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.

Translin is a multifunctional DNA/RNA binding protein involved in DNA repair and RNA metabolism. It has two basic regions and involvement of some residues in these regions in nucleic acid binding is established experimentally. Here we report the functional role of four residues of basic region II, Y85, R86, H88, R92 and one residue of C terminal region, K193 in nucleic acid binding using substitution mutant variants. CD analysis of the mutant proteins showed that secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Octameric state was maintained in all the mutants of basic region as evidenced by TEM, DLS, native PAGE and gel filtration analyses. However, K193G mutation completely abolished the octameric state of Translin protein and consequently its ability to bind ssDNA/ssRNA. The mutants of the basic region II exhibited a differential effect on nucleic acid binding, with R86A and R92G as most deleterious. Interestingly, H88A mutant showed higher nucleic acid binding affinity in comparison to the wild type Translin. An in silico analysis of the mutant variant sequences predicted all the mutations to be destabilizing, causing increase in flexibility and also leading to disruption of local interactions. The differential effect of mutations on DNA/RNA binding where octameric state is maintained could be attributed to these predicted disturbances.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3'-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson-Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.

Alzheimer's disease is characterized by important patho-proteins, which being composed of Amyloid-β plaques and intracellular neurofibrillary tangles of Tau. Intrinsically disordered protein tau has several interacting partners, which are necessary for its normal functioning. Tau has been shown to interact with various proteins, nucleic acid, and lipids. α-Linolenic acid (ALA) a plant-based omega-3 fatty acid has been studied for its role as neuroprotective and beneficial fatty acid in the brain. In this study, we are focusing on the ability of ALA to induce spontaneous assembly in tau protein. ALA inhibited the Tau aggregation as indicated by reduced ThS fluorescence kinetics, which indicates no aggregation of Tau. Similarly, SDS-PAGE analysis supported that ALA exposure inhibited the aggregation as no higher-order tau species were observed. Along with its ability to impede the aggregation of Tau, ALA also maintains a native random coiled structure, which was estimated by CD spectroscopy. Finally, TEM analysis showed that the formation of Tau fibrils was found to be discouraged by ALA. Hence, conclusion of the study suggested that ALA profoundly inhibited aggregation of Tau and maintained it's the random-coil structure.

Nucleic acid sensing is a central process in the immune system, with far-reaching roles in antiviral defense, autoinflammation, and cancer. Z-DNA binding protein 1 (ZBP1) is a sensor for double-stranded DNA and RNA helices in the unusual left-handed Z conformation termed Z-DNA and Z-RNA. Recent research established ZBP1 as a key upstream regulator of cell death and proinflammatory signaling. Recognition of Z-DNA/RNA by ZBP1 promotes host resistance to viral infection but can also drive detrimental autoinflammation. Additionally, ZBP1 has interesting roles in cancer and other disease settings and is emerging as an attractive target for therapy.

Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand-duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form.

'Locked nucleic acids' (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an 'all LNA' duplex containing exclusively modified β-D-2'-O-4'C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern.

"Locked nucleic acids" (LNAs) belong to the backbone-modified nucleic acid family. The 2'-O,4'-C-methylene-β-D-ribofuranose nucleotides are used for single or multiple substitutions in RNA molecules and thereby introduce enhanced bio- and thermostability. This renders LNAs powerful tools for diagnostic and therapeutic applications. RNA molecules maintain the overall canonical A-type conformation upon substitution of single or multiple residues/nucleotides by LNA monomers. The structures of "all" LNA homoduplexes, however, exhibit significant differences in their overall geometry, in particular a decreased twist, roll and propeller twist. This results in a widening of the major groove, a decrease in helical winding, and an enlarged helical pitch. Therefore, the LNA duplex structure can no longer be described as a canonical A-type RNA geometry but can rather be brought into proximity to other backbone-modified nucleic acids, like glycol nucleic acids or peptide nucleic acids. LNA-modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery.

The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, 'compound 2' [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors.

In contrast to a number of studies on the humanization of non-human antibodies, the reshaping of a non-human antibody into a chicken antibody has never been attempted. Therefore, nothing is known about the animal species-dependent compatibility of the framework regions (FRs) that sustain the appropriate conformation of the complementarity-determining regions (CDRs). In this study, we attempted the reshaping of the variable domains of the mouse catalytic anti-nucleic acid antibody 3D8 (m3D8) into the FRs of a chicken antibody (“chickenization”) by CDR grafting, which is a common method for the humanization of antibodies. CDRs of the acceptor chicken antibody that showed a high homology to the FRs of m3D8 were replaced with those of m3D8, resulting in the chickenized antibody (ck3D8). ck3D8 retained the biochemical properties (DNA binding, DNA hydrolysis, and cellular internalizing activities) and three-dimensional structure of m3D8 and showed reduced immunogenicity in chickens. Our study demonstrates that CDR grafting can be applied to the chickenization of a mouse antibody, probably due to the interspecies compatibility of the FRs.

N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation.

