miRNA concentration-dependent, which obeyed Poisson statistics. Probing several such PRs under dark-field microscopy would provide precise determination of miRNA in vitro and in living cells, without photobleaching or blinking of the fluorophores. We believe the simple and practical approach on the basis of dynamic PRs with single-molecule sensitivity combined with statistical analysis hold promising potential to visualize native nucleic acids with short sequence and low-abundance.

A Single Amino Acid Substitution in Therminator DNA Polymerase Increases Incorporation Efficiency of Deoxyxylonucleotides.

Deoxyxylonucleic acid (dxNA) is a synthetic polymer that might have potential for heredity and evolution. Because of dxNA's unusual backbone geometry, sequence information stored in it is presumed to be inaccessible to natural nucleic acids or proteins. Despite a large structural similarity with natural nucleotides, incorporation of 2'-deoxyxylonucleotides (dxNTs) through the action of polymerases is limited. We present the identification of a mutant of the DNA polymerase Therminator with increased tolerance to deoxyxylose-induced backbone distortions. Whereas the original polymerase stops after incorporation of two consecutive dxNTs, the mutant is able to catalyse the extension of incorporated dxNTs with 2'-deoxyribonucleotides (dNTs) and the incorporation of up to four dxNTs alternates with dNTs, thereby translocating a highly distorted double helix throughout the entire polymerase. A single His-to-Arg substitution very close to the catalytic site residues is held to be responsible for interaction with the primer phosphate groups and for stabilizing nucleotide sugar-induced distortions during incorporation and translocation.

Therapeutic Role of Harmalol Targeting Nucleic Acids: Biophysical Perspective and in vitro Cytotoxicity.

Harmalol, a beta carboline alkaloid, shows remarkable importance in the contemporary biomedical research and drug discovery programs. With time, there is emerging interest in search for better anti-cancer drugs of plant origin with high activity and lower toxicity. Most of the chemotherapeutic agents due to their non-specific target and toxicity on active healthy cells, use is often restricted, necessitating search for newer drugs having greater potentiality. The review highlighted the interaction of harmalol with nucleic acids of different motifs as sole target biomolecules and in vitro cytotoxicity of the alkaloid in human cancer cell lines with special emphasis on its apoptotic induction ability. Binding study and in vitro cytotoxicity was performed using several biophysical techniques and biochemical assays, respectively. Data from competition dialysis, UV and fluorescence spectroscopic analysis, circular dichroism, viscometry and isothermal calorimetry shows binding and interaction of harmalol with several natural and synthetic nucleic acids, both DNA and RNA, of different motifs. Furthermore, apoptotic hallmarks like internucleosomal DNA fragmentation, membrane blebbing, cell shrinkage, chromatin condensation, change of mitochondrial membrane potential, comet tail formation and ROS (reactive oxygen species) dependent cytotoxicity being analyzed in the harmalol treated cancer cells. These results stating the therapeutic role of harmalol, will lead to the interesting knowledge on the cytotoxicity, mode, mechanism, specificity of binding and correlation between structural aspects and energetics enabling a complete set of guidelines for design of new drugs.

Progress on Detection of Metals Ions by Functional Nucleic Acids Biosensor

Abstract Functional nucleic acids are natural or artificial nucleic acid sequences with specific functions and special structures. A part of metal ions are essential trace elements of human health, but excessive metal ions will be harmful to human health. The functional nucleic acids are widely used for detection of metal ions because of its advantages such as easy modification, low price, high stability and strong specificity. This paper detailed the interaction between functional nucleic acids and metal ions, mainly including cutting type, link type, metal ion-mediated base pairing, click chemistry type, conformational change type, and other types. The biosensors based on the combination of functional nucleic acid with different signal output were then introduced. Finally, the research significance and existing problems of functional nucleic acid for metal ion detection were discussed. The future development trends and applications of functional nucleic acid biosensor were prospected.

Nucleic acids binding strategies of small molecules: Lessons from alkaloids

BackgroundNucleic acids are now important targets for therapeutic intervention. Alkaloids are an important class of molecules that have myriad therapeutic utility. Isoquinoline and benzophenanthridine alkaloids exhibit multiple pharmacological activities which are often related to their strong nucleic acid binding abilities. Therefore, a review of their interaction aspects with varying nucleic acid structures is essential for rational design and development as therapeutic agents. Scope of the reviewThis work reviews the interaction of various therapeutically important isoquinoline and benzophenanthridine alkaloids with nucleic acids. The review lends insights into the molecular aspects of the interaction that is critical from the perspective of designing better therapeutics. Major conclusionsThis review provides a concise report on the recent developments and advancements on the interaction of various alkaloids with natural and synthetic nucleic acids. The review focuses on the mode, mechanism, specificity, conformational aspects and energetics of the interaction that will be helpful in the design and synthesis nucleic acid targeted alkaloid analogs. General significanceThe molecular aspects of the interaction presented here will benefit the development of effective drugs for many diseases. The fundamental results discussed in this review can serve as a database for the design and development of futuristic nucleic acid based small molecule therapeutics.

