Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.

Two G-quadruplex forming sequences, 5'-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming oligonucleotides (QFOs) and the effect of LNA monomers in the context of biologically active QFOs. In addition, recent literature reports and our own studies on the gel retardation of the phosphodiester analogue of T30177 led to the conclusion that this sequence forms a parallel, dimeric G-quadruplex. Introduction of the 5'-phosphate inhibits dimerisation of this G-quadruplex as a result of negative charge-charge repulsion. Contrary to that, we found that attachment of the 5'-O-DMT-group produced a more active 17-mer sequence that showed signs of aggregation-forming multimeric G-quadruplex species in solution. Many of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed.

Nucleic acids are generally considered as efficient cation binders. Therefore, the likelihood that negatively charged ions might intrude their first hydration shell is rarely considered. Here, we show on the basis of (i) a survey of the Nucleic Acid Database, (ii) several structures extracted from the Cambridge Structural Database, and (iii) molecular dynamics simulations, that the nucleotide electropositive edges involving mainly amino, imino, and hydroxyl groups can cast specific anion binding sites. These binding sites constitute also good locations for the binding of the negatively charged groups of the Asp and Glu residues or the nucleic acid phosphate groups. Furthermore, it is observed in several instances that anions, like water molecules and cations, do mediate protein/nucleic acid interactions. Thus, anions as well as negatively charged groups are directly involved in specific recognition and folding phenomena involving polyanionic nucleic acids.

NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis, such methods are still in their infancy in the field of nucleic acids, especially when non-canonical structures and nucleic acid complexes are considered. Here, we present an ab initio approach for predicting proton chemical shifts of arbitrary nucleic acid structures based on state-of-the-art fragment-based quantum chemical calculations. We tested our prediction method on a diverse set of nucleic acid structures including double-stranded DNA, hairpins, DNA/protein complexes and chemically-modified DNA. Overall, our quantum chemical calculations yield highly/very accurate predictions with mean absolute deviations of 0.3-0.6 ppm and correlation coefficients (r(2)) usually above 0.9. This will allow for identifying misassignments and validating 3D structures. Furthermore, our calculations reveal that chemical shifts of protons involved in hydrogen bonding are predicted significantly less accurately. This is in part caused by insufficient inclusion of solvation effects. However, it also points toward shortcomings of current force fields used for structure determination of nucleic acids. Our quantum chemical calculations could therefore provide input for force field optimization.

Alternatives to efficient viral vectors in gene therapy are desired because of their poor safety profiles. Chitosan is a promising non-viral nucleotide delivery vector because of its biocompatibility, biodegradability, low immunogenicity and ease of manufacturing. Since the transfection efficiency of chitosan polyplexes is relatively low compared to viral counterparts, there is an impetus to gain a better understanding of the structure-performance relationship. Recent progress in preparation and characterisation has enabled coupling analysis of chitosans structural parameters that has led to increased TE by tailoring of chitosan's structure. In this review, we summarize the recent advances that have lead to a more rational design of chitosan polyplexes. We present an integrated review of all major areas of chitosan-based transfection, including preparation, chitosan and polyplexes physicochemical characterisation, in vitro and in vivo assessment. In each, we present the obstacles to efficient transfection and the strategies adopted over time to surmount these impediments.

Therapeutic nucleic acids represent a powerful class of drug molecules to control gene expression and protein synthesis. A major challenge in this field is that soluble oligonucleotides have limited serum stability, and the majority of nucleic acids that enter the cells are trapped within endosomes. Delivery efficiency can be improved using lipid scaffolds. One such example is the nanodisc (ND), a self-assembled nanostructure composed of phospholipids and peptides and modeled after high density lipoproteins (HDLs). Herein, we describe the development of the nanodiscoidal nucleic acid (NNA) which is a ND covalently modified with nucleic acids on the top and bottom lipid faces as well as the lateral peptide belt. The 13 nm ND was doped with thiolated phospholipids and thiol-containing peptides and coupled in a one-pot reaction with oligonucleotides to achieve ∼30 DNA/NNA nucleic acid density. NNAs showed superior nuclease resistance and enhanced cellular uptake that was mediated through the scavenger receptor B1. Time-dependent Förster resonance energy transfer (FRET) analysis of internalized NNA confirmed that NNAs display increased stability. NNAs modified with clinically validated antisense oligonucleotides (ASOs) that target hypoxia inducible factor 1-α (HIF-1-α) mRNA showed enhanced activity compared with that of the soluble DNA across multiple cell lines as well as a 3D cancer spheroid model. Lastly, in vivo experiments show that ASO-modified NNAs are primarily localized into livers and kidneys, and NNAs were potent in downregulating HIF-1-α using 5-fold lower doses than previously reported. Collectively, our results highlight the therapeutic potential for NNAs.

