Resonance Hyper-Raman Spectroscopy of Nucleotides and Polynucleotides.

We applied 532 nm-excited two-photon resonance hyper-Raman (RHR) spectroscopy to nucleotides (dA, dG, dT, and dC) to obtain fundamental knowledge about their spectral patterns. The RHR spectrum of each nucleotide exhibited various modes of the purine and pyrimidine rings, showing the ability to acquire the structural information on the chromophore. The band positions of the RHR spectrum and the 266 nm-excited one-photon UV-resonance Raman (UVRR) spectrum were identical, while the intensity patterns differed. The peak assignments of the RHR bands were given by analogy to the UVRR spectrum. In examining the polynucleotides, which form a double-stranded helix through intermolecular hydrogen bonds, some RHR bands were found to be available as structural markers. Moreover, several overtone and combination bands were detected above 2000 cm-1. The frequencies of dA and dG were accounted for by considering the involvement of the vibration of dA at 1579 cm-1 and that of dG at 1482 cm-1, respectively. Multiple vibronically active modes were seen for dT and dC. HR spectroscopy offers unique information on the fundamental, combination, and overtone modes of dA and dG, of which the multiple electronic states are involved in the resonance process.

Alkyne Activation in the Diversity Oriented Synthesis of sp2 -Rich Scaffolds: A Biased Library Approach for Targeting Polynucleotides (DNA/RNA).

Polynucleotides, DNA and RNA (mRNA and non-coding RNAs) are critically involved in the molecular pathways of disease. Small molecule binding interactions with polynucleotides can modify functional polynucleotide topologies and/or their interactions with proteins. Current approaches to library design (lead-like or fragment-like libraries) are based on protein-ligand interactions and often include careful consideration of the 3-dimensional orientation of binding motifs and exclude π-rich compounds (polyfused aromatics) to avoid off-target R/DNA interactions. In contrast to proteins, where π,π-interactions are weak, polynucleotides can form strong π,π-interactions with suitable π-rich ligands. To assist in designing a polynucleotide-biased library, a scaffold-divergent synthesis approach to polyfused aromatic scaffolds has been undertaken. Initial screening hits that form moderately stable polynucleotide-ligand-protein ternary complexes can be further optimized through judicious incorporation of substituents on the scaffold to increase protein-ligand interactions. An example of this approach is given for topoisomerase-1 (TOP1), generating a novel TOP1 inhibitory chemotype.

Programmable Site-Specific Functionalization of DNA Origami with Polynucleotide Brushes.

Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. We demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.

M6A-BiNP: predicting N6-methyladenosine sites based on bidirectional position-specific propensities of polynucleotides and pointwise joint mutual information.

N6-methyladenosine (m6A) plays an important role in various biological processes. Identifying m6A site is a key step in exploring its biological functions. One of the biggest challenges in identifying m6A sites is how to extract features comprising rich categorical information to distinguish m6A and non-m6A sites. To address this challenge, we propose bidirectional dinucleotide and trinucleotide position-specific propensities, respectively, in this paper. Based on this, we propose two feature-encoding algorithms: Position-Specific Propensities and Pointwise Mutual Information (PSP-PMI) and Position-Specific Propensities and Pointwise Joint Mutual Information (PSP-PJMI). PSP-PMI is based on the bidirectional dinucleotide propensity and the pointwise mutual information, while PSP-PJMI is based on the bidirectional trinucleotide position-specific propensity and the proposed pointwise joint mutual information in this paper. We introduce parameters α and ß in PSP-PMI and PSP-PJMI, respectively, to represent the distance from the nucleotide to its forward or backward adjacent nucleotide or dinucleotide, so as to extract features containing local and global classification information. Finally, we propose the M6A-BiNP predictor based on PSP-PMI or PSP-PJMI and SVM classifier. The 10-fold cross-validation experimental results on the benchmark datasets of non-single-base resolution and single-base resolution demonstrate that PSP-PMI and PSP-PJMI can extract features with strong capabilities to identify m6A and non-m6A sites. The M6A-BiNP predictor based on our proposed feature encoding algorithm PSP-PJMI is better than the state-of-the-art predictors, and it is so far the best model to identify m6A and non-m6A sites.

