Complex In Silico RNA Design with MoiRNAiFold.

In the advent of the RNA therapeutics and diagnostics era, it is of great relevance to introduce new and more efficient RNA technologies that prove to be effective tools in practical contexts. Moreover, it is of utmost importance to develop and provide access to computational tools capable of designing such RNA constructs. Here we introduce one such novel diagnostics technology (Apta-SMART) and show how to design (using MoiRNAiFold) and implement it, step by step. Moreover, we show how to combine this technique with well-known RNA amplification methods and briefly mention some encouraging results.

Advances in nucleic acid aptamer-based detection of respiratory virus and bacteria: a mini review.

Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.

Aptamers: precision tools for diagnosing and treating infectious diseases.

Infectious diseases represent a significant global health challenge, with bacteria, fungi, viruses, and parasitic protozoa being significant causative agents. The shared symptoms among diseases and the emergence of new pathogen variations make diagnosis and treatment complex. Conventional diagnostic methods are laborious and intricate, underscoring the need for rapid, accurate techniques. Aptamer-based technologies offer a promising solution, as they are cost-effective, sensitive, specific, and convenient for molecular disease diagnosis. Aptamers, which are single-stranded RNA or DNA sequences, serve as nucleotide equivalents of monoclonal antibodies, displaying high specificity and affinity for target molecules. They are structurally robust, allowing for long-term storage without substantial activity loss. Aptamers find applications in diverse fields such as drug screening, material science, and environmental monitoring. In biomedicine, they are extensively studied for biomarker detection, diagnostics, imaging, and targeted therapy. This comprehensive review focuses on the utility of aptamers in managing infectious diseases, particularly in the realms of diagnostics and therapeutics.

Highly Stable Biotemplated InP/ZnSe/ZnS Quantum Dots for In Situ Bacterial Monitoring.

Despite their unique optical and electrical characteristics, traditional semiconductor quantum dots (QDs) made of heavy metals or carbon are not ideally suited for biomedical applications. Cytotoxicity and environmental concerns are key limiting factors affecting the adoption of QDs from laboratory research to real-world medical applications. Recently, advanced InP/ZnSe/ZnS QDs have emerged as alternatives to traditional QDs due to their low toxicity and optical properties; however, bioconjugation has remained a challenge due to surface chemistry limitations that can lead to instability in aqueous environments. Here, we report water-soluble, biotemplated InP/ZnSe/ZnS-aptamer quantum dots (QDAPTs) with long-term stability and high selectivity for targeting bacterial membrane proteins. QDAPTs show fast binding reaction kinetics (less than 5 min), high brightness, and high levels of stability (3 months) after biotemplating in aqueous solvents. We use these materials to demonstrate the detection of bacterial membrane proteins on common surfaces using a hand-held imaging device, which attests to the potential of this system for biomedical applications.

Therapeutic delivery of siRNA for the management of breast cancer and triple-negative breast cancer.

Breast cancer is the leading cause of cancer-related deaths among women globally. The difficulties with anticancer medications, such as ineffective targeting, larger doses, toxicity to healthy cells and side effects, have prompted attention to alternate approaches to address these difficulties. RNA interference by small interfering RNA (siRNA) is one such tactic. When compared with chemotherapy, siRNA has several advantages, including the ability to quickly modify and suppress the expression of the target gene and display superior efficacy and safety. However, there are known challenges and hurdles that limits their clinical translation. Decomposition by endonucleases, renal clearance, hydrophilicity, negative surface charge, short half-life and off-target effects of naked siRNA are obstacles that hinder the desired biological activity of naked siRNA. Nanoparticulate systems such as polymeric, lipid, lipid-polymeric, metallic, mesoporous silica nanoparticles and several other nanocarriers were used for effective delivery of siRNA and to knock down genes involved in breast cancer and triple-negative breast cancer. The focus of this review is to provide a comprehensive picture of various strategies utilized for delivering siRNA, such as combinatorial delivery, development of modified nanoparticles, smart nanocarriers and nanocarriers that target angiogenesis, cancer stem cells and metastasis of breast cancer.

Breast cancer is the leading cause of cancer-related deaths among women globally. The difficulties with anticancer medications, have prompted attention to alternate approaches to address these difficulties. RNA interference (RNAi) by small interfering RNA (siRNA) is one such approach, which has several advantages, including the ability to quickly modify and suppress the expression of the target gene and display superior efficacy and safety. However, there are many hurdles that hinder the desired biological activity of naked siRNA. For addressing various problems pertaining to the delivery of naked siRNA, nanoparticles are employed to carry siRNA to the target cells. We have attempted to outline a broad variety of nanocarriers carrying various siRNAs developed for the therapy of breast cancer and triple-negative breast cancer.

Aptamer-decorated nanocarriers for viral adsorption: A special look at COVID-19.

