Natural Nucleic Acids Research Articles

Functional nucleic acid-based biosensors for virus detection

A growing number of viruses pose significant threats to human health, impact agricultural production and compromise food safety. Minimizing the impact of viral infections demands for effective technologies that can deliver sensitive viral detection under various settings. Functional nucleic acids (FNAs) – natural or synthetic nucleic acid molecules with catalytic and/or binding properties – have been increasingly explored as key components for setting up diagnostic tests for viral detection. FNA-based viral biosensors offer distinct advantages, such as the ability to achieve sensitive viral detection in complex biological samples, ease of use, scalability, and cost-effectiveness. This mini-review will be dedicated to appraising progress made in recent years in FNA-based biosensors for diagnosing diverse human and animal viruses. We will first describe virus structures and potential biomarkers that can be utilized as the targets for FNA probe engineering. This is followed by reviewing the latest advances in two classes of FNAs - DNAzyme and DNA aptamers, with focuses on DNAzyme- and DNA aptamer-based biosensors that employ diverse signal transduction mechanisms to achieve sensitive viral detections. Finally, we will discuss current challenges in the field and suggest future research directions that can address these challenges.

Synthesis of a glycan hairpin

The primary sequence of a biopolymer encodes the essential information for folding, permitting to carry out sophisticated functions. Inspired by natural biopolymers, peptide and nucleic acid sequences have been designed to adopt particular three-dimensional (3D) shapes and programmed to exert specific functions. In contrast, synthetic glycans capable of autonomously folding into defined 3D conformations have so far not been explored owing to their structural complexity and lack of design rules. Here we generate a glycan that adopts a stable secondary structure not present in nature, a glycan hairpin, by combining natural glycan motifs, stabilized by a non-conventional hydrogen bond and hydrophobic interactions. Automated glycan assembly enabled rapid access to synthetic analogues, including site-specific 13C-labelled ones, for nuclear magnetic resonance conformational analysis. Long-range inter-residue nuclear Overhauser effects unequivocally confirmed the folded conformation of the synthetic glycan hairpin. The capacity to control the 3D shape across the pool of available monosaccharides has the potential to afford more foldamer scaffolds with programmable properties and functions.

A Novel Approach for Screening Sericin-Derived Therapeutic Peptides Using Transcriptomics and Immunoprecipitation.

With the demand for more efficient and safer therapeutic drugs, targeted therapeutic peptides are well received due to their advantages of high targeting (specificity), low immunogenicity, and minimal side effects. However, the conventional methods of screening targeted therapeutic peptides in natural proteins are tedious, time-consuming, less efficient, and require too many validation experiments, which seriously restricts the innovation and clinical development of peptide drugs. In this study, we established a novel method of screening targeted therapeutic peptides in natural proteins. We also provide details for library construction, transcription assays, receptor selection, therapeutic peptide screening, and biological activity analysis of our proposed method. This method allows us to screen the therapeutic peptides TS263 and TS1000, which have the ability to specifically promote the synthesis of the extracellular matrix. We believe that this method provides a reference for screening other drugs in natural resources, including proteins, peptides, fats, nucleic acids, and small molecules.

Site-Specific Covalent Labeling of DNA Substrates by an RNA Transglycosylase.

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.

Modern semiempirical electronic structure methods and machine learning potentials for drug discovery: Conformers, tautomers, and protonation states.

Modern semiempirical electronic structure methods have considerable promise in drug discovery as universal "force fields" that can reliably model biological and drug-like molecules, including alternative tautomers and protonation states. Herein, we compare the performance of several neglect of diatomic differential overlap-based semiempirical (MNDO/d, AM1, PM6, PM6-D3H4X, PM7, and ODM2), density-functional tight-binding based (DFTB3, DFTB/ChIMES, GFN1-xTB, and GFN2-xTB) models with pure machine learning potentials (ANI-1x and ANI-2x) and hybrid quantum mechanical/machine learning potentials (AIQM1 and QDπ) for a wide range of data computed at a consistent ωB97X/6-31G* level of theory (as in the ANI-1x database). This data includes conformational energies, intermolecular interactions, tautomers, and protonation states. Additional comparisons are made to a set of natural and synthetic nucleic acids from the artificially expanded genetic information system that has important implications for the design of new biotechnology and therapeutics. Finally, we examine the acid/base chemistry relevant for RNA cleavage reactions catalyzed by small nucleolytic ribozymes, DNAzymes, and ribonucleases. Overall, the hybrid quantum mechanical/machine learning potentials appear to be the most robust for these datasets, and the recently developed QDπ model performs exceptionally well, having especially high accuracy for tautomers and protonation states relevant to drug discovery.

