A Visual Compendium of Principal Modifications within the Nucleic Acid Sugar Phosphate Backbone.

Nucleic acid chemistry is a huge research area that has received new impetus due to the recent explosive success of oligonucleotide therapy. In order for an oligonucleotide to become clinically effective, its monomeric parts are subjected to modifications. Although a large number of redesigned natural nucleic acids have been proposed in recent years, the vast majority of them are combinations of simple modifications proposed over the past 50 years. This review is devoted to the main modifications of the sugar phosphate backbone of natural nucleic acids known to date. Here, we propose a systematization of existing knowledge about modifications of nucleic acid monomers and an acceptable classification from the point of view of chemical logic. The visual representation is intended to inspire researchers to create a new type of modification or an original combination of known modifications that will produce unique oligonucleotides with valuable characteristics.

The Application of Microsampling Disks in Circular Dichroism Spectroscopy for Peptide and Nucleic Acid Drugs.

In recent years, there has been a growing focus on the development of medium-sized drugs based on peptides or nucleic acids owing to their potential therapeutic benefits. As some of these medium-sized drugs exert their therapeutic effects by adopting specific secondary structures, evaluating their conformational states is crucial to ensure the efficacy, quality, and safety of the drug products. It is important to assess the structural integrity of biomolecular therapeutics to guarantee their intended pharmacological activity and maintain the required standards for drug development and manufacturing. One widely utilized technique for quality evaluation is secondary structural analysis using circular dichroism (CD) spectroscopy. Given the higher production and quality control costs associated with medium-sized drugs compared with small-molecule drugs, developing analytical techniques that enable CD analysis with reduced sample volumes is highly desirable. Herein, we focused on a microsampling disk-type cell as a potential solution for reducing the required sample volume. We investigated whether CD spectral analysis using a microsampling disk could provide equivalent spectra compared with the standard cell (sample volume: approx. 300 µL). Our findings demonstrated that the microsampling disk (sample volume: 2-10 µL) could be successfully applied to CD spectral analysis of peptide and nucleic acid drugs, paving the way for more efficient and cost-effective quality evaluation processes.

Force Field X: A computational microscope to study genetic variation and organic crystals using theory and experiment.

Force Field X (FFX) is an open-source software package for atomic resolution modeling of genetic variants and organic crystals that leverages advanced potential energy functions and experimental data. FFX currently consists of nine modular packages with novel algorithms that include global optimization via a many-body expansion, acid-base chemistry using polarizable constant-pH molecular dynamics, estimation of free energy differences, generalized Kirkwood implicit solvent models, and many more. Applications of FFX focus on the use and development of a crystal structure prediction pipeline, biomolecular structure refinement against experimental datasets, and estimation of the thermodynamic effects of genetic variants on both proteins and nucleic acids. The use of Parallel Java and OpenMM combines to offer shared memory, message passing, and graphics processing unit parallelization for high performance simulations. Overall, the FFX platform serves as a computational microscope to study systems ranging from organic crystals to solvated biomolecular systems.

Recent advances in gene delivery nanoplatforms based on spherical nucleic acids.

Gene therapy is a therapeutic option for mitigating diseases that do not respond well to pharmacological therapy. This type of therapy allows for correcting altered and defective genes by transferring nucleic acids to target cells. Notably, achieving a desirable outcome is possible by successfully delivering genetic materials into the cell. In-vivo gene transfer strategies use two major classes of vectors, namely viral and nonviral. Both of these systems have distinct pros and cons, and the choice of a delivery system depends on therapeutic objectives and other considerations. Safe and efficient gene transfer is the main feature of any delivery system. Spherical nucleic acids (SNAs) are nanotechnology-based gene delivery systems (i.e., non-viral vectors). They are three-dimensional structures consisting of a hollow or solid spherical core nanoparticle that is functionalized with a dense and highly organized layer of oligonucleotides. The unique structural features of SNAs confer them a high potency in internalization into various types of tissue and cells, a high stability against nucleases, and efficay in penetrating through various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier). SNAs also show negligible toxicity and trigger minimal immune response reactions. During the last two decades, all these favorable physicochemical and biological attributes have made them attractive vehicles for drug and nucleic acid delivery. This article discusses the unique structural properties, types of SNAs, and also optimization mechanisms of SNAs. We also focus on recent advances in the synthesis of gene delivery nanoplatforms based on the SNAs.

