Interaction Of Oligonucleotides Research Articles

A comprehensive adsorption and desorption study on the interaction of DNA oligonucleotides with TiO2 nanolayer

The utilization of TiO2 nanolayer that possesses excellent biocompatibility and physical properties in DNA sensing and sequencing remains largely to be explored. To examine its applicability in gene sequencing, this...

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Optimized Coiled-Coil Interactions for Multiplexed Peptide-PAINT.

Super-resolution microscopy has revolutionized how researchers characterize samples in the life sciences in the last decades. Amongst methods employing single-molecule localization microscopy, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement method that uses the programmable and repetitive binding of dye-labeled DNA imager strands to their respective docking strands. Recently developed Peptide-PAINT replaces the interaction of oligonucleotides by short coiled-coil peptide sequences leading to an improved labeling scheme by reducing linkage errors to target proteins. However, only one coiled-coil pair is currently available for Peptide-PAINT, preventing multiplexed imaging. In this study, the initial Peptide-PAINT E/K coil is improved by modifying its length for optimized binding kinetics leading to improved localization precisions. Additionally, an orthogonal P3/P4 coil pair is introduced, enabling 2-plex Peptide-PAINT imaging and benchmarking its performance and orthogonality using single-molecule and DNA origami assays. Finally, the P3/P4 peptide pair is used to image the human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D and 3D at the single receptor level using genetically encoded peptide tags.

Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity Biosensing

The high affinity and/or selectivity of oligonucleotide-mediated binding offers a myriad of therapeutical and analytical applications, whose rational design implies an accurate knowledge of the involved molecular mechanisms, concurring equilibrium processes and key affinity parameters. Oligonucleotide-functionalized gold surfaces or nanostructures are regularly employed analytical platforms for the development of label-free optical or electrochemical biosensors, and recently, novel detection platform designs have been increasingly considering the synergistic effect of polyvalent binding, involving the simultaneous interaction of two or several oligonucleotide strands. Considering the general lack of studies involving ternary single-stranded DNA (ssDNA) interactions, a complementary analytical workflow involving capillary gel electrophoretic (CGE) mobility shift assay, microcalorimetry and computational modeling has been deployed for the characterization of a series of free and surface-bound binary and ternary oligonucleotide interactions. As a proof of concept, the DNA analogue of MicroRNA 21 (miR21), a well-known oncogenic short MicroRNA (miRNA) sequence, has been chosen as a target molecule, simulating limiting-case scenarios involved in dual molecular recognition models exploited in affinity (bio)sensing. Novel data for the characterization of oligonucleotide interacting modules is revealed, offering a fast and complete mapping of the specific or non-specific, often competing, binary and ternary order interactions in dynamic equilibria, occurring between various free and metal surface-bound oligonucleotides.

Transfer of blocker-based qPCR reactions for DNA methylation analysis into a microfluidic LoC system using thermal modeling.

Changes in the DNA methylation landscape are associated with many diseases like cancer. Therefore, DNA methylation analysis is of great interest for molecular diagnostics and can be applied, e.g., for minimally invasive diagnostics in liquid biopsy samples like blood plasma. Sensitive detection of local de novo methylation, which occurs in various cancer types, can be achieved with quantitative HeavyMethyl-PCR using oligonucleotides that block the amplification of unmethylated DNA. A transfer of these quantitative PCRs (qPCRs) into point-of-care (PoC) devices like microfluidic Lab-on-Chip (LoC) cartridges can be challenging as LoC systems show significantly different thermal properties than qPCR cyclers. We demonstrate how an adequate thermal model of the specific LoC system can help us to identify a suitable thermal profile, even for complex HeavyMethyl qPCRs, with reduced experimental effort. Using a simulation-based approach, we demonstrate a proof-of-principle for the successful LoC transfer of colorectal SEPT9/ACTB-qPCR from Epi Procolon® colorectal carcinoma test, by avoidance of oligonucleotide interactions.

Life on Minerals: Binding Behaviors of Oligonucleotides on Zirconium Silicate and Its Inhibitory Activity for the Self-Cleavage of Hammerhead Ribozyme.

The role of minerals in the chemical evolution of RNA molecules is an important issue when considering the early stage of the Hadean Earth. In particular, the interaction between functional ribozymes and ancient minerals under simulated primitive conditions is a recent research focus. We are currently attempting to design a primitive RNA metabolic network which would function with minerals, and believe that the simulated chemical network of RNA molecules would be useful for evaluation of the chemical evolution from a simple RNA mixture to an RNA-based life-like system. First, we measured the binding interactions of oligonucleotides with four types of minerals; Aerosil silica, zirconium silicate, sepiolite, and montmorillonite. Oligonucleotides bound zirconium silicate and montmorillonite in the presence of MgCl2, and bound sepiolite both in the presence and absence of MgCl2, but they did not bind Aerosil. Based on the binding behavior, we attempted the self-cleavage reaction of the hammerhead ribozyme from an avocado viroid. This reaction was strongly inhibited by zirconium silicate, a compound regarded as mineral evidence for the existence of water. The present study suggests that the chemical evolution of functional RNA molecules requires specific conformational binding, resulting in efficient ribozyme function as well as zirconium silicate for the chemical evolution of biomolecules.

Multi-Functionalized Heteroduplex Antisense Oligonucleotides for Targeted Intracellular Delivery and Gene Silencing in HeLa Cells.

Oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) are short synthetic nucleic acid molecules with a promising potential to treat a wide range of diseases. Despite considerable progress in the field, the development of safe and effective delivery systems that target organs and tissues other than the liver is challenging. While keeping possible off-target oligonucleotide interactions and toxicity related to chemical modifications in mind, innovative solutions for targeted intracellular delivery are highly needed. Herein, we report on the design, synthesis and testing of a novel multi-modified and multi-functionalized heteroduplex oligonucleotide (HDO) with respect to its intracellular delivery and its ability to silence genes in HeLa cells. Simultaneously, folic acid- and peptide- labeled HDO show proficient silencing of the green fluorescent protein (GFP) gene with an 84% reduction in the GFP fluorescence. In addition, the Bcl2 HDO achieved effective Bcl2 gene knockdown in the cells. The data show the proficiency of the multi-functionalization strategy and provide an example for advancing the design of safe and efficient forthcoming oligonucleotide therapeutics, such as HDO.

Oligonucleotic Probes and Immunosensors Based on Nanoporous Anodic Alumina for Screening of Diseases

AbstractThis paper is an overview of the recent advances in nanoporous anodic alumina sensing platforms based on the interaction of oligonucleotides and antibodies with bioelements (e.g., virus, bacteria, or biomarkers) that can cause or identify diseases. Nanoporous alumina is the common platform material for all these sensing devices that have an enormous potential for the next‐generation bioanalytical systems. Nanoporous alumina is a cost‐effective material with morphological flexibility that provides high sensitivity to the sensing systems due to its large surface‐to‐volume ratio. Immunosensors transduce the signal produced by the specific antibody–antigen interaction. They are widely used for clinical analysis due to the high selectivity and sensitivity of their immunoreaction, transduced by different methods (electrochemical, optical, piezoelectric, etc). Another important, outstanding and more recently type of biosensors are based on the oligonucleotic functionalization of nanoporous alumina. Oligonucleotides can bind to a wide variety of elements (small organic molecules, proteins, or cells) with high selectivity, specificity, and affinity, even higher to those of antibodies. The basic concepts and mechanisms of their sensing methods for health care biosensing are introduced. Their transduction methods and great potential applications will be evaluated and classified in detail, together with a discussion of possible future trends.

Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.

Using DNA to Control the Mechanical Response of Nanoparticle Superlattices.

Nanoparticle superlattice assembly has been proposed as an ideal means of programming material properties as a function of hierarchical organization of different building blocks. While many investigations have focused on electromagnetic, optical, and transport behaviors, nanoscale self-assembly via supramolecular interactions is also a potentially desirable method to program material mechanical behavior, as it allows the strength and three-dimensional organization of chemical bonds to be used as handles to manipulate how a material responds to external stress. DNA-grafted nanoparticles are a particularly promising building block for such hierarchically organized materials because of DNA's tunable and nucleobase sequence-specific complementary binding. Using nanoindentation, we show here that the programmability of oligonucleotide interactions allows the modulus of DNA-grafted nanoparticle superlattices to be easily tuned overly nearly 2 orders of magnitude. Additionally, we demonstrate that alterations to the supramolecular bond strength between particles can alter how a lattice deforms under applied mechanical force. As a result, the superlattices can be programmed either to reorganize their internal structures to dissipate mechanical energy or to completely recover their initial structure upon relaxation, independently of how the particles are arranged in 3D space. These behaviors are subsequently explained as a function of the hierarchical structure of the DNA-guided assemblies by using a simple truss-structure model. Altering the supramolecular DNA connections between particles therefore provides a simple and rational means of dictating different aspects of material mechanical response to produce tailorable properties that are not typically observed in conventional bulk materials. Ultimately, these studies enable control over the deformation behavior of future DNA-assembled nanomaterials and provide evidence that supramolecular chemistry is an effective tool in controlling the mechanical properties of nanomaterials as a function of their hierarchical design.

Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil Interactions.

Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-molecule-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, we here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. We benchmark and optimize its binding kinetics in a single-molecule assay and demonstrate its super-resolution capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, we prove the suitability of Peptide-PAINT for cellular super-resolution imaging by visualizing the microtubule and vimentin network in fixed cells.

The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents.

Recent progress in understanding phosphorothioate antisense oligonucleotide (PS-ASO) interactions with proteins has revealed that proteins play deterministic roles in the absorption, distribution, cellular uptake, subcellular distribution, molecular mechanisms of action, and toxicity of PS-ASOs. Similarly, such interactions can alter the fates of many intracellular proteins. These and other advances have opened new avenues for the medicinal chemistry of PS-ASOs and research on all elements of the molecular pharmacology of these molecules. These advances have recently been reviewed. In this Perspective article, we summarize some of those learnings, the general principles that have emerged, and a few of the exciting new questions that can now be addressed.

Physical Interactions of Biopolymers with Nanoparticles

Using fluorescence resonance energy transfer, the physical interaction of nanoparticles with biopolymers is studied by the example of the interaction of quantum dots (energy donor) with two-chain oligonucleotides labeled with a fluorescent organic dye BDP TR (energy acceptor). The high-efficiency resonance energy transfer from quantum dots to an organic dye not only makes it possible to study the dependence of the transfer efficiency on the donor-acceptor molar ratio but also represents an efficient tool for studying the interactions of oligonucleotides of different structures with nanoscale objects.

Posttranscriptional Inhibition of Protein Tyrosine Phosphatase Nonreceptor Type 23 by Staphylococcal Nuclease and Tudor Domain Containing 1: Implications for Hepatocellular Carcinoma.

Oncoprotein staphylococcal nuclease and tudor domain containing 1 (SND1) regulates gene expression at a posttranscriptional level in multiple cancers, including hepatocellular carcinoma (HCC). Staphylococcal nuclease (SN) domains of SND1 function as a ribonuclease (RNase), and the tudor domain facilitates protein–oligonucleotide interaction. In the present study, we aimed to identify RNA interactome of SND1 to obtain enhanced insights into gene regulation by SND1. RNA interactome was identified by immunoprecipitation (IP) of RNA using anti‐SND1 antibody from human HCC cells followed by RNA immunoprecipitation sequencing (RIP‐Seq). Among RNA species that showed more than 10‐fold enrichment over the control, we focused on the tumor suppressor protein tyrosine phosphatase nonreceptor type 23 (PTPN23) because its regulation by SND1 and its role in HCC are not known. PTPN23 levels were down‐regulated in human HCC cells versus normal hepatocytes and in human HCC tissues versus normal adjacent liver, as revealed by immunohistochemistry. In human HCC cells, knocking down SND1 increased and overexpression of SND1 decreased PTPN23 protein. RNA binding and degradation assays revealed that SND1 binds to and degrades the 3′‐untranslated region (UTR) of PTPN23 messenger RNA (mRNA). Tetracycline‐inducible PTPN23 overexpression in human HCC cells resulted in significant inhibition in proliferation, migration, and invasion and in vivo tumorigenesis. PTPN23 induction caused inhibition in activation of tyrosine‐protein kinase Met (c‐Met), epidermal growth factor receptor (EGFR), Src, and focal adhesion kinase (FAK), suggesting that, as a putative phosphatase, PTPN23 inhibits activation of these oncogenic kinases. Conclusion: PTPN23 is a novel target of SND1, and our findings identify PTPN23 as a unique tumor suppressor for HCC. PTPN23 might function as a homeostatic regulator of multiple kinases, restraining their activation.

A Kinetic Model of Oligonucleotide-Brush Interactions for the Rational Design of Gene Delivery Vectors.

Polymer brushes are attractive candidates for the design of gene delivery vectors as they allow the systematic study of the impact of structural (type, size, and shape of nanomaterials core) and physicochemical parameters (cationic monomer chemistry, brush thickness, and grafting density) on transfection efficiency. However, relatively little is known of their interactions of oligonucleotides. To study such interactions, we use surface plasmon resonance and developed a kinetic model of brush binding and infiltration. We identify the striking impact that brush grafting density and thickness have on oligonucleotide kinetics of infiltration, binding affinity, and maximum loading. Surprisingly, double-stranded RNA molecules are found to load at significantly higher levels compared to DNA molecules of identical sequence (apart from uracils/thymines). Furthermore, analysis of the kinetics of adsorption of these oligonucleotides indicates that the stoichiometry of binding (ratio of amine versus phosphate residues) is close to parity for the uptake of 20 bp double-stranded RNA. Finally, nanoparticles were designed to be used as gene transfection vectors and to quantify if the brush grafting density and thickness significantly impact transfection efficiencies in a small interfering RNA knockdown assay. Therefore, this study demonstrates the rational design of polymer brush-based nanoparticle vectors for efficient delivery of oligonucleotides. The model developed will allow to uncover how the refinement of the physicochemical and structural properties of polymer brushes enable the tuning of RNA binding and allow the systematic study of cationic vectors efficiency for RNA delivery.

Squaramide-Based 5'-Phosphate Replacements Bind to the DNA Repair Exonuclease SNM1A.

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of N‐oxyamide and squaramide modifications at the 5’‐end of oligonucleotides as phosphate replacements and their biological evaluation using the 5’‐exonuclease SNM1A. The squaryl diamide modification showed minimal recognition as a 5’‐phosphate mimic; however, modest inhibition of SNM1A, postulated to occur through metal coordination at the active site, was observed. Their facile incorporation after solid‐phase synthesis and recognition by the exonuclease makes squaryl diamides attractive neutral 5’‐phosphate replacements for oligonucleotides. This work is the first example of squaryl diamide modifications at the 5’‐terminal position of oligonucleotides and of the potential use of modified oligonucleotides to bind to the metal center of SNM1A.

Secondary Structure-Dependent Physicochemical Interaction of Oligonucleotides with Gold Nanorod and Photothermal Effect for Future Applications: A New Insight.

We investigate the physicochemical interactions of gold nanorod (GNR) with single-stranded, double-stranded, and hairpin DNA structures to improve the biological compatibility as well as the therapeutic potential, including the photothermal effect of the conjugates. Studies have demonstrated that different DNA secondary structures, containing thiol group, have different patterns of physicochemical interaction. Conjugation efficiency of paired oligonucleotides are significantly higher than that of oligonucleotides with naked bases. Furthermore, hairpin-shaped DNA structures are most efficient in terms of conjugation and increased dispersion, with least interference on GNR near-infrared absorbance and photothermal effect. Our conjugation method can successfully exchange the overall coating of the GNR, attaching the maximum number of DNA molecules, thus far reported. Chemical mapping depicted uniform attachment of thiolated DNA molecules without any topological preference on the GNR surface. Hairpin DNA-coated GNR are suitable for intracellular uptake and remain dispersed in the cellular environment. Finally, we conjugated GNR with 5-fluoro-2′-deoxyuridine-containing DNA hairpin and the conjugate demonstrated significant cytotoxic activity against human cervical cancer cell line (KB). Thus, hairpin DNA structures could be utilized for optimal dispersion and photothermal effect of GNR, along with the delivery of cytotoxic nucleotides, developing the concept of multimodality approach.

Study on Interaction of G-rich Oligonucleotides Modified Gold Nanoparticles with Cells

Abstract Herein, two kinds of ssDNA-GNPs (termed as PolyG 1 -GNPs and PolyG 2 -GNPs, respectively) were synthesized by modification of two different G-rich single-strand oligonucleotides (PolyG 1 and PolyG 2 ) onto 13-nm citrate-protected gold nanoparticles (GNPs) surface through formation of the sulfur-gold covalent bond, respectively. The as-prepared PolyG 1 -GNPs and PolyG 2 -GNPs had reasonable colloidal stability in complex dispersing matrixes. The effect of ssDNA sequence on the interaction between ssDNA-GNPs and cells was systematically investigated by UV-visible absorption spectroscopy, cellular transmission electronic microscopy and inductively coupled plasma-mass spectrometry. The experimental results showed that both of the as-prepared PolyG 1 -GNPs and PolyG 2 -GNPs had low cytotoxicity and exhibited energy related endocytosis. In addition, the cellular internalization amount and dispersibility of ssDNA-GNPs were strongly affected by the sequences of immobilized ssDNAs. PolyG 2 with G-quadruplex (G4) secondary structure could significantly increase the cellular internalization amount and intracellular stability of PolyG 2 -GNPs.

Myristoylation and Oligonucleotide Interaction Modulate Peptide and Protein Surface Properties: The Case of the HIV-1 Matrix Domain.

Myristoylated proteins typically develop a tight association with membranes. One example is the matrix domain (MA) of the HIV-1 Gag protein. In addition, MA is able to bind the Sel25 RNA sequence, a ligand that can act as a competitor for the interaction with the membrane. These properties make HIV-1 MA an attractive molecule to understand how protein and peptide surface properties can be controlled by myristoylation and oligonucleotide interaction. In this line, we analyzed the stability, thermodynamics, and the topography of Langmuir monolayers composed of the myristoylated or unmyristoylated versions of MA in the presence or the absence of a single-strand DNA (ssDNASel25) analogue of the Sel25 RNA sequence. With a similar approach, we compared the MA surface properties with those obtained from monolayers of myristoylated and unmyristoylated MA-derived peptides (first 21 residues of the MA sequence). Our results show that the protein or peptide films are destabilized by the presence of ssDNASel25, inducing solubilization of the monolayer components into the bulk phase. In addition, the oligonucleotide affects the protein-protein or peptide-peptide lateral interactions, provoking interfacial topography changes of the monolayers, visualized by Brewster angle microscopy. Furthermore, we also show how the myristoyl group has major effects on the lateral stability and the elasticity of the monolayers. Altogether, here we propose a general model considering the effect of myristoylation and the interaction with oligonucleotides on the interfacial properties of MA and derived peptides. In this model, we introduce a new role of the core region of MA (sequence of MA after the 21st residue) that confers higher lateral interfacial stability to the protein.

Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.