Hybrid extracellular vesicles for drug delivery

Extracellular vesicles (EVs) are expected to serve as interesting drug delivery vectors as they may offer unique and new properties for drug delivery. Their natural origin, protein and nucleic acid composition, and intrinsic pleiotropic therapeutic effects could enable new possibilities in the field of drug delivery. Here, we aimed to review the methods used to produce Hybrid EVs, a recently emerged type of EV-based vector made from both EVs and synthetic vectors to exploit their respective properties. Hybrid EV/synthetic objects can be obtained by incubation, electrostatic interactions, polyethylene glycol (PEG)-mediated fusion, co-extrusion, freeze-thawing, or simple EV surface modification, leading to different types of objects. We also opted to review the properties of these vectors, and specifically compared them with those of other drug delivery vectors. It has to be noticed that only a limited number of study report loading metrics that allow cross article comparison. Based on this critical analysis, we attempted to draw the pith and marrow from these relatively difficult-to-compare studies and integrate them into the more general context of opportunities in drug delivery and drug development, with a particular focus on oncology.

Development of Nitroso-Based Probes for Labeling and Regulation of RAS Proteins in Cancer Cells via Sequential Ene-Ligation and Oxime Condensation.

Prenyl functionalities have been widely discovered in natural products, nucleic acids, and proteins with significant biological roles in both healthy and diseased cells. In this work, we develop a series of new nitroso-based probes for the labeling, enrichment, and regulation of prenylated RAS protein, which is highly associated with ∼20% of human cancers and used to be regarded as an "undruggable" target via a sequential ene-ligation and oxime condensation (SELOC) process. We found that these nitroso species can rapidly react with prenyl-containing molecules through ene-ligation and install a molecular tag for functional applications under physiological conditions. We first investigated this ligation process on two peptide models and demonstrated its labeling efficiency on various proteins such as myoglobin, lysozyme, RNase A, BSA, and HSP40. We further coupled this reactive platform with proteolysis-targeting chimera technology targeting to increase its efficiency and accuracy, as well as to expand its application range. Using the prenylated RAS protein as the model, we demonstrated that RAS could be efficiently decorated with our nitroso probes, which further condensate with oxime and rapidly react with a pomalidomide-containing hydroxylamine probe for protein degradation. As a result, the RAS protein in both HeLa and A549 cell lines has been determined to be efficiently degraded both in vitro and in vivo. This is the first case targeting post-translational modification other than ligand-protein interaction to degrade and regulate RAS proteins. We envision that our SELOC strategy will have great potential in studying the fundamental structures and functions of prenylated biomolecules and developing new drugs based on these unique cellular pathways.

Virus-like Particles for TEM Regulation and Antitumor Therapy.

Tumor development and metastasis are intimately associated with the tumor microenvironment (TME), and it is difficult for vector-restricted drugs to act on the TME for long-term cancer immunotherapy. Virus-like particles (VLPs) are nanocage structures self-assembled from nucleic acid free viral proteins. Most VLPs range from 20-200 nm in diameter and can naturally drain into lymph nodes to induce robust humoral immunity. As natural nucleic acid nanocarriers, their surfaces can also be genetically or chemically modified to achieve functions such as TME targeting. This review focuses on the design ideas of VLP as nanocarriers and the progress of their research in regulating TME.

Design, Synthesis and Actual Applications of the Polymers Containing Acidic P-OH Fragments: Part 1. Polyphosphodiesters.