Impact of N-(2-aminoethyl) Glycine Unit on Watson-Crick Base Pairs

Abstract We aim to understand the structure and stability of the backbone tailored Watson-Crick base pairs, Guanine-Cytosine (GC), Adenine-Thymine (AT) and Adenine-Uracil (AU) by incorporating N-(2-aminoethyl) glycine units (linked by amide bonds) at the purine and pyrimidine sites of the nucleobases. Density functional theory (DFT) is employed in which B3LYP/6-311++G∗ ∗ level of theory has been used to optimize all the structures. The peptide attached base pairs are compared with the natural deoxyribose nucleic acid (DNA)/ribonucleic acid (RNA) base pairs and the calculations are carried out in both the gas and solution phases. The structural propensities of the optimized base pairs are analyzed using base pair geometries, hydrogen bond distances and stabilization energies and, compared with the standard reference data. The structural parameters were found to correlate well with the available data. The addition of peptide chain at the back bone of the DNA/RNA base pairs results only with a minimal distortion and hence does not alter the structural configuration of the base pairs. Also enhanced stability of the base pairs is spotted while adding peptidic chain at the purine site rather than the pyrimidine site of the nucleobases. The stability of the complexes is further interpreted by considering the hydrogen bonded N–H stretching frequencies of the respective base pairs. The discrimination in the interaction energies observed in both gas and solution phases are resulted due to the existence of distinct lowest unoccupied molecular orbitals (LUMO) in the solution phase. The reactivity of the base pairs is also analyzed through the in-depth examinations on the highest occupied molecular orbital (HOMO)-LUMO orbitals.

Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers.

Although several synthetic or xenobiotic nucleic acids (XNAs) have been shown to be viable genetic materials in vitro, major hurdles remain for their in vivo applications, particularly orthogonality. The availability of XNAs that do not interact with natural nucleic acids and are not affected by natural DNA processing enzymes, as well as specialized XNA processing enzymes that do not interact with natural nucleic acids, is essential. Here, we report 3'-2' phosphonomethyl-threosyl nucleic acid (tPhoNA) as a novel XNA genetic material and a prime candidate for in vivo XNA applications. We established routes for the chemical synthesis of phosphonate nucleic acids and phosphorylated monomeric building blocks, and we demonstrated that DNA duplexes were destabilized upon replacement with tPhoNA. We engineered a novel tPhoNA synthetase enzyme and, with a previously reported XNA reverse transcriptase, demonstrated that tPhoNA is a viable genetic material (with an aggregate error rate of approximately 17 × 10-3 per base) compatible with the isolation of functional XNAs. In vivo experiments to test tPhoNA orthogonality showed that the E. coli cellular machinery had only very limited potential to access genetic information in tPhoNA. Our work is the first report of a synthetic genetic material modified in both sugar and phosphate backbone moieties and represents a significant advance in biorthogonality toward the introduction of XNA systems in vivo.

Chemistry of Artificial Nucleic Acid and Oligonucleotide Therapeutics Based on Natural Nucleic Acids

Chemistry of Artificial Nucleic Acid and Oligonucleotide Therapeutics Based on Natural Nucleic Acids

The ULTIMATE Reagent: A Universal Photocleavable and Clickable Reagent for the Regiospecific and Reversible End Labeling of Any Nucleic Acid.

There is a need for methods to chemically incorporate photocleavable labels into synthetic and biologically sourced nucleic acids in a chemically defined and reversible manner. We have previously demonstrated that the light-cleaved diazo di-methoxy nitro phenyl ethyl (diazo-DMNPE) group has a remarkable regiospecificity for modifying terminally phosphorylated siRNA. Building on this observation, we have identified conditions under which a diazo-DMNPE reagent that we designed (diazo-DMNPE-azide or DDA) is able to singly modify any nucleic acid (RNA, DNA, single-stranded, double-stranded, 3' or 5' phosphate). It can then be modified with any clickable reagent to incorporate arbitrary labels such as fluorophores into the nucleic acid. Finally, native nucleic acid can be regenerated directly through photolysis of the reagent. Use of the described approach should allow for the tagging of any nucleic acid, from any source-natural or unnatural-while allowing for the light-induced regeneration of native nucleic acid.

