Natural Nucleic Acids Research Articles

Anti-syn Unnatural Base Pair Enables Alphabet-Expanded DNA Self-Assembly.

Self-assembly properties and diversity in higher-order structures of DNA enable programmable tools to be used to construct algorithms at the molecular level. However, the utility of DNA-based programmable tools is hampered by the low orthogonality to natural nucleic acids, especially in complex molecular systems. To address this challenge, we report here the orthogonal regulation of DNA self-assembly by using an unnatural base pair (UBP) formation. Our newly designed UBP AnN:SyN is formed in combination with anti and unusual syn glycosidic conformation with high thermal stability and selectivity. Furthermore, AnC worked as a pH-sensitive artificial nucleobase, which forms a strong base pair with cytosine under a weak acidic condition (pH 6.0). The orthogonal AnN:SyN base pair functioned as a trigger for hybridization chain reaction to provide long nicked double-stranded DNA (ca. 1000 base pairs). This work represents the first example of the orthogonal DNA self-assembly that is nonreactive to natural four-letter alphabets DNA trigger and expands the types of programmable tools that work in a complex environment.

Logic devices based on nucleic acid self‐assembly

AbstractNucleic acids are natural macromolecules with the ability to store and transmit information based on the strict base‐pairing principle. Beyond the natural nucleic acid double helixes, various DNA/RNA nanostructures with customized geometries and functionalities have been fabricated. Featured with programmability and sequence‐dependent responsiveness, DNA/RNA nanostructures have been employed for the rational design and construction of logic devices. When stimulated by internal molecular triggers and/or external stimuli, these logic gate devices can operate at nanoscale level in complex biological environments, performing logic operations and producing corresponding outputs. In this minireview, we summarize the recent advances of nucleic acid logic devices, which are responsive to various stimuli, including DNA/RNA strands, metal ions, small molecules, peptides, proteins, photo‐irradiation, pH changes, and so forth. The applications of these devices in biosensing and biofunction regulation are also included. In the last part of the present study, we discuss the remaining challenges and perspectives of nucleic acid logic devices.image

Novel peptide (RATH) mediated delivery of peptide nucleic acids for antiviral interventions.

The peptide nucleic acid (PNA) is a chimeric molecule with the nucleobases connected by peptide bonds. This chimeric nature gives the PNA certain therapeutic advantages over natural antisense nucleic acid molecules. The PNA probes are known for its better and stronger complementation with target nucleic acids. However, cellular delivery of PNA is a major hurdle due to the charge-neutral nature of the PNA. For cellular delivery of PNA, peptide-PNA conjugates are used. This approach may face some practical limitation in terms of PNA antisense activity. In this study, we propose a novel RATH-2 peptide-based non-covalent PNA delivery mechanism. We observed RATH-2 shows a favorable molecular interaction with PNA at 16:1 (peptide:PNA) molar ratio resulting in co-centric nanoparticle formation. With this combination, we could achieve as high as 93% cellular delivery of the PNA. The proposed non-covalent RATH:PNA delivery model showed endocytic entrapment free delivery of PNA. The study further demonstrated the therapeutic application of PNA with in vitro antiviral intervention model. Using RATH-2 non-covalent PNA delivery system, we could inhibit 69.5% viral load. The present study demonstrates a cell-penetrating peptide:PNA interaction can lead to nanoparticle formations that facilitated cellular delivery of PNA.Key points• A novel cell-penetrating peptide (RATH-2) was identified for non-covalent delivery of PNA.• RATH-2 and PNA formed co-centric nanoparticles at appropriate molar combination.• PNA delivered through the RATH-2 inhibited the viral gene expression and reduced the viral load.