Among natural and synthetic polymers, main-chain phosphorus-containing polyacids (PCPAs) (polyphosphodiesters), stand in a unique position at the intersection of chemistry, physics, biology and medicine. The structural similarity of polyphosphodiesters PCPAs to natural nucleic and teichoic acids, their biocompatibility, mimicking to biomolecules providing the 'stealth effect', high bone mineral affinity of polyphosphodiesters resulting in biomineralization at physiological conditions, and adjustable hydrolytic stability of polyphosphodiesters are the basis for various biomedical, industrial and household applications of this type of polymers. In the present review, we discuss the synthesis, properties and actual applications of polyphosphodiesters.

DNA-PAINT Super-Resolution Imaging for Characterization of Nucleic Acid Nanostructures.

Numerous nucleic acid nanostructures of unique addressability and programmability have been fabricated for emerging applications. Structural characterization with atomic force microscopy and electron microscopy can provide information on the structural morphology and precision of these nanostructures. However, either structural information of native nucleic acid nanostructures in hydrated environment or the availability of addressable sites on these nanostructures could not be determined. Alternatively, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) enables direct optical visualization of nucleic acid nanostructures in native forms, as well as evaluation of the accessibility of addressable sites on them. In this Review, the working principle of DNA-PAINT is introduced, followed by the summary on advances of DNA-PAINT characterization of various nucleic acid nanostructures. Finally, the current challenges and prospects for DNA-PAINT characterization are presented. We envision DNA-PAINT to be a potent characterization tool for functional nanomaterials.

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.

Sequence-Specific Recognition of Double-Stranded DNA by Peptide Nucleic Acid Forming Double-Duplex Invasion Complex

Peptide nucleic acid (PNA) is an analog of natural nucleic acids, where the sugar-phosphate backbone of DNA is replaced by an electrostatically neutral N-(2-aminoethyl)glycine backbone. This unique peptide-based backbone enables PNAs to form a very stable duplex with the complementary nucleic acids via Watson–Crick base pairing since there is no electrostatic repulsion between PNA and DNA·RNA. With this high nucleic acid affinity, PNAs have been used in a wide range of fields, from biological applications such as gene targeting, to engineering applications such as probe and sensor developments. In addition to single-stranded DNA, PNA can also recognize double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is hard to achieve with other artificial nucleic acids and is expected to be a promising method to recognize dsDNA in cellula or in vivo since the invasion does not require the prior denaturation of dsDNA. In this paper, we provide basic knowledge of PNA and mainly focus on the research of PNA invasion.

Halogen Bonds Exist between Noncovalent Ligands and Natural Nucleic Acids.

Because of their strong electron-rich properties, nucleic acids (NAs) can theoretically serve as halogen bond (XB) acceptors. From a PDB database survey, Kolář found that no XBs are formed between noncovalent ligands and NAs. Through statistical database analysis, quantum-mechanics/molecular-mechanics (QM/MM) optimizations, and energy calculations, we find that XBs formed between natural NAs and noncovalent ligands are primarily underestimated and that NAs can serve as XB acceptors to interact with noncovalent halogen ligands. Finally, through energy calculations, natural bond orbital analysis, and noncovalent interaction analysis, XBs are confirmed in 13 systems, among which two systems (445D and 4Q9Q) have relatively strong XBs. In addition, on the basis of energy scanning of four model systems, we explore the geometric rule for XB formation in NAs. This work will inspire researchers to utilize XBs in rational drug design targeting NAs.

Hoechst 33258 aggregation and binding to DNA studied by surface‐enhanced Raman spectroscopy

AbstractInteractions of Hoechst 33258 (H33258) with two synthetic DNA polynucleotides, poly dA–poly dT and poly dG–poly dC, as well as natural calf thymus (ct) DNA were studied using surface‐enhanced Raman spectroscopy (SERS). Given variety of stoichiometry dependent interactions, complexes were prepared in [H33258]/[DNA] molar ratios of 1/1, 1/5, and 1/10 in the silver colloidal suspension. Prior to analysis of binding modes with DNA polynucleotides, aggregation tendency of the dye in the silver colloid was studied in the concentration range 1 × 10−6–1 × 10−4 mol/L. The SERS spectra distinctive of aggregated and monomeric H33258 species were obtained, additionally indicating the form of the molecules bound to DNA. The obtained SERS spectra of H33258 with homo‐polymeric polynucleotides implied a deep insertion of the molecules into the AT minor groove, and a partial intercalation between the GC base pairs achieved by the access through the GC major groove. Spectral characteristics of the dye dominated in the spectra of the complexes with ct‐DNA, most likely due to random AT and GC sequences of the long natural nucleic acid, affecting uniform binding of the dye and consequently the adsorption of the H33258/DNA complex onto the enhancing metallic surface.