Electrochemical Synthesis of Azanucleosides

Recently, the nucleic acid medicine receiving a lot of attention because of various drug targets and few side effects compared with conventional low molecular medicine. The numerous nucleoside analogues have been reported. Especially, azanucleosides, in which the oxygen atom of furanose ring has been replaced with a nitrogen atom, show anticancer, antivirus, and antileukemia activity. Furthermore, azanucleosides exhibit tolerance towards nucleases when their incorporate into oligonucleotides. However, the traditional synthetic strategy requires multiple steps and harsh conditions, thereby limiting to obtain desired product with high yield. We developed mild condition and efficient synthesis of azanucleosides from prolinol derivative using by an electrochemical reaction in a 1.0 M lithium perchlorate-nitroethane and small amount of acetic acid mixture medium with a glassy carbon anode and platinum cathode. In this reaction, intermediate iminium cation was generated through anodic oxidation of N-α position. The nitroalkane solvent is suitable for cation-pool method because the cation intermediate species are stabilized at nearly room temperature. After 2.6 F of electricity, the nucleophiles were added into reaction medium and stirred overnight. We used each nucleobases (N6-benzoyl adenine: A(Bz), N4-benzoyl cytosine: C(Bz), N2-isobutyryl guanine: G(Ib)) as nucleophiles. The coupling with thymine (T) was carried out through silyl Hilbert-Johnson reaction from other prolinol derivative that has hydroxyl group in N-α position. It was also prepared by anodic oxidative C-H activation. As a result, desired deoxyribo-azanucleosides was obtained. However, the desired azanucleosides was generated as α and β anomer by equally ratio in this reaction. The natural nucleic acids are composed by only β-anomer nucleosides. In this reason, to regulate the anomeric selectivity is very important. Now, we are challenging the stereoselective synthesis of these deoxyribo-azanucleosides. Furthermore, we are applying our electrochemical method for the synthesis of ribo-azanucleosides.

Fluorogenic PNA probes

Fluorogenic oligonucleotide probes that can produce a change in fluorescence signal upon binding to specific biomolecular targets, including nucleic acids as well as non-nucleic acid targets, such as proteins and small molecules, have applications in various important areas. These include diagnostics, drug development and as tools for studying biomolecular interactions in situ and in real time. The probes usually consist of a labeled oligonucleotide strand as a recognition element together with a mechanism for signal transduction that can translate the binding event into a measurable signal. While a number of strategies have been developed for the signal transduction, relatively little attention has been paid to the recognition element. Peptide nucleic acids (PNA) are DNA mimics with several favorable properties making them a potential alternative to natural nucleic acids for the development of fluorogenic probes, including their very strong and specific recognition and excellent chemical and biological stabilities in addition to their ability to bind to structured nucleic acid targets. In addition, the uncharged backbone of PNA allows for other unique designs that cannot be performed with oligonucleotides or analogues with negatively-charged backbones. This review aims to introduce the principle, showcase state-of-the-art technologies and update recent developments in the areas of fluorogenic PNA probes during the past 20 years.

Fluorescent nucleobase analogues for base-base FRET in nucleic acids: synthesis, photophysics and applications.

Förster resonance energy transfer (FRET) between a donor nucleobase analogue and an acceptor nucleobase analogue, base–base FRET, works as a spectroscopic ruler and protractor. With their firm stacking and ability to replace the natural nucleic acid bases inside the base-stack, base analogue donor and acceptor molecules complement external fluorophores like the Cy-, Alexa- and ATTO-dyes and enable detailed investigations of structure and dynamics of nucleic acid containing systems. The first base–base FRET pair, tCO–tCnitro, has recently been complemented with among others the adenine analogue FRET pair, qAN1–qAnitro, increasing the flexibility of the methodology. Here we present the design, synthesis, photophysical characterization and use of such base analogues. They enable a higher control of the FRET orientation factor, κ2, have a different distance window of opportunity than external fluorophores, and, thus, have the potential to facilitate better structure resolution. Netropsin DNA binding and the B-to-Z-DNA transition are examples of structure investigations that recently have been performed using base–base FRET and that are described here. Base–base FRET has been around for less than a decade, only in 2017 expanded beyond one FRET pair, and represents a highly promising structure and dynamics methodology for the field of nucleic acids. Here we bring up its advantages as well as disadvantages and touch upon potential future applications.