Nucleic acids have been widely recognized as potential targets in drug discovery and aptamer selection. Quantifying the interactions between small molecules and nucleic acids is critical to discover lead compounds and design novel aptamers. Scoring function is normally employed to quantify the interactions in structure-based virtual screening. However, the predictive power of nucleic acid-ligand scoring functions is still a challenge compared to other types of biomolecular recognition. With the rapid growth of experimentally determined nucleic acid-ligand complex structures, in this work, we develop a knowledge-based scoring function of nucleic acid-ligand interactions, namely SPA-LN. SPA-LN is optimized by maximizing both the affinity and specificity of native complex structures. The development strategy is different from those of previous nucleic acid-ligand scoring functions which focus on the affinity only in the optimization. The native conformation is stabilized while non-native conformations are destabilized by our optimization, making the funnel-like binding energy landscape more biased toward the native state. The performance of SPA-LN validates the development strategy and provides a relatively more accurate way to score the nucleic acid-ligand interactions.

In the investigation of interaction of aurintricarboxylic acid (ATA) with four biologically important proteins we observed inhibition of enzymatic activity of DNase I, RNase A, M-MLV reverse transcriptase and Taq polymerase by ATA in vitro assay. As the telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase holoenzyme, we also monitored effect of ATA on telomerase activity in vivo and observed dose-dependent inhibition of telomerase activity in Chinese hamster V79 cells treated with ATA. Direct association of ATA with DNase I (K(d)=9.019 microM)), RNase A (K(d)=2.33 microM) reverse transcriptase (K(d)=0.255 microM) and Taq polymerase (K(d)=81.97 microM) was further shown by tryptophan fluorescence quenching studies. Such association altered the three-dimensional conformation of DNase I, RNase A and Taq polymerase as detected by circular dichroism. We propose ATA inhibits enzymatic activity of the four proteins through interfering with DNA or RNA binding to the respective proteins either competitively or allosterically, i.e. by perturbing three-dimensional structure of enzymes.

Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy.

PCI domain proteins play important roles in post-transcriptional gene regulation. In the TREX-2 complex, PCI domain-containing Sac3 and Thp1 proteins and accessory Sem1 protein form a ternary complex required for mRNA nuclear export. In contrast, structurally related Thp3-Csn12-Sem1 complex mediates pre-mRNA splicing. In this study, we determined the structure of yeast Thp3186-470-Csn12-Sem1 ternary complex at 2.9 Å resolution. Both Thp3 and Csn12 structures have a typical PCI structural fold, characterized by a stack of α-helices capped by a C-terminal winged-helix (WH) domain. The overall structure of Thp3186-470-Csn12-Sem1 complex has an inverted V-shape with Thp3 and Csn12 forming the two sides. A fishhook-shaped Sem1 makes extensive contacts on Csn12 to stabilize its conformation. The overall structure of Thp3186-470-Csn12-Sem1 complex resembles the previously reported Sac3-Thp1-Sem1 complex, but also has significant structural differences. The C-terminal WH domains of Thp3 and Csn12 form a continuous surface to bind different forms of nucleic acids with micromolar affinity. Mutation of the basic residues in the WH domains of Thp3 and Csn12 affects nucleic acid binding in vitro and mRNA splicing in vivo. The Thp3-Csn12-Sem1 structure provides a foundation for further exploring the structural elements required for its specific recruitment to spliceosome for pre-mRNA splicing.

Cyclohexene nucleic acids (CeNA), which are characterized by the presence of a cyclohexene moiety instead of a natural (deoxy)ribose sugar, are known to increase the thermal and enzymatic stability when incorporated in RNA oligonucleotides. As it has been demonstrated that even a single cyclohexenyl nucleoside, when incorporated in an oligonucleotide, can have a profound effect on the biological activity of the oligonucleotide, further research is warranted to study the complex of such oligonucleotides with target proteins. In order to analyse the influence of CeNA residues onto the helix conformation and hydration of natural nucleic acid structures, a cyclohexenyl-adenine building block (xAr) was incorporated into the Dickerson sequence CGCGA(xAr)TTCGCG. The crystal structure of this sequence determined to a resolution of 1.90 A. The global helix belongs to the B-type family and shows a water spine, which is partially broken up by the apolar cyclohexene residue. The cyclohexene ring adopts the (2)E-conformation allowing a better incorporation of the residue in the dodecamer sequence. The crystal packing is stabilized by cobalt hexamine residues and belongs to space group P222(1), never before reported for nucleic acids.

Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5'-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3'-endo conformation which in fact helps better orienting within the active site.

The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue into the vacant spot within the G-quartet. The remaining two G-rich tracts of three residues each adopt parallel orientation and are linked with edgewise and propeller loops. Both 5' with 3 nt and 3' with 4 nt overhangs display well-defined conformations, with latter adopting a basket handle topology. Locked residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G-quadruplexes provides means for the improvement of vascular endothelial growth factor aptamers and advances our insights into driving nucleic acid structure by locking or unlocking the conformation of sugar moieties of nucleotides in general.

Heterochromatin protein 1α (HP1α) is a crucial element of chromatin organization. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can undergo phase separation upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. Here, we combine computational and experimental approaches to elucidate the molecular interactions that drive these processes. In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged HP1α peptide ligands and disrupted by the addition of negatively charged or neutral peptides. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in HP1α LLPS where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine-tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.