Natural DNA assisted white light generation and stimuli responsive colour tuning

The unique structure of a natural nucleic acid, calf thymus DNA, which can provide an appropriate scaffold for an efficient cascaded energy transfer among organic chromophores, has been used for the generation of bright and pure white light on UV light excitation. Two most commonly used DNA stains, 4′,6-diamidino-2-phenylindole (DAPI) and ethidium bromide (EB) have been used as a part of the donor-acceptor pairs. We have judiciously selected 10-anthracene-10-yl-3-methylbenzothiazol-3-ium chloride (AnMBTZ), an ultrafast molecular rotor, to act as a bridge between DNA bound DAPI and EB for the cascaded flow of energy. The unique molecular rotor properties of AnMBTZ and its exceptional binding ability with natural DNA help to form a distinct tri-chromophoric system in DNA template which can produce bright and pure white light on UV excitation. Detailed flow of energy from photoexcited DAPI to EB via AnMBTZ has been explored using steady state and time-resolved emission spectroscopy. Further, unique binding nature of AnMBTZ with DNA molecules has been used to modulate the colour of the emission from the present tri-chromophoric system by external stimuli, like salt and temperature. Such unique stimuli responsive multi-chromophoric system in a bio-template has great potential for different lightening applications.

Exogenous Plant gma-miR-159a, Identified by miRNA Library Functional Screening, Ameliorated Hepatic Stellate Cell Activation and Inflammation via Inhibiting GSK-3β-Mediated Pathways.

PurposePlant-derived exogenous microRNAs (miRNAs) regulate human physiological functions by blocking the translation of target mRNAs. Although several computational approaches have been developed to elucidate the interactions of cross-species miRNAs and their targets in mammals, the number of verified plant miRNAs is still limited, and the biological roles of most exogenous plant miRNAs remain unknown.MethodsA miRNA mimic library-based phenotypic screening, which contained 8394 plant mature miRNAs published in the official database miRbase, was performed to identify more novel bioactive plant miRNAs for the prevention of hepatic fibrosis. Inhibition of candidates for the activation of hepatic stellate cells (HSCs) and the underlying mechanisms were evaluated in TGF-β1- and PDGF-exposed HSC models. The protective effects of the candidates against CCl4-induced liver fibrosis were evaluated in a mouse model.ResultsAmong the 8394 plant mature miRNAs reported in the official database miRBase, five candidates were found to effectively inhibit the differentiation of HSCs. gma-miR-159a (miR159a) exerted the strongest inhibitory activities on both TGF-β1- and PDGF-induced HSC activation and proliferation by inhibiting the GSK-3β-mediated NF-κB and TGF-β1 pathways. Moreover, miR159a was mainly accumulated in the liver after intravenous injection, and it reduced CCl4-induced hepatic fibrosis and inflammation in mice.ConclusionResults indicated that miR159a has the therapeutic potential for preventing hepatic fibrosis. This study provides a novel strategy for achieving natural nucleic acid drugs.

Isolation of Natural DNA Aptamers for Challenging Small-Molecule Targets, Cannabinoids.

Aptamers are nucleic acid-based affinity reagents that are isolated via an in vitro process known as systematic evolution of ligands by exponential enrichment (SELEX). Despite their great potential for a wide range of analytical applications, there are relatively few high-quality small-molecule binding aptamers, especially for "challenging" targets that have low water solubility and/or limited moieties for aptamer recognition. The use of libraries containing chemically modified bases may improve the outcome of some SELEX experiments, but this approach is costly and yields inconsistent results. Here, we demonstrate that a thoughtfully designed SELEX procedure with natural DNA libraries can isolate aptamers with high affinity and specificity for challenging small molecules, including targets for which such selections have previously failed. We first isolate a DNA aptamer with nanomolar affinity and high specificity for (-)-trans-Δ9-tetrahydrocannabinol (THC), a target previously thought to be unsuitable for SELEX with natural DNA libraries. We subsequently isolate aptamers that exhibit high affinity and cross-reactivity to two other challenging targets, synthetic cannabinoids UR-144 and XLR-11, while maintaining excellent specificity against a wide range of non-target interferents. Our findings demonstrate that natural nucleic acid libraries can yield high-quality aptamers for small-molecule targets, and we outline a robust workflow for isolating other such aptamers in future selection efforts.

