Single-stranded Nucleic Acids Research Articles

Responsive Nucleic Acid-Based Organosilica Nanoparticles.

The development of smart nanoparticles (NPs) that encode responsive features in the structural framework promises to extend the applications of NP-based drugs, vaccines, and diagnostic tools. New nanocarriers would ideally consist of a minimal number of biocompatible components and exhibit multiresponsive behavior to specific biomolecules, but progress is limited by the difficulty of synthesizing suitable building blocks. Through a nature-inspired approach that combines the programmability of nucleic acid interactions and sol-gel chemistry, we report the incorporation of synthetic nucleic acids and analogs, as constitutive components, into organosilica NPs. We prepared different nanomaterials containing single-stranded nucleic acids that are covalently embedded in the silica network. Through the incorporation of functional nucleic acids into the organosilica framework, the particles respond to various biological, physical, and chemical inputs, resulting in detectable physicochemical changes. The one-step bottom-up approach used to prepare organosilica NPs provides multifunctional systems that combine the tunability of oligonucleotides with the stiffness, low cost, and biocompatibility of silica for different applications ranging from drug delivery to sensing.

A Universal Hydrogel Microneedle for on-Site Detection of Small Molecules, Proteins, and Ribonucleic Acids

Point-of-care testing (POCT) of clinical biomarkers is critical to health monitoring and timely treatment, yet biosensing assays capable of detecting biomarkers without the need for costly external equipment and reagents are limited. Blood-based assays are, specifically, challenging as blood collection is invasive, and several processing steps are required. A promising alternative to blood is interstitial fluid (ISF), which originates from blood and fills the extracellular space; thus, it has common biomarkers with plasma/serum while also containing biomarkers unique to the local cells. A wide range of metabolites, including amino acids, lipids, nucleotides, and protein biomarkers, can be detected in ISF, emphasizing the potential of ISF for health monitoring. Microneedle (MN)-based biosensors have recently emerged as a promising approach for investigating the ISF. They enable minimally invasive penetration through the skin for ISF access. Specifically, Hydrogel-based MNs have two main advantages; i) their fabrication is cost-effective and simple, ii) they are highly biocompatible. Therefore, they overcome the challenges with conventional solid MNs. However, Hydrogel Microneedles (HMN) capable of both extracting ISF and sensing an analyte on-microneedle (without any need for post-sampling processing) have been limited. Here, we report a versatile assay that employs HMNs to both extract (ISF) and perform on-MN sensing of a specific analyte/biomarker in a minimally invasive manner. Our assay incorporates graphene oxide-nucleic acid (GO.NA) optical sensors for sensing. The GO.NA optical sensor consists of GO nanosheets conjugated to fluorophore-modified nucleic acid (NA), in which GO acts as a quencher. Single-stranded NAs have high affinity for binding to GO, therefore, in the absence of the biomarker of interest the NAs are tightly bound to GO; in turn, their fluorophore tag is quenched. However, in the presence of a specific biomarker, the NAs bind to their target, inducing a conformational change that distances the fluorophore tag from GO, leading to fluorescence recovery and generation of an optical signal. GO also acts as a substrate for NA immobilization; therefore, it protects the NA from degradation and prevents any potential NA release from HMN-GO.NA. To enable microscope-free patch visualization and optical measurements for POCT, we developed a miniaturized smartphone-based system that captures fluorescence images of the HMN-GO.NA patches (Scheme 1A and 1B), which are then analyzed using freely available software (ImageJ). Our system could successfully measure six clinically important biomarkers (glucose, uric acid, insulin, and serotonin as well as microribonucleic acid 210 and 21) in-vitro (using phantom gel) and ex-vivo (using porcine skin). Further, we have been able to accurately detect glucose and uric acid in live diabetic animal models, proving the efficacy of our system. Figure 1

Fluorescent nanostructured carbon dot-aptasensor for chlorpyrifos detection: Elucidating the interaction mechanism for an environmentally hazardous pollutant