Interaction of double-stranded polynucleotide poly(A:U) with graphene/graphene oxide.

Hybrids formed by DNA/RNA and graphene family nanomaterials are considered as potentially useful multifunctional agents in biosensing and nanomedicine. In this work, we study the noncovalent interaction between double-stranded (ds) RNA, polyadenylic:polyuridylic acids (poly(A:U)) and graphene oxide/graphene (GO/Gr) using UV absorption spectroscopy and molecular dynamics (MD) simulations. RNA melting showed that relatively long ds-RNA is adsorbed onto GO (at an ionic strength of [Formula: see text]) at that a large fraction of RNA maintains the duplex structure. It was revealed that this fraction decreases over long time (during a few days), indicating a slow adsorption process of the long polymer. MD simulations showed that the adsorption of duplex (rA)[Formula: see text]: (rU)[Formula: see text] or (rA)[Formula: see text]: (rU)[Formula: see text] on graphene starts with the interaction between [Formula: see text]-systems of graphene and base pairs located at a duplex tail. In contrast to relatively long duplex (rA)[Formula: see text]: (rU)[Formula: see text] which keeps parallel arrangement along the graphene surface, the shorter one ((rA)[Formula: see text]: (rU)[Formula: see text]) always adopts a perpendicular orientation relative to graphene even in case of the initial parallel orientation. It was found out that (rA)[Formula: see text]: (rU)[Formula: see text] forms the stable hybrid with graphene keeping essential fraction of the duplex, while (rA)[Formula: see text]: (rU)[Formula: see text] demonstrates the duplex unzipping into two single strands with time. The interaction energies between adenine/uracil stacked with graphene as well between nucleotides in water environment were determined.

Gene-PROBER - a tool to design polynucleotide probes for targeting microbial genes.

Recent developments in fluorescence in situ hybridization (FISH) methods allow the detection and visualization of the genes/genomic regions of bacteria, archaea and infecting viruses at the single cell level. These methods use mixtures of polynucleotides as probes to specifically detect the target of interest. Gene-PROBER enables the design of polynucleotide mixtures for targeting genes or genomic regions in microorganisms. It has four workflows, depending on the availability of non-target sequences and the choice of probe synthesis, either by chemical synthesis or by PCR. It outputs polynucleotides that are spread along the target sequence and have similar melting properties. Therefore, such a polynucleotide mixture can be used as a single probe, in a single hybridization reaction. Gene-PROBER is a freely available web service that can be accessed at http://gene-prober.icbm.de/, and is implemented in the R language using the Shiny package.

Sequence dependent phase separation of protein-polynucleotide mixtures elucidated using molecular simulations.

Ribonucleoprotein (RNP) granules are membraneless organelles (MLOs), which majorly consist of RNA and RNA-binding proteins and are formed via liquid-liquid phase separation (LLPS). Experimental studies investigating the drivers of LLPS have shown that intrinsically disordered proteins (IDPs) and nucleic acids like RNA and other polynucleotides play a key role in modulating protein phase separation. There is currently a dearth of modelling techniques which allow one to delve deeper into how polynucleotides play the role of a modulator/promoter of LLPS in cells using computational methods. Here, we present a coarse-grained polynucleotide model developed to fill this gap, which together with our recently developed HPS model for protein LLPS, allows us to capture the factors driving protein-polynucleotide phase separation. We explore the capabilities of the modelling framework with the LAF-1 RGG system which has been well studied in experiments and also with the HPS model previously. Further taking advantage of the fact that the HPS model maintains sequence specificity we explore the role of charge patterning on controlling polynucleotide incorporation into condensates. With increased charge patterning we observe formation of structured or patterned condensates which suggests the possible roles of polynucleotides in not only shifting the phase boundaries but also introducing microscopic organization in MLOs.

