Nucleic Acid Interactions Research Articles

Deciphering molecular interactions by proximity labeling.

Many biological processes are executed and regulated through the molecular interactions of proteins and nucleic acids. Proximity labeling (PL) is a technology for tagging the endogenous interaction partners of specific protein 'baits', via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. Here we review the development of PL technologies and highlight studies that have applied PL to the discovery and analysis of molecular interactions. In particular, we focus on the use of PL for mapping protein-protein, protein-RNA and protein-DNA interactions in living cells and organisms.

Recent developments in deuterium solid-state NMR for the detection of slow motions in proteins

Slow timescale dynamics in proteins are essential for a variety of biological functions spanning ligand binding, enzymatic catalysis, protein folding and misfolding regulations, as well as protein–protein and protein–nucleic acid interactions. In this review, we focus on the experimental and theoretical developments of 2H static NMR methods applicable for studies of microsecond to millisecond motional modes in proteins, particularly rotating frame relaxation dispersion (R1ρ), quadrupolar Carr–Purcell–Meiboom–Gill (QCPMG) relaxation dispersion, and quadrupolar chemical exchange saturation transfer NMR experiments (Q-CEST). With applications chosen from amyloid-β fibrils, we show the complementarity of these approaches for elucidating the complexities of conformational ensembles in disordered domains in the non-crystalline solid state, with the employment of selective deuterium labels. Combined with recent advances in relaxation dispersion backbone measurements for 15N/13C/1H nuclei, these techniques provide powerful tools for studies of biologically relevant timescale dynamics in disordered domains in the solid state.

HISNAPI: a bioinformatic tool for dynamic hot spot analysis in nucleic acid-protein interface with a case study.

Protein–nucleic acid interactions play essential roles in many biological processes, such as transcription, replication and translation. In protein–nucleic acid interfaces, hotspot residues contribute the majority of binding affinity toward molecular recognition. Hotspot residues are commonly regarded as potential binding sites for compound molecules in drug design projects. The dynamic property is a considerable factor that affects the binding of ligands. Computational approaches have been developed to expedite the prediction of hotspot residues on protein–nucleic acid interfaces. However, existing approaches overlook hotspot dynamics, despite their essential role in protein function. Here, we report a web server named Hotspots In silico Scanning on Nucleic Acid and Protein Interface (HISNAPI) to analyze hotspot residue dynamics by integrating molecular dynamics simulation and one-step free energy perturbation. HISNAPI is capable of not only predicting the hotspot residues in protein–nucleic acid interfaces but also providing insights into their intensity and correlation of dynamic motion. Protein dynamics have been recognized as a vital factor that has an effect on the interaction specificity and affinity of the binding partners. We applied HISNAPI to the case of SARS-CoV-2 RNA-dependent RNA polymerase, a vital target of the antiviral drug for the treatment of coronavirus disease 2019. We identified the hotspot residues and characterized their dynamic behaviors, which might provide insight into the target site for antiviral drug design. The web server is freely available via a user-friendly web interface at http://chemyang.ccnu.edu.cn/ccb/server/HISNAPI/ and http://agroda.gzu.edu.cn:9999/ccb/server/HISNAPI/.

Crosslinker-modified nucleic acid probes for improved target identification and biomarker detection.

Understanding the intricate interaction pattern of nucleic acids with other molecules is essential to gain further insight in biological processes and disease mechanisms. To this end, a multitude of hybridization-based assays have been designed that rely on the non-covalent recognition between complementary nucleic acid sequences. However, the ephemeral nature of these interactions complicates straightforward analysis as low efficiency and specificity are rule rather than exception. By covalently locking nucleic acid interactions by means of a crosslinking agent, the overall efficiency, specificity and selectivity of hybridization-based assays could be increased. In this mini-review we highlight methodologies that exploit the use of crosslinker-modified nucleic acid probes for interstrand nucleic acid crosslinking with the objective to study, detect and identify important targets as well as nucleic acid sequences that can be considered relevant biomarkers. We emphasize on the usefulness and advantages of crosslinking agents and elaborate on the chemistry behind the crosslinking reactions they induce.

Gel-Based Analysis of Protein-Nucleic Acid Interactions.