A Repurposed Drug Interferes with Nucleic Acid to Inhibit the Dual Activities of Coronavirus Nsp13.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.

Lipoplexes' Structure, Preparation, and Role in Managing Different Diseases.

Lipid-based vectors are becoming promising alternatives to traditional therapies over the last 2 decades specially for managing life-threatening diseases like cancer. Cationic lipids are the most prevalent non-viral vectors utilized in gene delivery. The increasing number of clinical trials about lipoplex-based gene therapy demonstrates their potential as well-established technology that can provide robust gene transfection. In this regard, this review will summarize this important point. These vectors however have a modest transfection efficiency. This limitation can be partly addressed by using functional lipids that provide a plethora of options for investigating nucleic acid-lipid interactions as well as in vitro and in vivo nucleic acid delivery for biomedical applications. Despite their lower gene transfer efficiency, lipid-based vectors such as lipoplexes have several advantages over viral ones: they are less toxic and immunogenic, can be targeted, and are simple to produce on a large scale. Researchers are actively investigating the parameters that are essential for an effective lipoplex delivery method. These include factors that influence the structure, stability, internalization, and transfection of the lipoplex. Thorough understanding of the design principles will enable synthesis of customized lipoplex formulations for life-saving therapy.

Molecular Docking Approach for Biological Interaction of Green Synthesized Nanoparticles.

In recent years, significant progress has been made in the subject of nanotechnology, with a range of methods developed to synthesize precise-sized and shaped nanoparticles according to particular requirements. Often, the nanoparticles are created by employing dangerous reducing chemicals to reduce metal ions into uncharged nanoparticles. Green synthesis or biological approaches have been used recently to circumvent this issue because biological techniques are simple, inexpensive, safe, clean, and extremely productive. Nowadays, much research is being conducted on how different kinds of nanoparticles connect to proteins and nucleic acids using molecular docking models. Therefore, this review discusses the most recent advancements in molecular docking capacity to predict the interactions between various nanoparticles (NPs), such as ZnO, CuO, Ag, Au, and Fe3O4, and biological macromolecules.

Probing of nucleic acid compaction using low-frequency Raman spectroscopy.

Compaction of nucleic acids, namely DNA and RNA, determines their functions and involvement in vital cell processes including transcription, replication, DNA repair and translation. However, experimental probing of the compaction of nucleic acids is not straightforward. In this study, we suggest an approach for this probing using low-frequency Raman spectroscopy. Specifically, we show theoretically, computationally and experimentally the quantifiable correlation between the low-frequency Raman intensity from nucleic acids, magnitude of thermal fluctuations of atomic positions, and the compaction state of biomolecules. Noteworthily, we highlight that the LF Raman intensity differs by an order of magnitude for different samples of DNA, and even for the same sample in the course of long-term storage. The feasibility of the approach is further shown by assessment of the DNA compaction in the nuclei of plant cells. We anticipate that the suggested approach will enlighten compaction of nucleic acids and their dynamics during the key processes of the cell life cycle and under various factors, facilitating advancement of molecular biology and medicine.

DNA Anchoring Strength Directly Correlates with Spherical Nucleic Acid-Based HPV E7 Cancer Vaccine Potency.

Vaccination for cancers arising from human papillomavirus (HPV) infection holds immense potential, yet clinical success has been elusive. Herein, we describe vaccination studies involving spherical nucleic acids (SNAs) incorporating a CpG adjuvant and a peptide antigen (E711-19) from the HPV-E7 oncoprotein. Administering the vaccine to humanized mice induced immunity-dependent on the oligonucleotide anchor chemistry (cholesterol vs (C12)9). SNAs containing a (C12)9-anchor enhanced IFN-γ production >200-fold, doubled memory CD8+ T-cell formation, and delivered more than twice the amount of oligonucleotide to lymph nodes in vivo compared to a simple admixture. Importantly, the analogous construct with a weaker cholesterol anchor performed similar to admix. Moreover, (C12)9-SNAs activated 50% more dendritic cells and generated T-cells cytotoxic toward an HPV+ cancer cell line, UM-SCC-104, with near 2-fold greater efficiency. These observations highlight the pivotal role of structural design, and specifically oligonucleotide anchoring strength (which correlates with overall construct stability), in developing efficacious therapeutic vaccines.