Chlorpyrifos (CPF) is a commonly used insecticide found in many water sources and is related to several health and environmental effects. Biosensors based on aptamers (single-stranded nucleic acid oligonucleotides) are promising alternatives to achieve the detection of CPF and other pesticides in natural waters. However, several challenges need to be addressed to promote the real application of functional aptasensing devices. In this work, an ssDNA aptamer (S1) is combined with carbon quantum dots (CD) and graphene oxide (GO) to produce a stable fluorescent aptasensor characterized through spectrophotometric and electrophoretic techniques. For a deeper understanding of the system, the mechanism of molecular interaction was studied through docking modeling using free bioinformatic tools like HDOCK, showing that the stem-loops and the higher guanine (G) content are crucial for better interaction. The model also suggests the possibility of generating a truncated aptamer to improve the binding affinity. The biosensor was evaluated for CPF detection, showing a low LOD of 0.01 μg L−1 and good specificity in tap water, even compared to other organophosphates pesticides (OPs) like profenofos. Finally, the recovery of the proposed aptasensor was evaluated in some natural water using spiked samples and compared with UPLC MS-MS chromatography as the gold standard, showing a good recovery above 2.85 nM and evidencing the need of protecting ssDNA aptamers from an erratic interaction with the aromatic groups of dissolved organic matter (humic substances). This work paves the way for a better aptasensors design and the on-site implementation of novel devices for environmental monitoring.

Surfactant-mediated colorimetric assay assisted with in-situ rolling circle amplification on magnetic beads

Gold nanoparticles (AuNPs) with localized surface plasmon resonance effect have been widely used for colorimetric detection based on the interparticle plasmon coupling during AuNPs aggregation. However, it is still challenging to develop portable and quantitative methods with good sensitivity and excellent selectivity. In this study, a smartphone-based colorimetric assay is developed on the principle of surfactant-mediated AuNPs aggregation assisted with rolling circle amplification (RCA) on magnetic beads (MBs). The detection of adenosine is demonstrated as an example. The cetyl trimethyl ammonium bromide (CTAB) causes the negatively charged AuNPs to aggregate, which results in the color change from red to blue. When adenosine is in solution, the RCA process is triggered on the MBs because of specific adenosine-aptamer recognition, resulting in prolongation of single-stranded nucleic acid (ssDNA). The solution color remains red due to the electrostatic interaction between CTAB and ssDNA. Using this method, the limit of detection (LOD) for adenosine can be as low as 16 pM. Besides, it also works well in human serum. In addition, a portable device integrated with in-situ RGB analysis software is developed for the detection with a smartphone. This study offers a new strategy to improve the sensitivity and selectivity for the AuNPs-based colorimetric assay, taking advantages of specific aptamer recognition, in-situ RCA on MBs, magnetic separation, and smartphone-based portable device.

Exploring the Potential of Aptamers in Targeting Neuroinflammation and Neurodegenerative Disorders: Opportunities and Challenges.

Neuroinflammation is the precursor for several neurodegenerative diseases (NDDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Targeting neuroinflammation has emerged as a promising strategy to address a wide range of CNS pathologies. These NDDs still present significant challenges in terms of limited and ineffective diagnosis and treatment options, driving the need to explore innovative and novel therapeutic alternatives. Aptamers are single-stranded nucleic acids that offer the potential for addressing these challenges through diagnostic and therapeutic applications. In this review, we summarize diagnostic and therapeutic aptamers for inflammatory biomolecules, as well as the inflammatory cells in NDDs. We also discussed the potential of short nucleotides for Aptamer-Based Targeted Brain Delivery through their unique features and modifications, as well as their ability to penetrate the blood-brain barrier. Moreover, the unprecedented opportunities and substantial challenges of using aptamers as therapeutic agents, such as drug efficacy, safety considerations, and pharmacokinetics, are also discussed. Taken together, this review assesses the potential of aptamers as a pioneering approach for target delivery to the CNS and the treatment of neuroinflammation and NDDs.

Blended Block Polycation Micelles Enhance Antisense Oligonucleotide Delivery.

Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which are short single-stranded nucleic acids that downregulate protein production by binding to mRNA. However, ASOs cannot enter the cell without a delivery vehicle. Diblock polymers containing cationic and hydrophobic blocks self-assemble into micelles that have shown improved delivery compared to linear nonmicelle variants. Yet synthetic and characterization bottlenecks have hindered rapid screening and optimization. In this study, we aim to develop a method to increase throughput and discovery of new micelle systems by mixing diblock polymers together to rapidly form new micelle formulations. We synthesized diblocks containing an n-butyl acrylate block chain extended with cationic moieties amino ethyl acrylamide (A), dimethyl amino ethyl acrylamide (D), or morpholino ethyl acrylamide (M). These diblocks were then self-assembled into homomicelles (A100, D100, and M100)), mixed micelles comprising 2 homomicelles (MixR%+R'%), and blended diblock micelles comprising 2 diblocks blended into one micelle (BldR%R'%) and tested for ASO delivery. Interestingly, we observed that mixing or blending M with A (BldA50M50 and MixA50+M50) did not improve transfection efficiency compared to A100; however, when M was mixed with D, there was a significant increase in transfection efficacy for the mixed micelle MixD50+M50 compared to D100. We further examined mixed and blended D systems at different ratios. We observed a large increase in transfection and minimal change in toxicity when M was mixed with D at a low percentage of D incorporation in mixed diblock micelles (i.e., BldD20M80) compared to D100 and MixD20+M80. To understand the cellular mechanisms that may result in these differences, we added proton pump inhibitor Bafilomycin-A1 (Baf-A1) to the transfection experiments. Formulations that contain D decreased in performance in the presence of Baf-A1, indicating that micelles with D rely on the proton sponge effect for endosomal escape more than micelles with A. This result supports our conclusion that M is able to modulate transfection of D, but not with A. This research shows that polymer blending in a manner similar to that of lipids can significantly boost transfection efficiency and is a facile way to increase throughput of testing, optimization, and successful formulation identification for polymeric nucleic acid delivery systems.

Application and Immunogenicity of Aptamers in Cancer Therapy

Aptamers are short single-stranded nucleic acid (DNA or RNA) or peptide sequences with a unique three-dimensional structure that bind to their targets with high specificity and affinity. Aptamers’ small size, fast and cheap production process, low immunogenicity, and high stability have made them attractive molecules in some fields of research, especially in the design of new diagnostic and therapeutic pathways. This review article discusses aptamers’ applications and immunogenic properties in the pharmaceutical sciences.

Using Exonucleases for Aptamer Characterization, Engineering, and Sensing.

ConspectusAptamers are short, single-stranded nucleic acids that have been selected from random libraries to bind specific molecules with high affinity via an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). They have been generated for diverse targets ranging from metal ions to small molecules to proteins and have demonstrated considerable promise as biorecognition elements in sensors for applications including medical diagnostics, environmental monitoring, food safety, and forensic analysis. While aptamer sensors have made great strides in terms of sensitivity, specificity, turnaround time, and ease of use, several challenges have hindered their broader adoption. These include inadequate sensitivity, bottlenecks in aptamer binding characterization, and the cost and labor associated with aptamer engineering. In this Account, we describe our successes in using nuclease enzymes to address these problems. While working with nucleases to enhance the sensitivity of split aptamer sensors via enzyme-assisted target recycling, we serendipitously discovered that the digestion of DNA aptamers by exonucleases is inhibited when an aptamer is bound to a ligand. This finding served as the foundation for the development of three novel aptamer-related methodologies in our laboratory. First, we used exonucleases to truncate nonessential nucleotides from aptamers to generate structure-switching aptamers in a single step, greatly simplifying the aptamer engineering process. Second, we used exonucleases to develop a label-free aptamer-based detection platform that can utilize aptamers directly obtained from in vitro selection to detect analytes with ultralow background and high sensitivity. Through this approach, we were able to detect analytes at nanomolar levels in biological samples, with the capacity for achieving multiplexed detection by using molecular beacons. Finally, we used exonucleases to develop a high throughput means of characterizing aptamer affinity and specificity for a variety of ligands. This approach has enabled more comprehensive analysis of aptamers by greatly increasing the number of aptamer candidates and aptamer-ligand pairs that can be tested in a single experiment. We have also demonstrated the success of this method as a means for identifying new mutant aptamers with augmented binding properties and for quantifying aptamer-target affinity. Our enzymatic technologies can greatly streamline the aptamer characterization and sensor development process, and with the adoption of robotics or liquid handling systems in the future, it should be possible to rapidly identify the most suitable aptamers for a particular application from hundreds to thousands of candidates.