Electrophoretic mobility shift assays (EMSAs) are among the most frequently used and straightforward experiments for studying protein-nucleic acid interactions. EMSAs rely on the principle that protein-nucleic acid complexes have reduced electrophoretic mobility in a native gel matrix compared to free nucleic acid due to their larger size and reduced negative charge. Therefore, bands for the protein-nucleic acid complexes are shifted in a gel and can be distinguished from free nucleic acids. EMSAs remain a popular technique since they do not require specialist equipment and the complexes formed are easily visualized. Furthermore, the technique can be adapted to enable various aspects of protein-nucleic acid interactions to be investigated, including sequence specificity, estimated binding affinity, and binding stoichiometry.

Assessing Nucleic Acid: Cationic Nanoparticle Interaction for Gene Delivery.

The assessment of the efficient binding between a nucleic acid and its associated nanoparticle is crucial for gene delivery. Emerging from the extensive search for versatile gene carriers, are complexes formed between nucleic acids and nonviral nanocarriers that promise to be viable alternatives to the predominantly viral-based gene delivery vehicles. However, much is still to be known about the exact structure and physico-chemical properties of such nanocomplexes. This chapter will concentrate on cationic lipid, polymer, and functionalized metal nanoparticles and their interaction with nucleic acids by direct conjugation or electrostatic interaction. Methods commonly employed to evaluate the nature and extent of nucleic acid interactions with cationic nanocarriers, such a nucleic acid binding, nuclease protection, and dye displacement assays will be described. In addition, the ultrastructural morphology, size, and zeta potential of these nanocomplexes, which are crucial for their cellular uptake and intracellular trafficking, will be assessed using electron microscopy, fluorescent detection, and nanoparticle tracking analysis (NTA). These assays have the ability to visualize and quantify the interaction and can also be used to complement each other, in addition to providing confirmation of the formation of the relevant nanocomplexes.

High-Throughput Protein-Nucleic Acid Interaction Assay Based on Protein-Induced Fluorescence Enhancement.

Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions is crucial for delineating the mechanisms governing transcription, genome duplication, and translation. Here we describe a detailed protocol for the quantitative analysis of protein-nucleic acid interactions based on protein-induced fluorescence enhancement (PIFE). While PIFE has mainly been used in single-molecule studies, we modified its application for bulk measurement of protein-nucleic acid interactions in microwell plates using standard fluorescent plate readers. The microwell plate PIFE assay (mwPIFE) is simple, does not require laborious protein labeling, and is high throughput. These properties predispose mwPIFE to become a method of choice for routine applications that require multiple parallel measurements such as buffer optimization, competition experiments, or screening chemical libraries for binding modulators.

Lighting Up the Thrombin-Binding Aptamer G-Quadruplex with an Internal Cyanine-Indole-Quinolinium Nucleobase Surrogate. Direct Fluorescent Intensity Readout for Thrombin Binding without Topology Switching.

Fluorescent nucleobases represent an important class of molecular reporters of nucleic acid interactions. In this work, the advantages of utilizing a noncanonical fluorescent nucleobase surrogate for monitoring thrombin binding by the 15-mer thrombin binding aptamer (TBA) is presented. TBA folds into an antiparallel G-quadruplex (GQ) with loop thymidine (T) residues interacting directly with the protein in the thrombin-TBA complex. In the free GQ, T3 is solvent-exposed and does not form canonical base-pairs within the antiparallel GQ motif. Upon thrombin binding, T3 interacts directly with a hydrophobic protein binding pocket. Replacing T3 with a cyanine-indole-quinolinium (4QI) hemicyanine dye tethered to an acyclic 1,2-propanediol linker is shown to have minimal impact on GQ stability and structure with the internal 4QI displaying a 40-fold increase in emission intensity at 586 nm (excitation 508 nm) compared to the free dye in solution. Molecular dynamics (MD) simulations demonstrate that the 4QI label π-stacks with T4 and T13 within the antiparallel GQ fold, which is supported by strong energy transfer (ET) fluorescence from the GQ (donor) to the 4QI label (acceptor). Thrombin binding to 4QI-TBA diminishes π-stacking interactions between 4QI and the GQ structure to cause a turn-off emission intensity response with an apparent dissociation constant (Kd) of 650 nM and a limit of detection (LoD) of 150 nM. These features highlight the utility of internal noncanonical fluorescent surrogates for monitoring protein binding by GQ-folding aptamers in the absence of DNA topology switching.