SOFB is a comprehensive ensemble deep learning approach for elucidating and characterizing protein-nucleic-acid-binding residues.

Proteins and nucleic-acids are essential components of living organisms that interact in critical cellular processes. Accurate prediction of nucleic acid-binding residues in proteins can contribute to a better understanding of protein function. However, the discrepancy between protein sequence information and obtained structural and functional data renders most current computational models ineffective. Therefore, it is vital to design computational models based on protein sequence information to identify nucleic acid binding sites in proteins. Here, we implement an ensemble deep learning model-based nucleic-acid-binding residues on proteins identification method, called SOFB, which characterizes protein sequences by learning the semantics of biological dynamics contexts, and then develop an ensemble deep learning-based sequence network to learn feature representation and classification by explicitly modeling dynamic semantic information. Among them, the language learning model, which is constructed from natural language to biological language, captures the underlying relationships of protein sequences, and the ensemble deep learning-based sequence network consisting of different convolutional layers together with Bi-LSTM refines various features for optimal performance. Meanwhile, to address the imbalanced issue, we adopt ensemble learning to train multiple models and then incorporate them. Our experimental results on several DNA/RNA nucleic-acid-binding residue datasets demonstrate that our proposed model outperforms other state-of-the-art methods. In addition, we conduct an interpretability analysis of the identified nucleic acid binding residue sequences based on the attention weights of the language learning model, revealing novel insights into the dynamic semantic information that supports the identified nucleic acid binding residues. SOFB is available at https://github.com/Encryptional/SOFB and https://figshare.com/articles/online_resource/SOFB_figshare_rar/25499452 .

Nucleic acid-responsive smart systems for controlled cargo delivery.

Stimulus-responsive delivery systems allow controlled, highly regulated, and efficient delivery of various cargos while minimizing side effects. Owing to the unique properties of nucleic acids, including the ability to adopt complex structures by base pairing, their easy synthesis, high specificity, shape memory, and configurability, they have been employed in autonomous molecular motors, logic circuits, reconfigurable nanoplatforms, and catalytic amplifiers. Moreover, the development of nucleic acid (NA)-responsive intelligent delivery vehicles is a rapidly growing field. These vehicles have attracted much attention in recent years due to their programmable, controllable, and reversible properties. In this work, we review several types of NA-responsive controlled delivery vehicles based on locks and keys, including DNA/RNA-responsive, aptamer-responsive, and CRISPR-responsive, and summarize their advantages and limitations.

Detecting 2'-5'-adenosine linked nucleic acids via acylation of secondary hydroxy functionality.

2'-5'-Adenosine linked nucleic acids are crucial components in living cells that play significant roles, including participating in antiviral defense mechanisms by facilitating the breakdown of viral genetic material. In this report, we present a chemical derivatization method employing 5-fluoro-2-pyridinoyl-imidazole as the acylation agent, a strategy that can be effectively combined with advanced analytical tools, including Nuclear Magnetic Resonance spectroscopy and Liquid Chromatography-Mass Spectrometry, to enhance the characterization and detection capabilities. This marks the first instance of a simple method designed to detect 2'-5'-adenosine linked nucleic acids. The new method is characterized by its time-saving nature, simplicity, and relative accuracy compared to previous methods.

High-content tailoring strategy to improve the multifunctionality of functional nucleic acids.