Sulfur modification in natural RNA and therapeutic oligonucleotides.

Sulfur modifications have been discovered on both DNA and RNA. Sulfur substitution of oxygen atoms at nucleobase or backbone locations in the nucleic acid framework led to a wide variety of sulfur-modified nucleosides and nucleotides. While the discovery, regulation and functions of DNA phosphorothioate (PS) modification, where one of the non-bridging oxygen atoms is replaced by sulfur on the DNA backbone, are important topics, this review focuses on the sulfur modification in natural cellular RNAs and therapeutic nucleic acids. The sulfur modifications on RNAs exhibit diversity in terms of modification location and cellular function, but the various sulfur modifications share common biosynthetic strategies across RNA species, cell types and domains of life. The first section reviews the post-transcriptional sulfur modifications on nucleobases with an emphasis on thiouridine on tRNA and phosphorothioate modification on RNA backbones, as well as the functions of the sulfur modifications on different species of cellular RNAs. The second section reviews the biosynthesis of different types of sulfur modifications and summarizes the general strategy for the biosynthesis of sulfur-containing RNA residues. One of the main goals of investigating sulfur modifications is to aid the genomic drug development pipeline and enhance our understandings of the rapidly growing nucleic acid-based gene therapies. The last section of the review focuses on the current drug development strategies employing sulfur substitution of oxygen atoms in therapeutic RNAs.

Recent advances and prospects in the Zn-catalysed Mannich reaction.

Zn-catalysed reactions are ubiquitously important due to their inexpensive, generally less toxic and atom-economic nature. According to the modern criteria of sustainability, their use in a catalytic manner is a highly desirable goal, especially when using chiral ligands. Considering the relevance of well-established zinc-mediated C–C bond formation reactions, it is relatively surprising that the use of Zn as a catalyst is still underdeveloped, especially in comparison with other transition metals. The vast majority of natural molecules, including proteins, nucleic acids and most biologically active compounds, contain nitrogen. Consequently, developing new synthetic methods for the construction of nitrogenous molecules receives great attention from organic chemists. The Mannich reaction is a very basic and very useful platform for the development of several such nitrogen-containing molecules. In this review, we summarise the recent advancements in the Zn-catalysed Mannich reaction, covering the literature from 2011 to 2020.

Nucleic acid-metal complex and its application in atomic-scale manufacturing

Atomic-scale and close-to-atomic scale manufacturing, a frontier hot issue in international academic research, is a cutting-edge manufacturing technique in which atoms are directly used as the manipulation object and atomic-scale structures with specific functions are established to meet the requirements for mass productions. This review focuses on precise atomic-scale manufacturing technology of nucleic acid materials. Firstly, the basic structures and functions of nucleic acid materials are introduced, and the basic principles of the interaction between DNA and metal atoms are discussed. Then the development process and breakthrough progress of nucleic acid materials-mediated precise atomic-scale manufacturing are introduced from the aspects of natural nucleic acid materials, artificial base “molecular elements”, and nucleic acid nanostructures. Finally, the challenges and opportunities in this field are systematically summarized and some suggestions for future development are given.

Evolution of peptide nucleic acid with modifications of its backbone and application in biotechnology.

Peptide nucleic acids (PNAs) are getting prodigious interest currently in the biomedical and diagnostic field as an extremely powerful tool because of their potentiality to hybridize with natural nucleic acids. Although PNA has strong affinity and sequence specificity to DNA/RNA, there is a considerable ongoing effort to further enhance their special chemical and biological properties for potential application in numerous fields, notably in the field of therapeutics. The toolbox for backbone modified PNAs synthesis has been extended substantially in recent decades, providing a more efficient synthesis of peptides with numerous scaffolds and modifications. This paper reviews the various strategies that have been developed so far for the modification of the PNA backbone, challenging the search for new PNA systems with improved chemical and physical properties lacking in the original aegPNA backbone. The various practical issues and limitations of different PNA systems are also summarized. The focus of this review is on the evolution of PNA by its backbone modification to improve the cellular uptake, sequence specificity, and compatibility of PNA to bind to DNA/RNA. Finally, an insight was also gained into major applications of backbone modified PNAs for the development of biosensors.