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GENE THERAPY Reactions ShareShare onFacebookTwitterWechatLinked InRedditEmail C&EN, 2023, 101 (18), p 3June 5, 2023Cite this:C&EN 101, 18, 3(Credit: C&EN)Figure1of2(Credit: C&EN)Figure1of2Letters to the editorPeptide nucleic acids targeting noncanonical DNA and RNAAs a molecular biologist, I always enjoy reading articles concerning DNA and RNA. Your excellent article in the April 3, 2023, issue of C&EN (page 18) titled “The Promise of Peptide Nucleic Acids” discussed an important topic relating to gene therapy—that is, antisense therapeutics and gene- editing tools. Peptide nucleic acids (PNAs) can open a double-stranded (ds) DNA molecule and inhibit its gene expression. PNAs bind to DNA and RNA with high specificity and selectivity, resulting in PNA-DNA and PNA-RNA hybrid structures. Therefore, they have exciting potential as diagnostic tools and therapeutic agents.However, only briefly discussed in the article is the possibility of developing PNAs to target nucleic acid secondary structures such as RNA hairpins. More focus needs to be placed on all the other nucleic acid secondary structures, going beyond canonical right-handed ds-B-DNA, right-handed ds-A-RNA, and single-stranded (such as messenger RNA) nucleic acids based on nucleotide sequences—that is, the hybridization of base pairs.Researchers need to consider targeting exotic, noncanonical nucleic acids—that is, alternative and multistranded DNA and RNA structures. Local transitions in the structure of canonical ds-B-DNA are functionally significant biological molecules, regulating many cellular processes, such as transcription and translation. These exotic nucleic acid structures have also been connected to genetic instability in the presence or deficiency of DNA damage, with or without DNA replication. DNA and RNA possess dynamic structural variability resulting in a variety of unusual nucleic acid structures, such as cruciform DNA, DNA-RNA hybrids, slipped-strand DNA, and parallel-stranded DNA.Expanding the therapeutic capabilities of PNAs beyond canonical B-DNA would expand PNAs’ use in therapeutics. The expansion should include alternative ds-DNA structures such as left-handed ds-Z-DNA and left-handed ds-Z-RNA. It should also include multistranded DNA structures such as triplex DNA (intermolecular and intramolecular, like H-DNA), G4-quadruplex DNA, and i-motif DNA.If possible, targeting noncanonical nucleic acid structures with PNAs would increase the chance of developing novel therapeutics that can target all genomic DNA and RNA structures in genes and telomeres. We are witnessing the transition of gene medicine from an experimental technology into a real-world, practical approach for developing nucleic acid–based therapeutics, such as used for gene silencing or gene editing (like CRISPR) of pathological conditions.Using PNAs as therapeutic tools for targeting both canonical and noncanonical nucleic acids will allow for their full potential and optimal expansion of gene therapy.Claude GagnaBronxville, New YorkRecognizing nitrogen-fixing bacteria’s potential to reduce fertilizer useIn the article titled “Growing More Food with Less Chemicals,” which appeared in the May 8, 2023, issue of C&EN (page 28), there was a glaring omission.What was left out was the research and development of genetically modified nitrogen-fixing bacteria that can be applied to seed, foliage, or soil to reduce fertilizer use.Herbert B. ScherMoraga, California

Inhaled aptamer therapy: A hopeful therapy for lung disease

Inhaled aptamer therapy: A hopeful therapy for lung disease

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

RPA engages telomeric G-quadruplexes more effectively than CST.