Functional nucleic acids (FNAs) have attracted increasing attention in recent years due to their diverse physiological functions. The understanding of their conformational recognition mechanisms has advanced through nucleic acid tailoring strategies and sequence optimization. With the development of the FNA tailoring techniques, they have become a methodological guide for nucleic acid repurposing. Therefore, it is necessary to systematize the relationship between FNA tailoring strategies and the development of nucleic acid multifunctionality. This review systematically categorizes eight types of FNA multifunctionality, and introduces the traditional FNA tailoring strategy from five aspects, including deletion, substitution, splitting, fusion and elongation. Based on the current state of FNA modification, a new generation of FNA tailoring strategy, called the high-content tailoring strategy, was unprecedentedly proposed to improve FNA multifunctionality. In addition, the multiple applications of rational tailoring-driven FNA performance enhancement in various fields were comprehensively summarized. The limitations and potential of FNA tailoring and repurposing in the future are also explored in this review. In summary, this review introduces a novel tailoring theory, systematically summarizes eight FNA performance enhancements, and provides a systematic overview of tailoring applications across all categories of FNAs. The high-content tailoring strategy is expected to expand the application scenarios of FNAs in biosensing, biomedicine and materials science, thus promoting the synergistic development of various fields.

Split T7 switch-mediated cell-free protein synthesis system for detecting target nucleic acids.

Cell-free protein synthesis (CFPS) reactions can be used to detect nucleic acids. However, most CFPS systems rely on a toehold switch and exhibit the following critical limitations: (i) off-target signals due to leaky translation in the absence of target nucleic acids, (ii) a suboptimal detection limit of approximately 30 nM without pre-amplification, and (iii) labor-intensive screening processes due to sequence constraints for the target nucleic acids. To overcome these shortcomings, we developed a new split T7 switch-mediated CFPS system in which the split T7 promoter was applied to a three-way junction structure to selectively initiate transcription-translation only in the presence of target nucleic acids. Both fluorescence and colorimetric detection systems were constructed by employing different reporter proteins. Notably, we introduced the self-complementation of split fluorescent proteins to streamline preparation of the proposed system, enabling versatile applications. Operation of this one-pot approach under isothermal conditions enabled the detection of target nucleic acids at concentrations as low as 10 pM, representing more than a thousand times improvement over previous toehold switch-based approaches. Furthermore, the proposed system demonstrated high specificity in detecting target nucleic acids and compatibility with various reporter proteins encoded in the expression region. By eliminating issues associated with the previous toehold switch system, our split T7 switch-mediated CFPS system could become a core platform for detecting various target nucleic acids.

Lung-selective nucleic acid vectors generated by in vivo lung-targeting-protein decoration of polyplexes.

Nucleic acid drugs show immense therapeutic potential, but achieving selective organ targeting (SORT) for pulmonary disease therapy remains a formidable challenge due to the high mortality rate caused by pulmonary embolism via intravenous administration or the mucus barrier in the respiratory tract via nebulized delivery. To meet this important challenge, we propose a new strategy to prepare lung-selective nucleic-acid vectors generated by in vivo decoration of lung-targeting proteins on bioreducible polyplexes. First, we synthesized polyamidoamines, named pabol and polylipo, to encapsulate and protect nucleic acids, forming polyamidoamines/mRNA polyplexes. Second, bovine serum albumin (BSA) was coated on the surface of these polyplexes, called BSA@polyplexes, including BSA@pabol polyplexes and BSA@polylipo polyplexes, to neutralize excess positive charge, thereby enhancing biosafety. Finally, after subcutaneous injection, proteins, especially vitronectin and fibronectins, attached to the polyplexes, resulting in the formation of lung-selective nucleic-acid vectors that achieve efficient lung targeting.

Templating Peptide Chemistry with Nucleic Acids: Toward Artificial Ribosomes, Cell-Specific Therapeutics, and Novel Protein-Mimetic Architectures.

In biology, nanomachines like the ribosome use nucleic acid templates to synthesize polymers in a sequence-specific, programmable fashion. Researchers have long been interested in using the programmable properties of nucleic acids to enhance chemical reactions via colocalization of reagents using complementary nucleic acid handles. In this review, we describe progress in using nucleic acid templates, handles, or splints to enhance the covalent coupling of peptides to other peptides or oligonucleotides. We discuss work in several areas: creating ribosome-mimetic systems, synthesizing bioactive peptides on DNA or RNA templates, linking peptides into longer molecules and bioactive antibody mimics, and scaffolding peptides to build protein-mimetic architectures. We close by highlighting the challenges that must be overcome in nucleic acid-templated peptide chemistry in two areas: making full-length, functional proteins from synthetic peptides and creating novel protein-mimetic architectures not possible through macromolecular folding alone.