Nucleic Acids and Their Analogues for Biomedical Applications.

Nucleic acids are emerging as powerful and functional biomaterials due to their molecular recognition ability, programmability, and ease of synthesis and chemical modification. Various types of nucleic acids have been used as gene regulation tools or therapeutic agents for the treatment of human diseases with genetic disorders. Nucleic acids can also be used to develop sensing platforms for detecting ions, small molecules, proteins, and cells. Their performance can be improved through integration with other organic or inorganic nanomaterials. To further enhance their biological properties, various chemically modified nucleic acid analogues can be generated by modifying their phosphodiester backbone, sugar moiety, nucleobase, or combined sites. Alternatively, using nucleic acids as building blocks for self-assembly of highly ordered nanostructures would enhance their biological stability and cellular uptake efficiency. In this review, we will focus on the development and biomedical applications of structural and functional natural nucleic acids, as well as the chemically modified nucleic acid analogues over the past ten years. The recent progress in the development of functional nanomaterials based on self-assembled DNA-based platforms for gene regulation, biosensing, drug delivery, and therapy will also be presented. We will then summarize with a discussion on the advanced development of nucleic acid research, highlight some of the challenges faced and propose suggestions for further improvement.

Recovery of Information Stored in Modified DNA with an Evolved Polymerase

DNA is increasingly being explored as an alternative medium for digital information storage, but the potential information loss from degradation and associated issues with error during reading challenge its wide-scale implementation. To address this, we propose an atomic-scale encoding standard for DNA, where information is encoded in degradation-resistant analogues of natural nucleic acids (xNAs). To better enable this approach, we used directed evolution to create a polymerase capable of transforming 2'-O-methyl templates into double-stranded DNA. Starting from a thermophilic, error-correcting reverse transcriptase, RTX, we evolved an enzyme (RTX-Ome v6) that relies on a fully functional proofreading domain to correct mismatches on DNA, RNA, and 2'-O-methyl templates. In addition, we implemented a downstream analysis strategy that accommodates deletions that arise during phosphoramidite synthesis, the most common type of synthesis error. By coupling and integrating new chemistries, enzymes, and algorithms, we further enable the large-scale use of nucleic acids for information storage.

Stimuli-responsive assembly of bilingual peptide nucleic acids.

Peptide nucleic acids (PNAs) are high-affinity synthetic nucleic acid analogs capable of hybridization with native nucleic acids. PNAs synthesized having amino acid sidechains installed at the γ-position along the backbone provide a template for a single biopolymer to simultaneously encode nucleic acid and amino acid sequences. Previously, we reported the development of “bilingual” PNAs through the synthesis of an amphiphilic sequence featuring separate blocks of hydrophobic and hydrophilic amino acid functional groups. These PNAs combined the sequence-specific binding activity of nucleic acids with the structural organization properties of peptides. Like other amphiphilic compounds, these γ-PNAs were observed to assemble spontaneously into micelle-like nanostructures in aqueous solutions and disassembly was induced through hybridization to a complementary sequence. Here, we explore whether assembly of these bilingual PNAs is possible by harnessing the nucleic acid code. Specifically, we designed an amphiphile-masking duplex system in which spontaneous amphiphile assembly is prevented through hybridization to a nucleic acid masking sequence. We show that the amphiphile is displaced upon introduction of a releasing sequence complementary to the masking sequence through toehold mediated displacement. Upon release, we observe that the amphiphile proceeds to assemble in a fashion consistent with our previously reported structures. Our approach represents a novel method for controlled stimuli-responsive assembly of PNA-based nanostructures.

Perspectives on conformationally constrained peptide nucleic acid (PNA): insights into the structural design, properties and applications.