Cyclohexenyl nucleic acid (CeNA) is a nucleic acid mimic, where the (deoxy)ribose sugar has been replaced by cyclohexenyl moieties. In order to study the conformation of cyclohexenyl nucleosides by NMR, the HexRot program was developed to calculate conformations from scalar coupling constants of cyclohexenyl compounds, analogous to the methods applied for (deoxy)ribose nucleosides. The conformational equilibria and the values of the thermodynamic parameters are very similar between a cyclohexenyl nucleoside [energy difference between 2H3 (N-type) and 2H3 (S-type) is 1.8 kJ/mol and equilibrium occurs via the eastern hemisphere with a barrier of 10.9 kJ/mol] and a natural ribose nucleoside (energy difference between N-type and S-type is 2 kJ/mol and equilibrium occurs via the eastern hemisphere with a barrier of 4-20 kJ/mol). The flexibility of the cyclohexenyl nucleoside was demonstrated by the fast equilibrium between two conformational states that was observed in a CeNA-U monomer, combined with the 2H3 conformation of the cyclohexene moiety when incorporated into a Dickerson dodecamer and the 2H3 conformation when incorporated in a d(5'-GCGT*GCG-3')/d(5'-CGCACGC-3') duplex, as determined by the NMR spectroscopy. This represents the first example of a synthetic nucleoside that adopts different conformations when incorporated in different double-stranded DNA sequences.

The efficient transfection of functional proteins into cells can serve as a means for regulating cellular processes toward solving fundamental challenges in biology and medicine. However, the use of proteins as effective intracellular agents is hindered by their low cellular uptake and susceptibility to degradation. Over the past 15 years, our group has been developing spherical nucleic acids (SNAs), nanoparticles functionalized with a dense radially oriented shell of nucleic acids. These structures actively enter cells and have opened new frontiers in chemical sensing, biodiagnostics and therapeutics. Recently, we have shown that proteins can be used as structurally precise and homogeneous nanoparticle cores in SNAs. The resultant protein SNAs (ProSNAs) allow previously cell-impermeable proteins to actively enter cells, exhibit high degrees of stability and activity both in cell culture and in vivo, and show enhanced pharmacokinetics. Consequently, these modular structures constitute a plug-and-play platform in which the protein core and nucleic acid shell can be independently varied to achieve a desired function. Here, we describe the synthesis of ProSNAs through the chemical modification of solvent-accessible surface residues (3-5 d). We also discuss design considerations, strategies for characterization, and applications of ProSNAs in cellular transfection, biological sensing and functional enzyme delivery in vivo.

DNA and RNA G-quadruplexes have gained increasing attention due to their potential role in a wide range of biological functions. The majority of functional studies characterize the influence of quadruplexes in gene expression including transcription and translation. Many of these studies have used reporter assays to elucidate the effect of quadruplexes at certain positions in promoters and untranslated mRNA regions (UTRs). Reporter assays are the preferred method to ascertain the biological function of DNA or RNA G-quadruplexes intracellularly due to their ready availability, fast cloning and experimental setup and reproducibility. Moreover, these reporter assays are also helpful to compare or screen for selectivity and efficacy of small molecules that target DNA and RNA G-quadruplexes in the cellular context. Here we briefly discuss various aspects of reporter assays followed by a review of available studies using reporter assays to understand the role and functions of DNA and RNA quadruplexes in gene expression.

The high-resolution structure of the DNA (5'-GTGTACA-C-3') with the selenium derivatization at the 2'-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 A resolution) with the 2'-Se modification in the minor groove is isomorphorous to the native structure (2.0 A). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 A resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization.

Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques.

Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation.

We describe Curves+, a new nucleic acid conformational analysis program which is applicable to a wide range of nucleic acid structures, including those with up to four strands and with either canonical or modified bases and backbones. The program is algorithmically simpler and computationally much faster than the earlier Curves approach, although it still provides both helical and backbone parameters, including a curvilinear axis and parameters relating the position of the bases to this axis. It additionally provides a full analysis of groove widths and depths. Curves+ can also be used to analyse molecular dynamics trajectories. With the help of the accompanying program Canal, it is possible to produce a variety of graphical output including parameter variations along a given structure and time series or histograms of parameter variations during dynamics.