Tunable multiphase dynamics of arginine and lysine liquid condensates.

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.

Biomolecular uptake effects on chitosan/tripolyphosphate micro- and nanoparticle stability.

Colloidal chitosan/tripolyphosphate (TPP) particles have attracted significant attention as potential delivery vehicles for drugs, genes and vaccines. Yet, there have been several fundamental studies that showed these particles to disintegrate at physiological pH and ionic strength levels. To reconcile these findings with the published drug, gene and vaccine delivery research where chitosan/TPP particle disintegration was not reported, it has been postulated that the particles could be stabilized by their bioactive payloads. To test this hypothesis, here we examine whether the association of chitosan/TPP particles with model anionic proteins, α-lactalbumin (α-LA) and bovine serum albumin (BSA), and polynucleotides (DNA) enhances chitosan/TPP particle stability at physiological ionic strengths, using 150 mM NaCl (pH 5.5) and 1× PBS (pH 6.0) as the dissolution media. Light scattering and UV-vis spectroscopy revealed that anionic protein uptake had no impact on particle stability, likely due to the relatively weak protein/particle binding at near-physiological ionic strengths, which caused the protein to be rapidly released. This result occurred regardless of whether the protein was loaded during or after particle formation. Conversely, DNA uptake (at least at some compositions) increased the chitosan fractions persisting in a complexed/particulate form in model dissolution media, with the DNA remaining largely complexed to the chitosan at all investigated conditions. Collectively, these findings suggest that, while most bioactive payloads do not interact with chitosan strongly enough to stabilize chitosan/TPP particles, these chitosan particles can be stabilized to dissolution through the incorporation of polyanions.

Conformation-dependent restraints for polynucleotides: the sugar moiety.

Stereochemical restraints are commonly used to aid the refinement of macromolecular structures obtained by experimental methods at lower resolution. The standard restraint library for nucleic acids has not been updated for over two decades and needs revision. In this paper, geometrical restraints for nucleic acids sugars are derived using information from high-resolution crystal structures in the Cambridge Structural Database. In contrast to the existing restraints, this work shows that different parts of the sugar moiety form groups of covalent geometry dependent on various chemical and conformational factors, such as the type of ribose or the attached nucleobase, and ring puckering or rotamers of the glycosidic (χ) or side-chain (γ) torsion angles. Moreover, the geometry of the glycosidic link and the endocyclic ribose bond angles are functionally dependent on χ and sugar pucker amplitude (τm), respectively. The proposed restraints have been positively validated against data from the Nucleic Acid Database, compared with an ultrahigh-resolution Z-DNA structure in the Protein Data Bank, and tested by re-refining hundreds of crystal structures in the Protein Data Bank. The conformation-dependent sugar restraints presented in this work are publicly available in REFMAC, PHENIX and SHELXL format through a dedicated RestraintLib web server with an API function.

Polynucleotide differentiation using hybrid solid-state nanopore functionalizing with α-hemolysin.

We report results from full atomistic molecular dynamics simulations on the properties of biomimetic nanopores. This latter result was obtained through the direct insertion of an α-hemolysin protein inside a hydrophobic solid-state nanopore. Upon translocation of different DNA strands, we demonstrate here that the theoretical system presents the same discrimination properties as the experimental one obtained previously. This opens an interesting way to promote the stability of a specific protein inside a solid nanopore to develop further biomimetic applications for DNA or protein sequencing.

Interaction of small molecules with polynucleotide repeats and frameshift site RNA.

This mini-review describes the interaction between small molecules and RNA, in addition to its application either in treating RNA-associated diseases or detecting target molecules. In the case of RNA-associated disease treatment, the designed small molecules interact with RNA sites, forming adducts and providing successful therapeutic strategies over oligonucleotides. On the other hand, synthetically designed RNA moieties (aptamers) interact with target molecules like toxins, drugs, hormones; these interactions are useful in the detection, quantification or separation of these target moieties.