Understanding DNA interactions in crowded environments with a coarse-grained model.

Nucleic acid interactions under crowded environments are of great importance for biological processes and nanotechnology. However, the kinetics and thermodynamics of nucleic acid interactions in a crowded environment remain poorly understood. We use a coarse-grained model of DNA to study the kinetics and thermodynamics of DNA duplex and hairpin formation in crowded environments. We find that crowders can increase the melting temperature of both an 8-mer DNA duplex and a hairpin with a stem of 6-nt depending on the excluded volume fraction of crowders in solution and the crowder size. The crowding induced stability originates from the entropic effect caused by the crowding particles in the system. Additionally, we study the hybridization kinetics of DNA duplex formation and the formation of hairpin stems, finding that the reaction rate kon is increased by the crowding effect, while koff is changed only moderately. The increase in kon mostly comes from increasing the probability of reaching a transition state with one base pair formed. A DNA strand displacement reaction in a crowded environment is also studied with the model and we find that rate of toehold association is increased, with possible applications to speeding up strand displacement cascades in nucleic acid nanotechnology.

Evidence for Tethering of Human Cytomegalovirus Genomes to Host Chromosomes.

Tethering of viral genomes to host chromosomes has been recognized in a variety of DNA and RNA viruses. It can occur during both the productive cycle and latent infection and may impact viral genomes in manifold ways including their protection, localization, transcription, replication, integration, and segregation. Tethering is typically accomplished by dedicated viral proteins that simultaneously associate with both the viral genome and cellular chromatin via nucleic acid, histone and/or non-histone protein interactions. Some of the most prominent tethering proteins have been identified in DNA viruses establishing sustained latent infections, including members of the papillomaviruses and herpesviruses. Herpesvirus particles have linear genomes that circularize in infected cell nuclei and usually persist as extrachromosomal episomes. In several γ-herpesviruses, tethering facilitates the nuclear retention and faithful segregation of viral episomes during cell division, thus contributing to persistence of these viruses in the absence of infectious particle production. However, it has not been studied whether the genomes of human Cytomegalovirus (hCMV), the prototypical β-herpesvirus, are tethered to host chromosomes. Here we provide evidence by fluorescence in situ hybridization that hCMV genomes associate with the surface of human mitotic chromosomes following infection of both non-permissive myeloid and permissive fibroblast cells. This chromosome association occurs at lower frequency in the absence of the immediate-early 1 (IE1) proteins, which bind to histones and have been implicated in the maintenance of hCMV episomes. Our findings point to a mechanism of hCMV genome maintenance through mitosis and suggest a supporting but non-essential role of IE1 in this process.

Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.

MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm-1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.

Metabolic Co-regulation of Protein and Nucleic Acid Interactions in the Biosynthetic Pathways of Bacteria and Fungi

Metabolic Co-regulation of Protein and Nucleic Acid Interactions in the Biosynthetic Pathways of Bacteria and Fungi

Dynamics of Ionic Interactions at Protein-Nucleic Acid Interfaces.

ConspectusMolecular association of proteins with nucleic acids is required for many biological processes essential to life. Electrostatic interactions via ion pairs (salt bridges) of nucleic acid phosphates and protein side chains are crucial for proteins to bind to DNA or RNA. Counterions around the macromolecules are also key constituents for the thermodynamics of protein–nucleic acid association. Until recently, there had been only a limited amount of experiment-based information about how ions and ionic moieties behave in biological macromolecular processes. In the past decade, there has been significant progress in quantitative experimental research on ionic interactions with nucleic acids and their complexes with proteins. The highly negatively charged surfaces of DNA and RNA electrostatically attract and condense cations, creating a zone called the ion atmosphere. Recent experimental studies were able to examine and validate theoretical models on ions and their mobility and interactions with macromolecules. The ionic interactions are highly dynamic. The counterions rapidly diffuse within the ion atmosphere. Some of the ions are released from the ion atmosphere when proteins bind to nucleic acids, balancing the charge via intermolecular ion pairs of positively charged side chains and negatively charged backbone phosphates. Previously, the release of counterions had been implicated indirectly by the salt-concentration dependence of the association constant.Recently, direct detection of counterion release by NMR spectroscopy has become possible and enabled more accurate and quantitative analysis of the counterion release and its entropic impact on the thermodynamics of protein–nucleic acid association. Recent studies also revealed the dynamic nature of ion pairs of protein side chains and nucleic acid phosphates. These ion pairs undergo transitions between two major states. In one of the major states, the cation and the anion are in direct contact and form hydrogen bonds. In the other major state, the cation and the anion are separated by water. Transitions between these states rapidly occur on a picosecond to nanosecond time scale. When proteins interact with nucleic acids, interfacial arginine (Arg) and lysine (Lys) side chains exhibit considerably different behaviors. Arg side chains show a higher propensity to form rigid contacts with nucleotide bases, whereas Lys side chains tend to be more mobile at the molecular interfaces. The dynamic ionic interactions may facilitate adaptive molecular recognition and play both thermodynamic and kinetic roles in protein–nucleic acid interactions.

