Nucleic Acid Aptamers Research Articles

Inhibition of SARS-CoV-2 infection in human airway epithelium with a xeno-nucleic acid aptamer

BackgroundSARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2.MethodsHere, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo.ResultsWe demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects.ConclusionsTogether, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

A smartphone-assisted colorimetric aptasensor based on aptamer and gold nanoparticles for visual, fast and sensitive detection of ZEN in maize.

A simple, fast, low cost, sensitive, intuitive, visual, label-free, and smartphone-assisted aptamer sensor based on colorimetric assay for the measurement of zearalenone was constructed. The nucleic acid aptamer of zearalenone was used as the recognition element and gold nanoparticles were used as the indicator. Several factors that could influence sensitivity, including the concentration of aptamer and NaCl, and incubation time, and specificity, have been investigated. The results showed that under the optimal conditions, the signal had a good linear relationship when zearalenone concentration is 5-300ng/mL. A linear regression equation is Y=0.0003X+0.5128 (R2=0.9989) and a limit of detection is 5ng/mL. The specificity of the sensor was good. Zearalenone in maize samples were successfully measured. The recoveries of Zearalenone are 81.3 %-96.4 %. The whole process takes only 15min to complete. The smartphone assisted colorimetric aptamer sensor can be used for the detection of zearalenone in maize.

Analysis on the in-silico ensemble methods for 3D modelling of ssDNA aptamers

Understanding the 3-D structure of nucleic acid aptamers is important for the rational design of aptamer-based constructs in various applications, including for developing aptasensors. Herein, a simple approach for 3D modelling of ssDNA aptamers through an ensemble of web applications has been described. The procedure utilized 30 aptamers whose 3D XRD or NMR experimental structures are available for validation. As a first step, the primary sequences of ssDNA aptamers were transformed into 2D structures using six widely used web applications: RNA fold, Vector builder, RNA Structure, UNA fold, Centroid fold, and IP Knot. The generated 2D structures were then passed through the RNA composer web application to generate 3D RNA structure, which in turn was converted to 3D DNA structures using various Visual Molecular Dynamics web applications that also include conversion of ribose sugar into deoxyribose sugar backbone and uracil to thiamine. The energy-minimized generated 3D structures were matched well with high accuracy to their experimental counterparts. This study identified that the Guanine residues are crucial in the aptamer 3D structure prediction and in algorithms that generate secondary structures. Further, the GC content (<50%), GC bond percentage (<60%), and G:C ratio (<1.12) act as limiting factors in predicting the 2D structures of aptamers. There were variations in the 2D structure predictions by the web applications, even though all these applications were a combination of the MFE, MEA, and McCaskill functions. Processing these structures through the web applications described above produced best-fit 3D structures with the experimental one, thus offering the present ensemble approach to reliably predict the 3D structure of aptamers for various applications.

AFB1 colorimetric aptamer sensor for the detection of AFB1 in ten different kinds of miscellaneous beans based on gold nanoparticles and smartphone imaging.

A simple, rapid, low-cost, sensitive, intuitive, visual, label-free, colorimetric smartphone-assisted assay was developed for the measurement of aflatoxin B1 in miscellaneous beans. Ten different kinds of miscellaneous beans were treated and measured by modified QuEChERS(Quick、Easy、Cheap、Effective、Rugged、Safe) method with aflatoxin B1 nucleic acid aptamer as a recognition element and gold nanoparticles as indicators. Several factors influencing its sensitivity were investigated, including consumes and NaCl concentrations, as well as incubation time and specificity. The results showed a good linear relationship between concentrations of 0.2-8.0ng/g under optimal conditions. With a detection limit of 0.08ng/g, the linear regression equation was Y=0.024X+0.4615 (R=0.9989). Sensor specificity is good. The content of aflatoxin B1 in bean samples was determined successfully. The recovery of aflatoxin B1 ranged from 87.18% to 110.24%. The whole thing took 15min. This smartphone-assisted colorimetric aptamer sensor can be used to detect aflatoxin B1 in beans.

Chemical Amplification-Enabled Topological Modification of Nucleic Acid Aptamers for Enhanced Cancer-Targeted Theranostics.