G-quadruplexes (G4s) are a set of stable secondary structures that form within guanine-rich regions of single-stranded nucleic acids that pose challenges for DNA maintenance. The G-rich DNA sequence at telomeres has a propensity to form G4s of various topologies. The human protein complexes Replication Protein A (RPA) and CTC1-STN1-TEN1 (CST) are implicated in managing G4s at telomeres, leading to DNA unfolding and allowing telomere replication to proceed. Here, we use fluorescence anisotropy equilibrium binding measurements to determine the ability of these proteins to bind various telomeric G4s. We find that the ability of CST to specifically bind G-rich ssDNA is substantially inhibited by the presence of G4s. In contrast, RPA tightly binds telomeric G4s, showing negligible changes in affinity for G4 structure compared to linear ssDNAs. Using a mutagenesis strategy, we found that RPA DNA-binding domains work together for G4 binding, and simultaneous disruption of these domains reduces the affinity of RPA for G4 ssDNA. The relative inability of CST to disrupt G4s, combined with the greater cellular abundance of RPA, suggests that RPA could act as a primary protein complex responsible for resolving G4s at telomeres.

Coordination-Driven One-Step Rapid Self-Assembly Synthesis of Dual-Functional Ag@Pt Nanozyme.

Realizing high-precise and adjustable regulation of engineering nanozyme is important in nanotechnology. Here, Ag@Pt nanozymes with excellent peroxidase-like and antibacterial effects are designed and synthesized by nucleic acid and metal ions coordination-driven one-step rapid self-assembly. The adjustable NA-Ag@Pt nanozyme is synthesized within 4min using single-stranded nucleic acid as templates, and peroxidase-like enhancing FNA-Ag@Pt nanozyme is received by regulating functional nucleic acids (FNA) based on NA-Ag@Pt nanozyme. Both Ag@Pt nanozymes that are developed not only has simple and general synthesis approaches, but also can produce artificial precise adjustment and possess dual-functional. Moreover, when lead ion-specific aptamers as FNA are introduced to NA-Ag@Pt nanozyme, the Pb2+ aptasensor is successfully constructed by increasing electron conversion efficiency and improving the specificity of nanozyme. In addition, both nanozyme has good antibacterial properties, with ~100% and ~85% antibacterial efficiency against Escherichia coli and Staphylococcus aureus, respectively. This work provides a synthesis method of novelty dual-functional Ag@Pt nanozymes and successful application in metal ions detection and antibacterial agents.

DNA-Based Nanomaterials as Drug Delivery Platforms for Increasing the Effect of Drugs in Tumors

DNA nanotechnology has significantly advanced and might be used in biomedical applications, drug delivery, and cancer treatment during the past few decades. DNA nanomaterials are widely used in biomedical research involving biosensing, bioimaging, and drug delivery since they are remarkably addressable and biocompatible. Gradually, modified nucleic acids have begun to be employed to construct multifunctional DNA nanostructures with a variety of architectural designs. Aptamers are single-stranded nucleic acids (both DNAs and RNAs) capable of self-pairing to acquire secondary structure and of specifically binding with the target. Diagnosis and tumor therapy are prospective fields in which aptamers can be applied. Many DNA nanomaterials with three-dimensional structures have been studied as drug delivery systems for different anticancer medications or gene therapy agents. Different chemical alterations can be employed to construct a wide range of modified DNA nanostructures. Chemically altered DNA-based nanomaterials are useful for drug delivery because of their improved stability and inclusion of functional groups. In this work, the most common oligonucleotide nanomaterials were reviewed as modern drug delivery systems in tumor cells.

Optical, structuraland antibacterial properties of silver nanoparticles and DNA-templated silver nanoclusters.

Silver nanoparticles (AgNPs) are increasingly considered for biomedical applications as drug-delivery carriers, imaging probesand antibacterial agents. Silver nanoclusters (AgNCs) represent another subclass of nanoscale silver. AgNCs are a promising tool for nanomedicine due to their small size, structural homogeneity, antibacterial activityand fluorescence, which arises from their molecule-like electron configurations. The template-assisted synthesis of AgNCs relies on organic molecules thatact as polydentate ligands. In particular, single-stranded nucleic acids reproducibly scaffold AgNCs to provide fluorescent, biocompatible materials thatare incorporable in other formulations. This mini review outlines the design and characterization of AgNPs and DNA-templated AgNCs, discusses factors that affect their physicochemical and biological properties, and highlights applications of these materials as antibacterial agents and biosensors.

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis.

The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improvement of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

Filament assembly underpins the double-stranded DNA specificity of AIM2-like receptors.

Upon sensing cytosolic- and/or viral double-stranded (ds)DNA, absent-in-melanoma-2 (AIM2)-like-receptors (ALRs) assemble into filamentous signaling platforms to initiate inflammatory responses. The versatile yet critical roles of ALRs in host innate defense are increasingly appreciated; however, the mechanisms by which AIM2 and its related IFI16 specifically recognize dsDNA over other nucleic acids remain poorly understood (i.e. single-stranded (ss)DNA, dsRNA, ssRNA and DNA:RNA hybrid). Here, we find that although AIM2 can interact with various nucleic acids, it preferentially binds to and assembles filaments faster on dsDNA in a duplex length-dependent manner. Moreover, AIM2 oligomers assembled on nucleic acids other than dsDNA not only display less ordered filamentous structures, but also fail to induce the polymerization of downstream ASC. Likewise, although showing broader nucleic acid selectivity than AIM2, IFI16 binds to and oligomerizes most readily on dsDNA in a duplex length-dependent manner. Nevertheless, IFI16 fails to form filaments on single-stranded nucleic acids and does not accelerate the polymerization of ASC regardless of bound nucleic acids. Together, we reveal that filament assembly is integral to nucleic acid distinction by ALRs.

In-silico selection employing rigiddocking and moleculardynamic simulation in selecting DNA aptamers against androgen receptor.

Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformationsandhighGCcontent. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA),whichincludesthemeasurementoftheir binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer,termedARapt1withtheestimatedKdvalueof5.5±3nm, was chosen as the mostprominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.

Antisense oligonucleotides to therapeutically target SARS-CoV-2 infection.

Although the COVID-19 pandemic began over three years ago, the virus responsible for the disease, SARS-CoV-2, continues to infect people across the globe. As such, there remains a critical need for development of novel therapeutics against SARS-CoV-2. One technology that has remained relatively unexplored in COVID-19 is the use of antisense oligonucleotides (ASOs)-short single-stranded nucleic acids that bind to target RNA transcripts to modulate their expression. In this study, ASOs targeted against the SARS-CoV-2 genome and host entry factors, ACE2 and TMPRSS2, were designed and tested for their ability to inhibit cellular infection by SARS-CoV-2. Using our previously developed SARS-CoV-2 bioassay platform, we screened 180 total ASOs targeting various regions of the SARS-CoV-2 genome and validated several ASOs that potently blocked SARS-CoV-2 infection in vitro. Notably, select ASOs retained activity against both the WA1 and B.1.1.7 (commonly known as alpha) variants. Screening of ACE2 and TMPRSS2 ASOs showed that targeting of ACE2 also potently prevented infection by the WA1 and B.1.1.7 SARS-CoV-2 viruses in the tested cell lines. Combined with the demonstrated success of ASOs in other disease indications, these results support further research into the development of ASOs targeting SARS-CoV-2 and host entry factors as potential COVID-19 therapeutics.

Top-down coarse-grained model for single stranded nucleic acids.

Though there exist several coarse-grained (CG) models for nucleic acids describing a range of resolutions, these generally give a poor reproduction of single-stranded nucleic acid properties, particularly RNA, and do not allow sequence specificity to be easily incorporated. Models that do tend to be higher in resolution and more computationally expensive to use. Since recent work has revealed the importance of both RNA and DNA in the formation of complex coacervates, as well as the role of proteins in forming a scaffold for DNA condensation in chromatin and spermatogenesis, we therefore adapt our existing 3-site model of nucleic acids to include sequence-specificity.