Peptide nucleic acid or PNA is a synthetic DNA mimic that contains a sequence of nucleobases attached to a peptide-like backbone derived from N-2-aminoethylglycine. The semi-rigid PNA backbone acts as a scaffold that arranges the nucleobases in a proper orientation and spacing so that they can pair with their complementary bases on another DNA, RNA, or even PNA strand perfectly well through the standard Watson–Crick base-pairing. The electrostatically neutral backbone of PNA contributes to its many unique properties that make PNA an outstanding member of the xeno-nucleic acid family. Not only PNA can recognize its complementary nucleic acid strand with high affinity, but it does so with excellent specificity that surpasses the specificity of natural nucleic acids and their analogs. Nevertheless, there is still room for further improvements of the original PNA in terms of stability and specificity of base-pairing, direction of binding, and selectivity for different types of nucleic acids, among others. This review focuses on attempts towards the rational design of new generation PNAs with superior performance by introducing conformational constraints such as a ring or a chiral substituent in the PNA backbone. A large collection of conformationally rigid PNAs developed during the past three decades are analyzed and compared in terms of molecular design and properties in relation to structural data if available. Applications of selected modified PNA in various areas such as targeting of structured nucleic acid targets, supramolecular scaffold, biosensing and bioimaging, and gene regulation will be highlighted to demonstrate how the conformation constraint can improve the performance of the PNA. Challenges and future of the research in the area of constrained PNA will also be discussed.

The Potential Contribution of Biopolymeric Particles in Lung Tissue Regeneration of COVID-19 Patients.

The lung is a vital organ that houses the alveoli, which is where gas exchange takes place. The COVID-19 illness attacks lung cells directly, creating significant inflammation and resulting in their inability to function. To return to the nature of their job, it may be essential to rejuvenate the afflicted lung cells. This is difficult because lung cells need a long time to rebuild and resume their function. Biopolymeric particles are the most effective means to transfer developing treatments to airway epithelial cells and then regenerate infected lung cells, which is one of the most significant symptoms connected with COVID-19. Delivering biocompatible and degradable natural biological materials, chemotherapeutic drugs, vaccines, proteins, antibodies, nucleic acids, and diagnostic agents are all examples of these molecules‘ usage. Furthermore, they are created by using several structural components, which allows them to effectively connect with these cells. We highlight their most recent uses in lung tissue regeneration in this review. These particles are classified into three groups: biopolymeric nanoparticles, biopolymeric stem cell materials, and biopolymeric scaffolds. The techniques and processes for regenerating lung tissue will be thoroughly explored.

Novel insights on nucleopeptide binding: A spectroscopic and in silico investigation on the interaction of a thymine-bearing tetrapeptide with a homoadenine DNA

Nucleopeptides are a class of molecules with numerous applications in the field of therapy, diagnostics and biomaterials development. Despite their nucleobase-decorated nature, their binding to natural nucleic acid targets does not necessarily involve all nucleopeptide bases, as we showed in this study. Here, we present a CD study on the interaction of a dithymine-functionalized tetra-L-serine with a homoadenine DNA (dA12) reporting an interpretation of the experimental data in light of our computational studies based on molecular docking and molecular dynamics (MD), as well as computer-assisted CD interpretation and simulation of the predicted complex structure. The stoichiometry of the complex, emerged by CD titration, accounted for a 1:2 T:A ratio. Hence, we supposed that binding did not involve a full pairing of the complementary bases but a partial thymines engagement. This hypothesis was sustained by the docking and MD simulations performed on the selected ligand and the complementary target of DNA and RNA, used for comparison. The nucleopeptide bound the DNA through a single A-T recognition involving complementary base-pairing, as well as by some interactions between its backbone (and in particular L-serine OH) and the nucleic acid. Overall, this confirmed that nucleopeptides can interact with nucleic acids leaving some of their nucleobases free for establishing further interactions with other biomolecules or for crosslinking in supramolecular structures in aqueous solution. Nevertheless, even though no typical DNA secondary structure is formed after nucleopeptide-binding, this ligand is able to induce a higher degree of structuration in the random deoxyoligonucleotide target as evidenced by CD, MD and CD simulation.