Evaluating Cationic Nanoparticles as Carriers of Antiviral Nucleic Acids.

Nanoparticle carriers enable the multivalent delivery of nucleic acids to cells and protect them from degradation. In this chapter, we present a comprehensive overview of four methodologies: electrophoretic mobility shift assay (EMSA), alamarBlue/CFDA-AM cell viability dyes, fluorescence microscopy, and antiviral assays, which collectively are tools to explore interactions between nucleic acids and nanoparticles, and their biological efficacy. These assays provide insights into binding potential, cytotoxicity, and antiviral efficacy of nucleic acid-based nanoparticle treatments furthering the development of effective antiviral therapeutics.

Nucleic acid mediated activation of a short prokaryotic Argonaute immune system.

A short prokaryotic Argonaute (pAgo) TIR-APAZ (SPARTA) defense system, activated by invading DNA to unleash its TIR domain for NAD(P)+ hydrolysis, was recently identified in bacteria. We report the crystal structure of SPARTA heterodimer in the absence of guide-RNA/target-ssDNA (2.66 Å) and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide-RNA/target-ssDNA at nominal 3.15-3.35 Å resolution. The crystal structure provides a high-resolution view of SPARTA, revealing the APAZ domain as equivalent to the N, L1, and L2 regions of long pAgos and the MID domain containing a unique insertion (insert57). Cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix αN) domains that reconfigure for nucleic-acid binding and decrypts regions/residues that reorganize to expose a positively charged pocket for higher-order assembly. The TIR domains amass in a parallel-strands arrangement for catalysis. We visualize SPARTA before and after RNA/ssDNA binding and uncover the basis of its active assembly leading to abortive infection.

Development and applications of lipid hydrophilic headgroups for nucleic acid therapy.

Nucleic acid therapy is currently the most promising method for treating tumors and genetic diseases and for preventing infectious diseases. However, the biggest obstacle to this therapy is delivery of the nucleic acids to the target site, which requires overcoming problems such as capture by the immune system, the need to penetrate biofilms, and degradation of nucleic acid performance. Designing suitable delivery vectors is key to solving these problems. Lipids-which consist of a hydrophilic headgroup, a linker, and a hydrophobic tail-are crucial components for the construction of vectors. The headgroup is particularly important because it affects the drug encapsulation rate, the vector cytotoxicity, and the transfection efficiency. Herein, we focus on various headgroup structures (tertiary amines, quaternary ammonium salts, peptides, piperazines, dendrimers, and several others), and we summarize and classify important lipid-based carriers that have been developed in recent years. We also discuss applications of cationic lipids with various headgroups for delivery of nucleic acid drugs, and we analyze how headgroup structure affects transport efficiency and carrier toxicity. Finally, we briefly describe the challenges of developing novel lipid carriers, as well as their prospects.

Design and synthesis of nucleic acid nano-environment interactome-targeting small molecule PROTACs and their anticancer activity.

Targeted protein degradation through PROteolysis TArgeting Chimeras (PROTACs) is a relatively new modality in cellular interventions. The minimum requirement for PROTACs to function is forming a tertiary complex of the protein of interest (POI), E3 ligase, and the molecular glue PROTAC. Here, we propose a new approach to modulate the nano-environment interactome of a non-protein target through a plausible quaternary complex of interactome-biomolecule of interest (BOI)-PROTAC and E3 ligase. We report nucleic acid-targeting PROTAC (NA-TAC) molecules by conjugating DNA-binding and E3 ligase ligands. We demonstrate that NA-TACs can target the G-quadruplex DNA and induce elevated DNA damage and cytotoxicity compared to the conventional G-quadruplex binding ligands. Our new class of NA-TACs lays the foundation for small molecule-based non-protein targeting PROTACs for interactome and nanoenvironment mapping and nucleic acid-targeted precision medicines.