C-halogen…pi interactions in nucleic acids: a database study

A Protein Data Bank study was conducted to check the role of C-halogen(X)…pi interactions (X = F, Cl, Br, I) in nucleic acids. The presence of halogens can be attributed to not only the modified residues of the nucleic acid but also the ones associated with the ligand. The study reports the presence of bromine amongst modified residues of the nucleic acid being the maximum than any other halogens. It is important to consider these interactions as they seem to be responsible for increasing the affinity of the ligand to the biomolecule concerned. The role of halogen-pi interactions becomes important to be assessed in order to design halogenated ligands that will certainly be more effective in increasing the binding of the corresponding ligand to the target nucleic acid. The study attempts at listing the halogens that are prominent in the existing nucleic acid and ligand interactions which are deposited in the PDB. Inter-atomic interactions based on C-X…pi were analyzed to be found in several PDB IDs and similar ligand halogenation can be carried out to effectively increase binding affinity of halogenated ligands to nucleic acid based targets in case of several diseases. SYNOPSIS A comprehensive analysis of C-halogen…pi interaction (C-X…pi) among available nucleic acid structures in RCSB PDB has been presented pointing to their application in ligand modification to improve interaction with biomolecules. Existing structures point to the presence of these non-bonded interactions in several diseases.

Recent advances in user-friendly computational tools to engineer protein function.

Progress in technology and algorithms throughout the past decade has transformed the field of protein design and engineering. Computational approaches have become well-engrained in the processes of tailoring proteins for various biotechnological applications. Many tools and methods are developed and upgraded each year to satisfy the increasing demands and challenges of protein engineering. To help protein engineers and bioinformaticians navigate this emerging wave of dedicated software, we have critically evaluated recent additions to the toolbox regarding their application for semi-rational and rational protein engineering. These newly developed tools identify and prioritize hotspots and analyze the effects of mutations for a variety of properties, comprising ligand binding, protein–protein and protein–nucleic acid interactions, and electrostatic potential. We also discuss notable progress to target elusive protein dynamics and associated properties like ligand-transport processes and allosteric communication. Finally, we discuss several challenges these tools face and provide our perspectives on the further development of readily applicable methods to guide protein engineering efforts.

The influence of myristoylation, liposome surface charge and nucleic acid interaction in the partition properties of HIV-1 Gag-N-terminal peptides to membranes

The group-specific antigen (GAG) polyprotein of HIV-1 is the main coordinator of the virus assembly process at the plasma membrane (PM) and is directed by its N-terminal matrix domain (MA). MA is myristoylated and possess a highly basic region (HBR) responsible for the interaction with the negative lipids of the PM, especially with PIP2. In addition, MA binds RNA molecules proposed as a regulatory step of the assembly process. Here we study the interaction of a synthetic peptide (N-terminal 21 amino acids of MA) and liposomes of different compositions using a variety of biophysical techniques. Particularly, we use the fluorescence properties of the single tryptophan of the peptide to analyze its partition to membranes, where we harness for first time the analytical ability of spectral phasors method to study this interaction. We found that electrostatic interactions play an important role for peptide partition to membranes and myristoylation reduces the free energy of the process. Interestingly, we observe that while the presence of PIP2 does not cause measurable changes on the peptide-membrane interaction, the interaction is favored by cholesterol. Additionally, we found that the partition process goes through a transition state involving peptide disaggregation and changes in the peptide secondary structure. On the other hand, we found that the presence of oligonucleotides competes with the interaction with lipids by increasing peptide solubility. In summary, we think that our results, in context of the current knowledge of the role of HIV-1 MA, contribute to a better molecular understanding of the membrane association process.