Spectroscopic and molecular docking investigation of polynucleotides and DNA binding affinity to Nile blue dye.

We used UV-vis absorption spectroscopy, fluorescence spectrophotometry and molecular docking calculations to investigate intermolecular interaction between the cationic dye, Nile blue (NB), and synthetic polynucleotides, poly(A-T), poly(G-C) and calf thymus DNA (Ct-DNA) at physiological pH. Strong hypsochromic absorbance and fluorescence quenching were observed that showed strong binding of NB to these polynucleotides and DNA. The binding affinity values derived from maximum absorption of the spectra of NB bound to various polynucleotides and Ct-DNA concentrations suggests that NB exhibits greater binding affinity to poly(G-C) than to poly(A-T). The thermodynamic parameters suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of NB to DNA. The molecular docking results suggested that NB was an intercalator of the stacked base pairs of Ct-DNA.

High-throughput evolution of near-infrared serotonin nanosensors.

Imaging neuromodulation with synthetic probes is an emerging technology for studying neurotransmission. However, most synthetic probes are developed through conjugation of fluorescent signal transducers to preexisting recognition moieties such as antibodies or receptors. We introduce a generic platform to evolve synthetic molecular recognition on the surface of near-infrared fluorescent single-wall carbon nanotube (SWCNT) signal transducers. We demonstrate evolution of molecular recognition toward neuromodulator serotonin generated from large libraries of ~6.9 × 1010 unique ssDNA sequences conjugated to SWCNTs. This probe is reversible and produces a ~200% fluorescence enhancement upon exposure to serotonin with a K d = 6.3 µM, and shows selective responsivity over serotonin analogs, metabolites, and receptor-targeting drugs. Furthermore, this probe remains responsive and reversible upon repeat exposure to exogenous serotonin in the extracellular space of acute brain slices. Our results suggest that evolution of nanosensors could be generically implemented to develop other neuromodulator probes with synthetic molecular recognition.

KnotGenome: a server to analyze entanglements of chromosomes.

The KnotGenome server enables the topological analysis of chromosome model data using three-dimensional coordinate files of chromosomes as input. In particular, it detects prime and composite knots in single chromosomes, and links between chromosomes. The knotting complexity of the chromosome is presented in the form of a matrix diagram that reveals the knot type of the entire polynucleotide chain and of each of its subchains. Links are determined by means of the Gaussian linking integral and the HOMFLY-PT polynomial. Entangled chromosomes are presented graphically in an intuitive way. It is also possible to relax structure with short molecular dynamics runs before the analysis. KnotGenome is freely available at http://knotgenom.cent.uw.edu.pl/.

THz spectra and corresponding vibrational modes of DNA base pair cocrystals and polynucleotides.

The generalized energy-based fragmentation (GEBF) approach has been applied to study the THz spectra and vibrational modes of base pair cocrystals under periodic boundary conditions (denoted as PBC-GEBF). Results of vibrational mode reveal that hydrogen bonds play a pivotal role in the pairing process of base crystals, where most NH and CH bonds stretch to some extent. We also found that hydrogen bonds of a self-made A:T cocrystal completely break in a transition from liquid to the solid state, while self-made C:G cocrystal is different and easier to form a cocrystal, as confirmed by X-ray diffraction (XRD) and terahertz (THz) spectra. Furthermore, we have studied DNA polynucleotides (in both A and B forms) found that the vibrational modes changed a lot during the process of their forming double strand. Despite the key role played by hydrogen bonds, the key contribution originates from collective motions of the main skeleton. A comparative study of the spectra of some stranded fragments suggests that different sequences or forms have similar spectra in THz band. They distinguish from each other mainly in the low-frequency regions, especially below 1 THz. This study would make great contributions to the molecular dynamics model based DNA long-chain structure simulation in the future study.