Intrastrand backbone-nucleobase interactions stabilize unwound right-handed helical structures of heteroduplexes of L-aTNA/RNA and SNA/RNA

Xeno nucleic acids, which are synthetic analogues of natural nucleic acids, have potential for use in nucleic acid drugs and as orthogonal genetic biopolymers and prebiotic precursors. Although few acyclic nucleic acids can stably bind to RNA and DNA, serinol nucleic acid (SNA) and L-threoninol nucleic acid (L-aTNA) stably bind to them. Here we disclose crystal structures of RNA hybridizing with SNA and with L-aTNA. The heteroduplexes show unwound right-handed helical structures. Unlike canonical A-type duplexes, the base pairs in the heteroduplexes align perpendicularly to the helical axes, and consequently helical pitches are large. The unwound helical structures originate from interactions between nucleobases and neighbouring backbones of L-aTNA and SNA through CH–O bonds. In addition, SNA and L-aTNA form a triplex structure via C:G*G parallel Hoogsteen interactions with RNA. The unique structural features of the RNA-recognizing mode of L-aTNA and SNA should prove useful in nanotechnology, biotechnology, and basic research into prebiotic chemistry.

Interaction of the putative anticancer alkaloid chelerythrine with nucleic acids: biophysical perspectives.

Alkaloids represent an important group of molecules that have immense pharmacological potential. Benzophenanthridine alkaloids are one such class of alkaloids known for their myriad pharmacological activities that include potential anticancer activities. Chelerythrine is a premier member of the benzophenanthridine family of the isoquinoline group. This alkaloid is endowed with excellent medicinal properties and exhibits antibacterial, antimicrobial and anti-inflammatory properties. The molecular basis of its therapeutic activity is considered due to its nucleic acid binding capabilities. This review focuses on consolidating the current status on the nucleic acid binding properties of chelerythrine that is essential for the rational design and development of this alkaloid as a potential drug. This work reviews the interaction of chelerythrine with different natural and synthetic nucleic acids like double- and single-stranded DNAs, heat-denatured DNA, quadruplex DNA, double- and single-stranded RNA, tRNA and triplex and quadruplexRNA. The review emphasizes on the mode, specificity, conformational aspects and energetics of the binding that is particularly helpful for developing nucleic acid targeted therapeutics. The fundamental results discussed in this review will greatly benefit drug development for many diseases and serve as a database for the design of futuristic benzophenanthridine-based therapeutics.

Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides.

In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides.

Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging.

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.

Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA-Gapmer Approach.

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a “gapmer” architecture comprised of one zwitterionic central segment (“gap”) containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA‐gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON‐RNA duplexes with base‐pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA‐gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents.

Energy Landscapes of Deoxyxylo- and Xylo-Nucleic Acid Octamers.

Artificial analogues of the natural nucleic acids have attracted interest as a diverse class of information storage molecules capable of self-replication. In this study, we use the computational potential energy landscape framework to investigate the structural and dynamical properties of xylo- and deoxyxylo-nucleic acids (XyNA and dXyNA), which are derived from their respective RNA and DNA analogues by inversion of a single chiral center in the sugar moiety of the nucleotides. For an octameric XyNA sequence and the analogue dXyNA, we observe facile conformational transitions between a left-handed helix, which is the free energy global minimum, and a ladder-type structure with approximately zero helicity. The competing ensembles are better separated in the dXyNA, making it a more suitable candidate for a molecular switch, whereas the XyNA exhibits additional flexibility. Both energy landscapes exhibit greater frustration than we observe in RNA or DNA, in agreement with the higher degree of optimization expected from the principle of minimal frustration in evolved biomolecules.

Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase

All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3'-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3'-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3'→P5' phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3'-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca2+ rather than Mg2+, the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA.

Kinetic and thermodynamic analyses of the corrosion inhibition of synthetic extracellular polymeric substances

Background Extracellular polymeric substances (EPS) extracted from waste activated sludge (WAS) have previously shown its potential in corrosion inhibition. The aim of this study is to design a synthetic EPS formulation as a surrogate of natural WAS EPS to overcome the corrosion inhibition inconsistency in WAS EPS. The adsorption behavior of the designed inhibitor was studied by kinetic and thermodynamic analyses. Methods Synthetic EPS is a bio-inspired material that was formulated based on the most typical chemical compositions of natural WAS EPS, that is, proteins, carbohydrates, humic substances, nucleic acids, and uronic acids, which was not optimized for corrosion inhibition performance. It is a mixture of glutamic acid, carboxymethylcellulose, humic acid, thymine, and alginic acid. Its corrosion inhibition performance was tested with carbon steel in 3.64% NaCl saturated with CO2, using the potentiodynamic polarization scanning technique. The resulted electrochemical parameters were used to evaluate the empirical corrosion kinetic and thermodynamic adsorption parameters. Results Addition of synthetic EPS showed significant decrease in corrosion rate as compared to the control. The inhibition efficiency improved with increasing inhibitor concentration and temperature. The optimum performance was 94% with 204 mg/L of inhibitor applied at 70 °C (343 K). The inhibition performance was controlled by both the concentration of inhibitor and temperature. Chemisorption of the inhibitor molecules contributed to the overall inhibition performance, reducing the contact of metal with the corrosive environment, thus, slowing down the overall corrosion rate.

Cucurbiturils in nucleic acids research.

During the past ten years, the importance of cucurbiturils (CB[n]) as macrocyclic hosts in supramolecular assemblies with various types of natural and synthetic nucleic acids (NAs) has increased explosively. As a component of such systems, CB[n] macrocycles can play a wide spectrum of roles from drug and gene delivery vehicles to catalysts/inhibitors of biochemical reactions and even building blocks for NA-based materials. The aim of this highlight article is to describe the development of the CB[n] applications in nucleic acids research and to outline the current situation and perspectives of this fascinating synergistic combination of supramolecular chemistry of CB[n] and NAs.

Simultaneous fluorescent detection of multiplexed miRNA of liver cancer based on DNA tetrahedron nanotags

The level of miRNA-21, miRNA-122, and miRNA-223 are always elevated when liver cancer is present at an early stage. In this paper, a novel assay to simultaneous detect miRNA-21, miRNA-122, and miRNA-223 was proposed based on DNA tetrahedron nanotags and fluorescence resonance energy transfer (FRET), which used a single laser stimulate wavelengh from one nucleic acid stain TOTO-1 to three diverse organic dyes (Cy3, Cy3.5, Cy5). In brief, a DNA tetrahedral nanostructure (DTN) was designed with three adaptor oligos on its vertices. TOTO-1, as a fluorescent donor, can imbed into native nucleic acid backbone of DTN. Three organic dye-functionalized strands (FRET oligos) are fluorescent receptors. In the presence of target miRNAs, they can be hybridized with FRET oligos and adaptor oligos on the vertices of DTN and the stable DNA tetrahedron nanotags are formed. As a result, the confinement of TOTO-1 is in close proximity to three fluorescence dyes, the FRET between TOTO-1 and three fluorescence dyes is generate efficiently in DNA tetrahedron nanotags. Point-of-care clinical applicability is demonstrated by sensitive multiplexed quantification of three miRNAs in 10% human serum samples.