Transducing Complex Biomolecular Interactions by Temperature-Output Artificial DNA Signaling Networks.

The requirement of special expensive instruments for quantitative information readout has significantly restricted sustainable development, from ideation to execution, of advanced artificial networks. Here we present a step toward a paradigm of evolutionary signaling networks that enable translating complex signaling information into easy-to-read temperature output. Combining DNA molecular engineering with basic optical mechanisms, a DNA/Hemin complex-derived versatile temperature-output transducer is established, which can be coupled with other functional modules to fabricate diverse portable DNA signaling networks by dynamic programming of DNA chemical reactions. Its versatility is successfully demonstrated by constructing self-amplified and logic-circuit-based DNA signaling networks to monitor trace and multibit nucleic acid interactions using a thermometer. This affordable yet powerful DNA signaling network design may portend an era of point-of-care signaling network methodology.

DIAZAPYRENES: INTERACTION WITH NUCLEIC ACIDS AND BIOLOGICAL ACTIVITY

The review summarizes data on the practical aspects of the interaction of nucleic acids with diazapyrene derivatives. The information on biological activity is given and the probable mechanisms underlying the action of diazapyrenes are analyzed. It contains 119 references.

ProbeRating: a recommender system to infer binding profiles for nucleic acid-binding proteins.

MotivationThe interaction between proteins and nucleic acids plays a crucial role in gene regulation and cell function. Determining the binding preferences of nucleic acid-binding proteins (NBPs), namely RNA-binding proteins (RBPs) and transcription factors (TFs), is the key to decipher the protein–nucleic acids interaction code. Today, available NBP binding data from in vivo or in vitro experiments are still limited, which leaves a large portion of NBPs uncovered. Unfortunately, existing computational methods that model the NBP binding preferences are mostly protein specific: they need the experimental data for a specific protein in interest, and thus only focus on experimentally characterized NBPs. The binding preferences of experimentally unexplored NBPs remain largely unknown.ResultsHere, we introduce ProbeRating, a nucleic acid recommender system that utilizes techniques from deep learning and word embeddings of natural language processing. ProbeRating is developed to predict binding profiles for unexplored or poorly studied NBPs by exploiting their homologs NBPs which currently have available binding data. Requiring only sequence information as input, ProbeRating adapts FastText from Facebook AI Research to extract biological features. It then builds a neural network-based recommender system. We evaluate the performance of ProbeRating on two different tasks: one for RBP and one for TF. As a result, ProbeRating outperforms previous methods on both tasks. The results show that ProbeRating can be a useful tool to study the binding mechanism for the many NBPs that lack direct experimental evidence. and implementationAvailability and implementationThe source code is freely available at <https://github.com/syang11/ProbeRating>.Supplementary information Supplementary data are available at Bioinformatics online.

5'-fluoro(di)phosphate-labeled oligonucleotides are versatile molecular probes for studying nucleic acid secondary structure and interactions by 19F NMR.

The high sensitivity of 19F nucleus to changes in the chemical environment has promoted the use of fluorine-labeled molecular probes to study structure and interactions of nucleic acids by 19F NMR. So far, most efforts have focused on incorporating the fluorine atom into nucleobase and ribose moieties using either monomer building blocks for solid-phase synthesis, or nucleoside triphosphates for enzymatic synthesis. Here, we report a simple and efficient synthesis of 5′-fluoromonophosphorylated and 5′-fluorodiphosphorylated oligodeoxyribonucleotides, which combines solid-phase and in-solution synthesis methods and requires only commercially available nucleoside phosphoramidites, followed by their evaluation as 19F NMR probes. We confirmed that the fluorine atom at the oligonucleotide 5′ end did not alter the secondary structure of DNA fragments. Moreover, at the same time, it enabled real-time 19F NMR monitoring of various DNA-related biophysical processes, such as oligonucleotide hybridization (including mismatch identification), G-quadruplex folding/unfolding and its interactions with thrombin, as well as formation of an i-motif structure and its interaction with small-molecule ligands.