Site-specific chemical conjugation has long been a challenging endeavor in the field of ligand-directed modification to produce homogeneous conjugates for precision medicine. Here, we develop a chemical amplification-enabled topological modification (Chem-ATM) methodology to establish a versatile platform for the programmable modification of nucleic acid aptamers with designated functionalities. Differing from conventional conjugation strategies, a three-dimensional artificial base is designed in Chem-ATM as a chemical amplifier, giving access to structurally and functionally diversified conjugation of aptamers, with precise control over loading capacity but in a sequence-independent manner. Meanwhile, the sp3 hybridized atom-containing amplifier enables planar-to-stereo conformational transformation of the entire conjugate, eliciting high steric hindrance against enzymatic degradation in complex biological environments. The versatility of Chem-ATM is successfully demonstrated by its delivery of anticancer drugs and imaging agents for enhanced therapy and high-contrast noninvasive tumor imaging in xenograft and orthotopic tumor models. This study offers a different perspective for ligand-directed chemical conjugation to enrich the molecular toolbox for bioimaging and drug development.

Electrochemical Biosensors for Clinical Analysis

In this talk, we will discuss the marriage of nucleic acids with electrochemical biosensors for the development of rapid clinical tests. These tests integrate both functional nucleic acids (aptamers and DNAzymes) and antibody/DNA complexes for developing barcode-based electrochemical biosensors. The barcode is a DNA strand modified with an electrochemical label that can be detected using an electrochemical chip. Following the presentation of different DNA barcoding methods, we will also present strategies for operating these biosensors in blood, saliva, and urine for direct analysis of clinical samples for the diagnosis of infectious diseases and cancer without the need for target enrichment methods such as growth cultures or nucleic acid amplification. These devices demonstrate the potential for rapid clinical testing at the point-of-need.

Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

A truncated DNA aptamer with high selectivity for estrogen receptor-positive breast cancer cells

The estrogen receptor-positive (ER+) breast cancers constitute more than 50 % of breast cancers, seriously threatening the health of women. Unfortunately, the detection and targeted therapy of ER+ breast cancers remain a challenge. Here, a novel nucleic acid aptamer S1–4 was developed to specifically target ER+ breast cancer MCF-7 cells by using Cell-SELEX and nucleic acid truncation strategies. The affinity dissociation constant of the binding of aptamer S1–4 to MCF-7 cells was 97.6 ± 7.5 nM in vitro. Compared with HER2+ breast cells SK-BR-3 and triple-negative breast cancer cells MDA-MB-231, MCF-7 cells were selectively recognized and targeted by aptamer S1–4. Fluorescence tracing in vivo results also indicated that aptamer S1–4 selectively targeted the cell membrane of tumor tissues in MCF-7- but not in SK-BR3 or MDB-MA-231-bearing mice. This selectively developed novel aptamer probe S1–4 with high affinity could be used for the diagnosis and treatment of ER+ breast cancers.

CRISPR-Cas12a-based aptasensor for sensitive and selective FB1 detection

Fumonisin B1 (FB1) is a common mycotoxin found in agricultural products. Therefore, establishing rapid detection methods is an important means to prevent the harm of FB1. This study was based on the clustered, regularly spaced short palindromic repeats (CRISPR)-Cas12a system with the introduction of aptamer F10. This study aimed to design CRISPR RNA (crRNA) to specifically recognize the aptamer F10, which formed the competitive relationship between the target molecule and crRNA for nucleic acid aptamer. Finally, a new type of biosensor was established using the trans-cleavage activity of Cas12a protein and amplifying the signal with a fluorescent probe. This study observed a linear relationship of FB1 concentrations in the range of 25–500 nM. The standard curve was y = −0.2586 × x + 775.3, R2 = 0.9922, and the limit of detection (LOD) was 16.84 nM. The recovery of the maize sample was 92.2–101.93% and 92.24–103.96%. The total assay time was 40 min. The biosensor showed high specificity and sensitivity for FB1 after the introduction of the aptamer F10, and the workflow was simple, convenient, and fast, which was suitable for point-of-care (POC) detection in the field. Our designed biosensor validated the principle of detecting small molecules using CRISPR-Cas12a systems, further facilitating its application in the diagnostic field.

Highly-efficient selection of aptamers for detecting various HPV subtypes in clinical samples

Nucleic acid aptamers are of great potentials in diagnostic and therapeutic applications because of their unique molecular recognition capabilities. However, satisfactory aptamers with high affinity and specificity are still in short supply. Herein, we have developed new selection methods allowing the free interactions between the targets and potential aptamers in solution. In our selection system, the protein targets (biotinylated randomly or site-specifically) were first incubated with the random DNA library, followed by the pull-down with the streptavidin magnetic beads or biolayer-interferometry (BLI) sensors. By comparing the two biotinylation strategies (random or site-specific) and two states of the targets (free or immobilized), we have found that the combination of the site-specific biotinylation and free-target strategies was most successful. Based on these highly-efficient selection strategies, HPV L1 aptamers were obtained. By designing the sandwich aptasensor assisted with RCA and CRISPR/Cas12a, we have diagnosed various HPV subtypes in clinical samples, such as easily-collected urine samples. In summary, our new strategy can allow efficient selection of aptamers with high affinity and specificity for clinical applications.

Characterization of an HNA aptamer suggests a non-canonical G-quadruplex motif.

Nucleic acids not only form the basis of heredity, but are increasingly a source of novel nano-structures, -devices and drugs. This has spurred the development of chemically modified alternatives (xeno nucleic acids (XNAs)) comprising chemical configurations not found in nature to extend their chemical and functional scope. XNAs can be evolved into ligands (XNA aptamers) that bind their targets with high affinity and specificity. However, detailed investigations into structural and functional aspects of XNA aptamers have been limited. Here we describe a detailed structure-function analysis of LYS-S8-19, a 1',5'-anhydrohexitol nucleic acid (HNA) aptamer to hen egg-white lysozyme (HEL). Mapping of the aptamer interaction interface with its cognate HEL target antigen revealed interaction epitopes, affinities, kinetics and hot-spots of binding energy similar to protein ligands such as anti-HEL-nanobodies. Truncation analysis and molecular dynamics (MD) simulations suggest that the HNA aptamer core motif folds into a novel and not previously observed HNA tertiary structure, comprising non-canonical hT-hA-hT/hT-hT-hT triplet and hG4-quadruplex structures, consistent with its recognition by two different G4-specific antibodies.

An Aptamer-Embedded Two-Dimensional DNA Nanoscale Material with the Property of Cells Recruitment.

Stem cells possess exceptional proliferation and differentiation abilities, making them highly promising for targeted recruitment research in tissue engineering and other clinical applications. DNA is a naturally water-soluble, biocompatible, and highly editable material that is widely used in cell recruitment research. However, DNA nanomaterials face challenges, such as poor stability, complex synthesis processes, and demanding storage conditions, which limit their potential applications. In this study, we designed a highly stable DNA nanomaterial that embeds nucleic acid aptamers in the single strand region. This material has the ability to specifically bind, recruit, and capture human mesenchymal stem cells. The synthesis process involves rolling circle amplification and topological isomerization, and it can be stored for extended periods under varying temperatures and humidity conditions. This DNA material offers high specificity, ease of fabrication, simple preservation, and low cost, providing a novel approach to stem cell recruitment strategies.

Fiber SPR biosensor sensitized by MOFs for MUC1 protein detection.

The concentration of tumor markers is low, which needs a highly sensitive, stable and fast detection method. In this paper, we proposed and demonstrated a U-shape fiber SPR biosensor sensitized by MOFs materials. The surface of the U-shape SPR sensor was modified with MOFs materials to enhance the sensitivity, and the nucleic acid aptamer was immobilized on the sensor surface because of the biocompatibility of MOFs materials. By the high specificity of the nucleic acid aptamer, the MUC1 protein was recognized and detected. The testing results indicate that the sensor has a logarithmic linear response in the MUC1 protein concentration detection range of 1 pg/ml-100μg/ml, its sensitivity and detection limit are 5.33 nm/log(μg/ml) and 0.16pg/ml respectively. After being sensitized by MOFs, the detection sensitivity of the sensor can be increased by 1.62 times，the LOD can be decreased by 0.75 times. The sensor has high sensitivity and specificity, which has broad application prospects in clinical detection of tumor markers.

Development of DNA Aptamer-Based PROTACs That Degrade the Estrogen Receptor.

Targeted protein degradation (TPD), using chimeric molecules such as proteolysis-targeting chimeras (PROTACs), has attracted attention as a strategy for selective degradation of intracellular proteins by hijacking the ubiquitin-proteasome system (UPS). However, it is often difficult to develop such degraders due to the absence of appropriate ligands for target proteins. In targeting proteins for degradation, the application of nucleic acid aptamers is considered to be effective because these can be explored using systematic evolution of ligand by exponential enrichment (SELEX) methods. In this study, we constructed chimeric molecules in which nucleic acid aptamers capable of binding to the estrogen receptor α (ERα) and E3 ubiquitin ligase ligands were linked via a linker. ERα aptamer-based PROTACs were found to degrade ERα via the UPS. These findings represent the development of novel aptamer-based PROTACs that target intracellular proteins and are potentially applicable to other proteins.

Automated screening of primary cell-based aptamers for targeting and therapy of pancreatic cancer

Although it has been developed for many years, nucleic acid aptamer screening technology still fails to be widely used, a considerable part of it is due to the variability of tumor cell morphology, which leads to the use of immortalized cell lines in the laboratory to screen nucleic acid aptamers for recognition ability of tumor cells in the diseased body. To address this, primary cells that can be stably passaged were isolated and extracted from spontaneous tumors of genetically engineered pancreatic ductal adenocarcinoma model mice in this study. Next, an automated screening instrument for nucleic acid aptamers developed autonomously by our group was used to perform efficient aptamer screening using a limited number of cells, and the obtained nucleic acid aptamers were affinity verified at the cellular level. Finally, to answer the question of the cell growth environment difference on the recognition ability of nucleic acid aptamers, we verified its targeting ability to tumors in vivo on a nude mice xenograft tumor model, and further used a common antitumor drug doxorubicin combined with nucleic acid aptamers to verify the drug loading ability of this aptamer combined with the targeting therapeutic ability.

The selection of a hydrophobic 7-phenylbutyl-7-deazaadenine-modified DNA aptamer with high binding affinity for the Heat Shock Protein 70

Nucleic acids aptamers often fail to efficiently target some proteins because of the hydrophilic character of the natural nucleotides. Here we present hydrophobic 7-phenylbutyl-7-deaadenine-modified DNA aptamers against the Heat Shock Protein 70 that were selected via PEX and magnetic bead-based SELEX. After 9 rounds of selection, the pool was sequenced and a number of candidates were identified. Following initial screening, two modified aptamers were chemically synthesised in-house and their binding affinity analysed by two methods, bio-layer interferometry and fluorescent-plate-based binding assay. The binding affinities of the modified aptamers were compared with that of their natural counterparts. The resulting modified aptamers bound with higher affinity (low nanomolar range) to the Hsp70 than their natural sequence (>5 µM) and hence have potential for applications and further development towards Hsp70 diagnostics or even therapeutics.

Advantages of Material Biofunctionalization Using Nucleic Acid Aptamers in Tissue Engineering and Regenerative Medicine.

Material engineering is a fundamental issue in the applications of materials in the medical field. One of the aspects of material engineering is incorporating recognition sites on the surface of biomaterials, which plays an essential role in increasing the efficiency of tissue engineering scaffolds in various aspects. The application of peptides and antibodies to establish the recognition and adhesion sites has limitations, such as fragility and instability under physical and chemical processes. Therefore, synthetic ligands such as nucleic acid aptamers have received much attention for easy synthesis, minimal immunogenicity, high specificity, and stability under processing. Due to the effective role of these ligands in increasing the efficiency of engineered constructs in this study, the advantages of nucleic acid aptamers in tissue engineering will be reviewed. Aptamer-functionalized biomaterials can attract endogenous stem cells to wounded areas and organize their actions to facilitate tissue regeneration. This approach harnesses the body's inherent regeneration potential to treat many diseases. Also, increased efficacy in controlled release, slow and targeted drug delivery are important issues in drug delivery for tissue engineering approaches which can be achieved by incorporating aptamers in drug delivery systems. Aptamer-functionalized scaffolds have very applications, such as diagnosis of cancer, hematological infections, narcotics, heavy metals, toxins, controlled release from the scaffolds, and in vivo cell tracing. Aptasensors, as a result of many advantages over other traditional assay methods, can replace older methods. Furthermore, their unique targeting mechanism also targets compounds with no particular receptors. Targeting cell homing, local and targeted drug delivery, cell adhesion efficacy, cytocompatibility and bioactivity of scaffolds, aptamer-based biosensor, and aptamer-functionalized scaffolds are the topics that will be examined in this review study.

Discovery of a Peptide Nucleic Acid (PNA) aptamer for cardiac troponin I: Substituting DNA with neutral PNA maintains picomolar affinity and improves performances for electronic sensing with graphene field-effect transistors (gFET)

DNA-based aptamers are widely employed as bioreceptors, owing to their tuneable affinity and specificity towards their targets. The use of peptide nucleic acids (PNAs) has instead proven challenging for this purpose, due to the absence of selection methods for the independent discovery of suitable receptors and due to the difficult mimicry of established DNA-based ones. Despite that PNAs exceed homologous DNA or RNA in terms of complementary base pairing, they can fail to reproduce alternative modes of binding because of their different structural features. The remarkable stability and charge distribution of PNAs could be beneficial to produce sensing bioreceptors, especially in the development of electronic devices such as field-effect transistor-based (FET) biosensors. We hereby report for the first time a high-affinity PNA aptamer for cardiac Troponin I (cTnI), a biomarker of acute myocardial infarction, able to interact with this specific protein in the picomolar range. The PNA aptamer was immobilized onto a graphene-based FET (gFET) transducer, and its ability in the direct detection of cTnI was compared with that of a DNA-based one of the same sequence. Similar dissociation constants were recorded for both receptors in 0.01 × PBS, as well as comparable detection limits of 6.0 ± 1.0 pg mL-1 (PNA aptamer) and 3.3 ± 0.7 pg mL-1 (DNA aptamer). Apart from the non-trivial demonstration that a PNA can behave as an aptamer, the tested receptor proved to be more consistent upon working in more complex biological matrices.

The Small RNA Landscape in NSCLC: Current Therapeutic Applications and Progresses

Non-small-cell lung cancer (NSCLC) is the second most diagnosed type of malignancy and the first cause of cancer death worldwide. Despite recent advances, the treatment of choice for NSCLC patients remains to be chemotherapy, often showing very limited effectiveness with the frequent occurrence of drug-resistant phenotype and the lack of selectivity for tumor cells. Therefore, new effective and targeted therapeutics are needed. In this context, short RNA-based therapeutics, including Antisense Oligonucleotides (ASOs), microRNAs (miRNAs), short interfering (siRNA) and aptamers, represent a promising class of molecules. ASOs, miRNAs and siRNAs act by targeting and inhibiting specific mRNAs, thus showing an improved specificity compared to traditional anti-cancer drugs. Nucleic acid aptamers target and inhibit specific cancer-associated proteins, such as "nucleic acid antibodies". Aptamers are also able of receptor-mediated cell internalization, and therefore, they can be used as carriers of secondary agents giving the possibility of producing very highly specific and effective therapeutics. This review provides an overview of the proposed applications of small RNAs for NSCLC treatment, highlighting their advantageous features and recent advancements in the field.

Aptamer base nanobiosensor for stress hormone measurement

Cortisol plays an important part in physiological procedures. Observing cortisol concentration as a stress indicator demonstrates the physiological mode for the diagnosis goal. The mechanism of the presented nano biosensor is LSPR and is performed based on the nucleic acid aptamer functionalized with gold nanoparticles which have specific interaction with cortisol. The interaction of aptamer particular measurement of cortisol was accomplished over the concentration from 0.05 -0.2 μg·mL-1 within 20 minutes. The detection limit is estimated to be 0.02 μg.mL-1.