Thermal stability and conformation of DNA and proteins under the confined condition in the matrix of hydrogels.

Spatially confined environments are seen in biological systems and in the fields of biotechnology and nanotechnology. The confinement restricts the conformational space of polymeric molecules and increasing the degree of molecular crowding. Here, we developed preparation methods for agarose and polyacrylamide gels applicable to UV spectroscopy that can evaluate the confinement effects on DNA and protein structures. Measurements of UV absorbance and CD spectra showed no significant effect of the confinement in the porous media of agarose gels on the base-pair stability of DNA polynucleotides [poly(dA)/poly(dT)] and oligonucleotides (hairpin, duplex, and triplex structures). On the other hand, a highly confined environment created by polyacrylamide gels at high concentrations increased the stability of polynucleotides while leaving that of oligonucleotides unaffected. The changes in the base-pair stability of the polynucleotides were accompanied by the perturbation of the helical conformation. The polyacrylamide gels prepared in this study were also used for the studies on proteins (lysozyme, bovine serum albumin, and myoglobin). The effects on the proteins were different from the effects on DNA structures, suggesting different nature of interactions within the gel. The experimental methods and results are useful to understand the physical properties of nucleic acids and proteins under confined conditions.

Structural Heterogeneity in Polynucleotide-Facilitated Assembly of Phenothiazine Dyes.

The assembly of stacked dyes on DNA is of interest for electron transfer, light harvesting, sensing, and catalysis applications. A combination of UV/vis absorption, linear dichroism (LD), and circular dichroism (CD) was applied to characterize thoroughly the aggregation with DNA of the phenothiazine dyes methylene blue, azure B, and thionine. Aggregates of each dye with [poly(dG-dC)]2, [poly(dA-dT)]2, and calf thymus DNA were explored at high dye:DNA binding ratios, where excess dye groove-binds after all intercalation sites are filled. The organization of the aggregates (dimers, trimers, and multimers) with polydeoxynucleotides displays a structural diversity that depends on DNA sequence, extent of methylation of dye exocyclic amine groups, and ionic strength. The dyes typically form right-handed H-aggregates having negative LD, consistent with stepped stacking along the minor groove. However, aggregates in some dye:DNA aggregates show left-handed chirality or positive LD, indicating unusual modes of aggregation such as formation of adventitious dimers between intercalated and minor groove bound dye. In terms of sequence-dependence, methylene blue shows more extensive aggregation with [poly(dA-dT)]2, while thionine aggregates more with [poly(dG-dC)]2. Azure B has distinctive behavior that is unlike either other dyes. Thus, although these phenothiazine dyes possess a common tricyclic framework, the organization of their polynucleotide-facilitated aggregates depends sensitively on the extent of methylation of the exocyclic amines.

Linear and circular dichroism characterization of thionine binding mode with DNA polynucleotides.

The binding mode of thionine (3,7-diamino-5-phenothiazinium) with alternating and non-alternating DNA polynucleotides at low binding ratios was conclusively determined using linear and circular dichroism spectroscopies. The binding to [poly(dG-dC)]2 and poly(dG)·poly(dC) was purely intercalative and was insensitive to ionic strength. Intercalative binding to [poly(dA-dT)]2 is observed at low ionic strength, but a shift of some dye to an non-intercalative mode is observed as the background salt concentration increases. With poly(dA)·poly(dT), intercalative binding is unfavourable, although some dye molecules may intercalate at low ionic strength, and groove binding is strongly promoted with increasing concentration of background salt. However, stacking with bases is observed with single-stranded poly(dA) and with triplex poly(dT)⁎poly(dA)·poly(dT) which suggests that the unusual structure of poly(dA)·poly(dT) precludes intercalation. Thionine behaves similarly to the related dye methylene blue, and small differences may be attributed either to the ability of thionine to form H-bonds that stabilize intercalation or to its improved stacking interactions in the basepair pocket on steric grounds.

Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures.

Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c7dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide.