Single-stranded Nucleic Acids Research Articles

Modulation of α-Synuclein Aggregation In Vitro by a DNA Aptamer.

Protein aggregation is an important problem for human health and biotechnology, with consequences in areas ranging from neurodegenerative diseases to protein production yields. Methods to modulate protein aggregation are therefore essential. One suggested method to modulate protein aggregation is the use of nucleic acid aptamers, that is, single-stranded nucleic acids that have been selected to specifically bind to a target. Previous studies in some systems have demonstrated that aptamers may inhibit protein aggregation, including for α-synuclein, a protein implicated in synucleinopathies. However, the mechanisms by which aptamers might affect or modulate aggregation have not been fully determined. In this study, we investigated the effect of an aptamer that binds α-synuclein oligomer, T-SO508, on α-synuclein aggregation in vitro using thioflavin T to monitor aggregation kinetics, and we performed atomic force microscopy, transmission electron microscopy, and analytical ultracentrifugation to characterize intermediate structures. The results indicated that T-SO508, but not control DNA sequences, extends the lag phase of aggregation and stabilizes formation of a small non-fibrillar aggregate complex. Attempts to use the aptamer-induced complexes to seed fibril formation did not in fact accelerate aggregation, indicating that these structures are off-pathway for aggregation. This study highlights a potential mechanism by which aptamers may modulate the aggregation properties of proteins.

Diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified electrode for electrochemical determination of cancer biomarker

Nucleic acid hybridization is occurred between the selective single-stranded nucleic acid sequence and its target sequence, which is one of the essential procedure for electrochemical detection of nucleic acid. microRNA-21 (miRNA-21) is known as a biomarker in various cancers. The determination of miRNA-21 was attained through by hybridization of inosine substituted miRNA-21 specific DNA probe (Pinosine) with its target miRNA-21. In this study, the surface of pencil graphite electrode (PGE) was firstly modified with halloysite nanoclay-ionic liquid (HNT/IL) nanocomposite. The characterization of surface was performed by Scanning Electron Microscope (SEM) images and Energy Dispersive X-Ray Analysis (EDX) analysis, and the differences at surface modifications were also shown by electrochemical methods with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For sensitive and selective determination of miRNA-21, Pinosine and target miRNA concentration, immobilization and hybridization time were optimized by using HNT/IL modified PGE in combination with differential pulse voltammetry (DPV). The detection limit was achieved as 0.17 μg/mL (equals to 23.69 nM) in the linear range of 0.25–2 μg/mL miRNA-21. The selectivity of voltammetric method based on HNT/IL-PGE developed for miRNA-21 was examined in the presence of mismatch (MM) and non-complementary (NC) sequences. Because miRNA-21 is over-expressed in cancer cells, it has been tested in total RNA samples isolated from cancer cell line (breast cancer cell line, MCF-7). In the total RNA samples obtained from MCF-7, the detection limit was calculated as 0.28 μg/mL in the linear range of 1–4 μg/mL. Besides, the healthy cell line (human embryonic kidney cell line, HEK-293) was used as a control group and the results obtained by MCF-7 total RNA samples were compared to the results using HEK-293 total RNA samples in terms of miRNA-21 level.

Towards Personalized Allele-Specific Antisense Oligonucleotide Therapies for Toxic Gain-of-Function Neurodegenerative Diseases.

Antisense oligonucleotides (ASOs) are single-stranded nucleic acid strings that can be used to selectively modify protein synthesis by binding complementary (pre-)mRNA sequences. By specific arrangements of DNA and RNA into a chain of nucleic acids and additional modifications of the backbone, sugar, and base, the specificity and functionality of the designed ASOs can be adjusted. Thereby cellular uptake, toxicity, and nuclease resistance, as well as binding affinity and specificity to its target (pre-)mRNA, can be modified. Several neurodegenerative diseases are caused by autosomal dominant toxic gain-of-function mutations, which lead to toxic protein products driving disease progression. ASOs targeting such mutations—or even more comprehensively, associated variants, such as single nucleotide polymorphisms (SNPs)—promise a selective degradation of the mutant (pre-)mRNA while sparing the wild type allele. By this approach, protein expression from the wild type strand is preserved, and side effects from an unselective knockdown of both alleles can be prevented. This makes allele-specific targeting strategies a focus for future personalized therapies. Here, we provide an overview of current strategies to develop personalized, allele-specific ASO therapies for the treatment of neurodegenerative diseases, such Huntington’s disease (HD) and spinocerebellar ataxia type 3 (SCA3/MJD).

The L1-ORF1p coiled coil enables formation of a tightly compacted nucleic acid-bound complex that is associated with retrotransposition.

Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.

Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociationand ssDNA binding kinetics.

SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart. Although phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (∼15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial dynamics in the C-terminal domain (which contains T592), which could be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.

CRISPR-Cas: the bacterial immunity that supports diagnostic in virology

CRISPR-Cas is an adaptive immune system that prevents bacteria and archea from nucleic acids invasion such as viral genomes. The ability of the CRISPR-Cas technology to effectively and precisely cut a targeted genomic DNA region was exploited to develop powerful genome editing tools that were adapted for a wide range of applications, revolutionizing biological sciences. The CRISPR-Cas system consists of a Cas endonuclease triggered by a RNA guide for highly specific cleavage of targeted DNA or RNA sequences. In addition to the target specific cleavage, some Cas enzymes, including Cas12a and Cas13a, display a collateral trans-cleavage activity that allows the cleavage of all surrounding single-stranded nucleic acids. These biosensing activities of CRISPR-Cas systems, based on target specific binding and cleavage, are promising tools to develop accurate diagnostic methods to detect specific nucleic acids. CRISPRCas could therefore be used to diagnose a wide variety of diseases. In the current review we propose to describe the more significant advances for virus detection based on CRISPR-Cas systems.

A versatile strategy for convenient circular bivalent functional nucleic acids construction.

Functional nucleic acids (FNAs), such as aptamers, nucleic acid enzymes and riboswitches play essential roles in various fields of life sciences. Tailoring of ingenious chemical moieties toward FNAs can enhance their biomedical properties and/or confer them with exogenic biological functions that, in turn, can considerably expand their biomedical applications, or even improve their clinical translations. Herein, we report the first example of a general chemical tailoring strategy that enables the divergent ligation of DNA sequences. By applying this technology, different types of aptamers and single-stranded nucleic acids of various lengths could be efficiently tailored to deliver the designed circular bivalent aptamers (CBApts) and cyclized DNA sequences with high yields. It is worth noting that CBApts exhibited significantly enhanced nuclease resistance, as well as considerably improved binding, targeting and tumor tissue enrichment abilities, which may pave the way for different investigations for biomedical purposes.

High sensitivity and fast response optical fiber nucleic acid sensor

A label-free optical fiber Mach-Zehnder interferometer (MZI) based biosensor was proposed for the specificity detection of single-stranded nucleic acid (NA). The MZI was made of a fiber sandwich structure by a core-offset fusion splicing means. The piranha solution, APTES (3-Aminopropyltriethoxylsilan) and GA (Glutaraldehyde) were used for the chemical modification of the optical fiber surface, and then the single-stranded probe NA with the known sequence was fixed on the optical fiber surface by covalent fixation method. The specificity of complementary target NA could be detected through the changing of the spectra of the MZI. Thanks to the high sensitivity of the MZI and the high fixation efficiency of the covalent fixation method, the optical fiber MZI based NA sensor showed a fast response of 30 s and a high sensitivity of 0.175 dB/nM with a minimum detection concentration of 1 nM.

Surface acoustic wave-assisted microfluidic isolation of aptamers.

Aptamers are synthetic single-stranded nucleic acid molecules that bind to biochemical targets with high affinity and specificity. The method of systematic evolution of ligands by exponential enrichment (SELEX) is widely used to isolate aptamers from randomized oligonucleotides. Recently, microfluidic technology has been applied to improve the efficiency and reduce the cost in SELEX processes. In this work, we present an approach that exploits surface acoustic waves to improve the affinity selection process in microfluidic SELEX. Acoustic streaming is used to enhance the interactions of the solution-based oligonucleotide molecules with microbead-immobilized target molecules, allowing the identification of high-affinity aptamer candidates in a more efficient manner. For demonstration, a DNA aptamer is isolated within three rounds of selection in 5 h to specifically bind to immunoglobulin E, a representative target protein, with an equilibrium dissociation constant of approximately 22.6 nM.

Single-stranded nucleic acid binding and coacervation by linker histone H1.

The H1 linker histone family is the most abundant group of eukaryotic chromatin-binding proteins. However, their contribution to chromosome structure and function remains incompletely understood. Here we use single-molecule fluorescence and force microscopy to directly visualize the behavior of H1 on various nucleic acid and nucleosome substrates. We observe that H1 coalesces around single-stranded DNA generated from tension-induced DNA duplex melting. Using a droplet fusion assay controlled by optical tweezers, we find that single-stranded nucleic acids mediate the formation of gel-like H1 droplets, whereas H1-double-stranded DNA and H1-nucleosome droplets are more liquid-like. Molecular dynamics simulations reveal that multivalent and transient engagement of H1 with unpaired DNA strands drives their enhanced phase separation. Using eGFP-tagged H1, we demonstrate that inducing single-stranded DNA accumulation in cells causes an increase in H1 puncta that are able to fuse. We further show that H1 and Replication Protein A occupy separate nuclear regions, but that H1 colocalizes with the replication factor Proliferating Cell Nuclear Antigen, particularly after DNA damage. Overall, our results provide a refined perspective on the diverse roles of H1 in genome organization and maintenance, and indicate its involvement at stalled replication forks.

Aptamer‑based therapy for targeting key mediators of cancer metastasis (Review).

Cancer-related deaths remain a challenging and devastating obstacle to defeat despite the tremendous advances in cancer treatment. Cancer metastasis is the major cause of these cancer-related deaths. Metastasis involves sequential steps during cancer cells' journey to a new site. These steps are coordinately regulated by specific intracellular regulators and cellular interactions between the cancer cells and the supporting microenvironment of the different organs. The development of aptamer-based therapeutics is a promising strategy to fight cancer metastasis as it holds potential advantages. Oligonucleotide and peptide aptamers are short sequences of single-stranded nucleic acids or amino acids, respectively, that target proteins, genetic materials, and cells. Antimetastatic aptamer-based therapeutics exert their pharmacological effect by direct interaction with the signaling pathways inside the cancer cells or the communications between cancer cells and the tumor microenvironment. In addition, aptamers have been utilized as a guiding ligand to deliver a therapeutic moiety to cancer cells or the supporting microenvironment. The selected aptamer possesses high specificity since it is designed to recognize and interact with its target. This review summarizes recent advances in the development of aptamer-based therapeutics targeting mediators of cancer metastasis. In addition, potential opportunities are discussed to inspire researchers in the field to develop novel aptamer-based antimetastatic treatments.

Nanocarrier based on halloysite and fluorescent probe for intracellular delivery of peptide nucleic acids

The development of systems able to deliver genetic material into a target site is a challenge for modern medicine. Single-stranded peptide nucleic acids have attracted attention as promising therapeutic molecules for diagnostic and gene therapy. However, their poor cell membrane permeability represents a drawback for biomedical applications. Halloysite nanotubes (HNTs) are emerging materials in drug delivery applications both for their ability to penetrate cell membranes and for enhancing the solubility of drugs in biological media. Herein, we report the first example of the use of a nanocarrier based on halloysite labelled with fluorescent switchable halochromic oxazine molecules, to deliver a single-stranded peptide nucleic acids tetramer (PNAts) into living cells. The PNAts is covalently attached to halloysite (HNTs-PNA), whereas the fluorescent probe supramolecularly interacts with HNTs. The ability of the nanomaterial to bind complementary single-stranded DNA was assessed by resonance light scattering measurements. Finally, studies of cellular uptake were carried out by confocal laser scanning microscopy on normal and tumoral cell lines. This work highlights the usefulness of the covalent approach to generate HNTs-PNA nanomaterials for the potential targeting of future specific nucleic acids in living cells, which could open the doorway to novel possibilities for theranostic and gene therapy applications.

Ultrasensitive multiplexed detection of small molecules and enzymes using stimuli-responsive nucleic acids

Small biomolecules and enzymes play essential roles in regulating various physiological functions and are highly correlated with many diseases, making them promising biomarkers for disease diagnosis. However, it remains challenging to achieve multiplexed detection of trace enzymes and small molecules due to the limited signal transformation and amplification capabilities in existing technologies. Here, we developed a signal transformation and amplification strategy based on stimuli-responsive nucleic acids (SR-NAs) by incorporating stimuli-responsive linker in single-stranded nucleic acid via click chemistry. The targets (e.g., enzymes, small molecules) could recognize the responsive linker and trigger the degradation of the SR-NAs, thus transforming the targets into DNA signals by generating new toehold nucleic acid strands, which were further augmented by nucleic acid amplification. We demonstrated that this strategy significantly improved the detection precision for small biomolecules (H2O2) and enzymes (esterase, β-galactosidase). Furthermore, the signal transformation function of SR-NA enables multiplexed detection via logical operation with multiple small molecules and enzymes as inputs, strand displacement reaction as Boolean logic-based algorithm, and fluorescence signal as output signal, as demonstrated by multiplexed detection of esterase and H2O2. More importantly, this method was capable of realizing ultrasensitive intracellular H2O2 detection. We envision that this SR-NA-based strategy could be expanded to detect other kinds of targets by designing self-immolative linkers, and thus provides a powerful toolbox for bioanalytical and biomedical applications.

Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Absorption, distribution, metabolism, and excretion (ADME) are the key biologic processes for determination of a drug's pharmacokinetic parameters, which have direct impacts on efficacy and adverse drug reactions (ADRs). The chemical structures, dosage forms, and sites and routes of administration are the principal determinants of ADME profiles and consequent impacts on their efficacy and ADRs. Newly developed large molecule biologic antisense oligonucleotide (ASO) drugs have completely unique ADME that is not fully defined. ASO-based drugs are single-stranded synthetic antisense nucleic acids with diverse modes of drug actions from induction of mRNA degradation, exon skipping and restoration, and interactions with proteins. ASO drugs have a great potential to treat certain human diseases that have remained untreatable with small molecule-based drugs. The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the 10 US Food and Drug Administration (FDA)-approved ASO drugs were selected: fomivirsen, pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. A meta-analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization through endocytosis and nucleolytic degradation by endonucleases and exonucleases are major ADME features of the ASO drugs that differ from small-molecule drugs. The information summarized here provides comprehensive ADME characteristics of FDA-approved ASO drugs, leading to a better understanding of their therapeutic efficacy and their potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distribution, are identified, and future perspectives and directions are discussed. SIGNIFICANCE STATEMENT: Through a systematic analysis of the existing information of absorption, distribution, metabolism, and excretion (ADME) parameters for 10 US Food and Drug Administration (FDA)-approved antisense oligonucleotide (ASO) drugs, this review provides an overall view of the unique ADME characteristics of ASO drugs, which are distinct from small chemical drug ADME. This knowledge is useful for discovery and development of new ASO drugs as well as clinical use of current FDA-approved ASO drugs.

In vitro Selection of High Affinity DNA and RNA Aptamers that Detect Hepatitis C Virus Core Protein of Genotypes 1 to 4 and Inhibit Virus Production in Cell Culture

Hepatitis C virus (HCV) core is a highly conserved and multifunctional protein that forms the viral capsid, making it an attractive target for HCV detection and inhibition. Aptamers are in vitro selected, single-stranded nucleic acids (RNA or ssDNA) with growing applicability in viral diagnostics and therapy. We have carried out DNA and RNA in vitro selection against six different variants of HCV core protein: two versions of the full-length protein of genotype 1, and the hydrophilic domain of genotypes 1 to 4. The aptamer populations obtained were analyzed by means of Ultra-Deep Sequencing (UDS), the most abundant sequences were identified and a number of highly represented sequence motifs were unveiled. Affinity (measured as the dissociation constant, Kd) of the most abundant DNA and RNA aptamers were quantified using Enzyme-Linked OligoNucleotide Assay (ELONA)-based methods. Some aptamers with nanomolar or subnanomolar Kd values (as low as 0.4 nM) were the common outcome of DNA and RNA selections against different HCV core variants. They were tested in sandwich and competitive biosensor assays, reaching a limit of detection for HCV core of 2 pM. Additionally, the two most prevalent and high affinity aptamers were assayed in Huh-7.5 reporter cell lines infected with HCV, where they decreased both the viral progeny titer and the extracellular viral RNA level, while increasing the amount of intracellular viral RNA. Our results suggest that these aptamers inhibit HCV capsid assembly and virion formation, thus making them good candidate molecules for the design of novel therapeutic approaches for hepatitis C.

Enhancing Peptide Nucleic Acid–Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects

Single-stranded peptide nucleic acid (PNA) probes interact strongly with several nanomaterials, and the interaction was diminished in the presence of complementary nucleic acid targets which forms the basis of many nucleic acid sensing platforms. As opposed to the negatively charged DNA probes, the charges on the PNA probes may be fine-tuned by incorporating amino acids with charged side chains. The contribution of electrostatic effects to the interaction between PNA probes and nanomaterials has been largely overlooked. This work reveals that electrostatic effects substantially enhanced the quenching of dye-labeled conformationally constrained pyrrolidinyl PNA probes by several nanomaterials including graphene oxide (GO), reduced graphene oxide, gold nanoparticles (AuNPs), and silver nanoparticles. The fluorescence quenching and the color change from red to purple in the case of AuNPs because of aggregation were inhibited in the presence of complementary nucleic acid targets. Thus, fluorescence and colorimetric assays for DNA and RNA that can distinguish even single-base-mismatched nucleic acids with improved sensitivity over conventional DNA probes were established. Both the GO- and AuNP-based sensing platforms have been successfully applied for the detection of real DNA and RNA samples in vitro and in living cells. This study emphasizes the active roles of electrostatic effects in the PNA-nanomaterial interactions, which paves the way toward improving the performance of PNA-nanomaterial based assays of nucleic acids.

Electrophoretic study of G-quadruplex aptamer interactions with different short single-strand complementary oligonucleotides

Aptamers are single-stranded nucleic acids, typically 20-80 nucleobases (nb) in length, which can bind different compounds with high affinity and selectively. Their ligand-binding properties can be attenuated by adding short complementary strands. These interactions open new opportunities for aptamer-based assays. Strong dependence between the length and electrophoretic mobility of short nucleic acids makes polyacrylamide gel electrophoresis a powerful tool for studying their complexes. The interactions between the 36 nb DNA G-quadruplex aptamer (5’-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3’) specific to ochratoxin A and 9 complementary single-stranded DNA (ssDNA) were studied. The length of ssDNA varied from 5 to 9 nb. To maintain ligand-binding conformation of the aptamer, a high ionic strength buffer was used. The best resolution between the aptamer and its complex was provided for the gel with 15% monomer and a monomer/cross-linker ratio of 15:1. Bands of free aptamer and ssDNA were observed for all studied variants. If the ssDNA length was less than 9 nb, the position of the aptamer’s band remained unchanged, independent of the aptamer/ssDNA ratios, and additional bands did not appear. The longest ssDNA (5’-CGC-CAC-CCA-3’) did not lead to the appearance of a new band, but it slowed the aptamer’s migration depending on the ssDNA concentration. Under a 27-fold excess of the given ssDNA, the relative mobility of the aptamer band changed from 0.566 to 0.468. Thus, electrophoresis visualizes aptamer-ssDNA interactions and can be used in the development of aptamer-based analytical systems.

Single-stranded nucleic acid sensing and coacervation by linker histone H1

The linker histone H1 family is the most abundant group of eukaryotic chromatin-binding proteins, in which H1 is known for its role as a nucleosome compactor and an essential component of higher order chromatin structure. However, the mechanism underlying the diverse physiological functions of H1 remains unclear. Here we used single-molecule fluorescence and force microscopy to observe the behavior of H1 on DNA under different tensions in real time. Unexpectedly, we observed that H1 preferentially coalesces around nascent single-stranded DNA, forming condensate-like complexes.

Recognition of single-stranded nucleic acids by small-molecule splicing modulators

Spinal muscular Atrophy (SMA) is one of the most lethal genetic disorders to manifest in children. Restoring proper splicing of the exon 7 of SMN2 gene to compensate for the loss of the SMN1 gene, serves a promising strategy in the direction of treating SMA using splicing modulators. Risdiplam is the first approved small-molecule splicing modulator for treating SMA. The analogues of risdiplam bind to two distinct binding sites on the exon 7 of the SMN pre-mRNA. A 5' splice site and a GA rich binding site located ∼24 nts located upstream to the 5'splice site.

Aptamer-Enabled Nanomaterials for Therapeutics, Drug Targeting and Imaging.

A wide variety of nanomaterials have emerged in recent years with advantageous properties for a plethora of therapeutic and diagnostic applications. Such applications include drug delivery, imaging, anti-cancer therapy and radiotherapy. There is a critical need for further components which can facilitate therapeutic targeting, augment their physicochemical properties, or broaden their theranostic applications. Aptamers are single-stranded nucleic acids which have been selected or evolved to bind specifically to molecules, surfaces, or cells. Aptamers can also act as direct biologic therapeutics, or in imaging and diagnostics. There is a rich field of discovery at the interdisciplinary interface between nanomaterials and aptamer science that has significant potential across biomedicine. Herein, we review recent progress in aptamer-enabled materials and discuss pending challenges for their future biomedical application.