Single-stranded nucleic acids (ssNAs) are ubiquitous in many key cellular functions. Their flexibility limits both the number of high-resolution structures available, leaving only a small number of protein-ssNA crystal structures, while forcing solution investigations to report ensemble averages. A description of the conformational distributions of ssNAs is essential to more fully characterize biologically relevant interactions. We combine small angle X-ray scattering (SAXS) with ensemble-optimization methods (EOM) to dynamically build and refine sets of ssNA structures. By constructing candidate chains in representative dinucleotide steps and refining the models against SAXS data, a broad array of structures can be obtained to match varying solution conditions and strand sequences. In addition to the distribution of large scale structural parameters, this approach reveals, for the first time, intricate details of the phosphate backbone and underlying strand conformations. Such information on unperturbed strands will critically inform a detailed understanding of an array of problems including protein-ssNA binding, RNA folding and the polymer nature of NAs. In addition, this scheme, which couples EOM selection with an iteratively refining pool to give confidence in the underlying structures, is likely extendable to the study of other flexible systems.

Antimicrobial resistance (AMR) is a growing concern because it causes microorganisms to develop resistance to drugs commonly used to treat infections. This results in increased difficulty in treating infections, leading to higher mortality rates and significant economic effects. Investing in new antimicrobial agents is, therefore, necessary to prevent and control AMR. Antimicrobial nucleic acids have arisen as potential key players in novel therapies for AMR infections. They have been designed to serve as antimicrobials and to act as adjuvants to conventional antibiotics or to inhibit virulent mechanisms. This new category of antimicrobial drugs consists of antisense oligonucleotides and oligomers, DNAzymes, and transcription factor decoys, differing in terms of structure, target molecules, and mechanisms of action. They are synthesized using nucleic acid analogs to enhance their resistance to nucleases. Because bacterial envelopes are generally impermeable to oligonucleotides, delivery into the cytoplasm typically requires the assistance of nanocarriers, which can affect their therapeutic potency. Given that numerous factors contribute to the success of these antimicrobial drugs, this review aims to provide a summary of the key advancements in the use of oligonucleotides for treating bacterial infections. Their mechanisms of action and the impact of factors such as nucleic acid design, target sequence, and nanocarriers on the antimicrobial potency are discussed.

Vesicular spherical nucleic acids are dynamic nucleic acid-based supramolecular structures that are held together via non-covalent bonds. They have promising applications as drug and nucleic acid delivery materials, diagnostic and imaging tools and platforms for development of various therapeutic schemes. In this contribution, we report on vesicular spherical nucleic acids, constructed from a non-phospholipid nucleolipid - an original hybrid biomacromolecule, composed of a hydrophobic residue, resembling that of the naturally occurring phospholipids, and a DNA oligonucleotide strand. The nucleolipid was synthesized by coupling of dibenzocyclooctyne-functionalized oligonucleotide and azidated 1,3-dihexadecyloxy-propane-2-ol via an azide-alkyne click reaction. In aqueous solution it spontaneously self-associated into nanosized supramolecular structures, identified as unilamellar vesicles composed of a self-closed interdigitated bilayer. Vesicular structures were also formed upon intercalation of the nucleolipid via its lipid-mimetic residue in the phospholipid bilayer membrane of liposomes prepared from readily available and FDA-approved lipids (1,2-dipalmitoyl-rac-glycero-3-phosphocholine and cholesterol). The vesicular structures are thoroughly investigated by light scattering (dynamic, static, and electrophoretic) and cryogenic TEM and the physical characteristics, in particular, number of strands per particle, grafting density, and conformation of the strands, were compared to those of reference spherical nucleic acids. Finally, the vesicular structures were shown to exhibit cellular internalization with no need of transfection agents and enhanced colloidal and nuclease stability.

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5'-5' manner based on different accessible tetrad stacking modes at the stacking interfaces of 5'-5' and 3'-3' stacked G-quadruplexes.

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.

To provide a new research direction for nerve regeneration and strategy for Alzheimer's disease treatment, tetrahedral DNA nanostructures (TDNs)-novel tetrahedral framework nucleic acid molecule nanoparticles (tFNA) that can inhibit the apoptosis of nerve cells are employed in the experiment.