Viral infections are one of the leading causes of death in the world. One main challenge in fighting against these diseases is the unavailability of effective eradicating drugs and specific treatments. Nanocarriers and aptamer-decorated nanocarriers are designed to attach to many targets, including viral particles. By lowering the viral infectivity and attachment capability, they add therapeutic values even without containing antiviral drugs. Nevertheless, the nanoparticles (NPs) with encapsulated antiviral drugs can display extra therapeutic effects. Furthermore, it has been shown that aptamers can bind to viral particles and nanocarriers, presenting promising approaches for the identification of viruses and treatment of viral infections. Although there is no satisfying literature revealing the strong therapeutic potential of nanotechnology against COVID-19, the following information can provide new perspectives for upcoming investigations pertaining to developing effective aptamer-nanocarrier agents against COVID-19.

Are Aptamer-Based Biosensors the Future of the Detection of the Human Gut Microbiome?-A Systematic Review and Meta-Analysis.

The gut microbiome is shaped early in life by dietary and lifestyle factors. Specific compounds in the gut affect the growth of different bacterial species and the production of beneficial or harmful byproducts. Dysbiosis of the gut microbiome has been linked to various diseases resulting from the presence of harmful bacteria and their byproducts. Existing methods for detecting microbial species, such as microscopic observation and molecular biological techniques, are costly, labor-intensive, and require skilled personnel. Biosensors, which integrate a recognition element, transducer, amplifier, signal processor, and display unit, can convert biological events into electronic signals. This review provides a comprehensive and systematic survey of scientific publications from 2018 to June 2024, obtained from ScienceDirect, PubMed, and Scopus databases. The aim was to evaluate the current state-of-the-art and identify knowledge gaps in the application of aptamer biosensors for the determination of gut microbiota. A total of 13 eligible publications were categorized based on the type of study: those using microbial bioreceptors (category 1) and those using aptamer bioreceptors (category 2) for the determination of gut microbiota. Point-of-care biosensors are being developed to monitor changes in metabolites that may lead to disease. They are well-suited for use in the healthcare system and offer an excellent alternative to traditional methods. Aptamers are gaining attention due to their stability, specificity, scalability, reproducibility, low production cost, and low immunogenicity. While there is limited research on using aptamers to detect human gut microbiota, they show promise for providing accurate, robust, and cost-effective diagnostic methods for monitoring the gut microbiome.

A DNA Molecular Logic Circuit for Precise Tumor Identification.

Tumor-associated antigens (TAAs) are not exclusively expressed in cancer cells, inevitably causing the "on target, off tumor" effect of molecular recognition tools. To achieve precise recognition of cancer cells, by using protein tyrosine kinase 7 (PTK7) as a model TAA, a DNA molecular logic circuit Aisgc8 was rationally developed by arranging H+-binding i-motif, ATP-binding aptamer, and PTK7-targeting aptamer Sgc8c in a DNA sequence. Aisgc8 output the conformation of Sgc8c to recognize PTK7 on cells in a simulated tumor microenvironment characterized by weak acidity and abundant ATP, but not in a simulated physiological environment. Through in vitro and in vivo results, Aisgc8 demonstrated its ability to precisely recognize cancer cells and, as a result, displayed excellent performance in tumor imaging. Thus, our studies produced a simple and efficient strategy to construct DNA logic circuits, opening new possibilities to develop convenient and intelligent precision diagnostics by using DNA logic circuits.

PD-L1 DNA aptamers isolated from agarose-bead SELEX.

Increased expression and activity of the PD-L1/PD-1 pathway suppresses the activation of cytotoxic T cells, which is vital in anti-tumour defence, allowing tumours to rise, expand and progress. Current strategies using antibodies to target PD-1/PD-L1 have been very effective in cancer therapeutics and companion diagnostics. Aptamers are a new class of molecules that offer an alternative to antibodies. Herein, the systematic evolution of ligands by exponential enrichment (SELEX) using agarose slurry beads was conducted to isolate DNA aptamers specific to recombinant human PD-L1 (rhPD-L1). Isolated aptamers were sequenced and analysed using MEGA X and structural features were examined using mFold. Three aptamer candidates (P33, P32, and P12) were selected for evaluation of binding affinity (dissociation constant, Kd) using ELONA and specificity and competitive inhibition assessment using the potentiostat-electrochemical method. Among those three, P32 displayed the highest specificity (8 nM) against PD-L1. However, P32 competes for the same binding site with the control antibody, 28-8. This study warrants further assessment of P32 aptamer as a potential, cost-effective alternative tool for targeting PD-L1.

Novel Aptamer Strategies in Combating Bacterial Infections: From Diagnostics to Therapeutics.

Bacterial infections and antimicrobial resistance are posing substantial difficulties to the worldwide healthcare system. The constraints of conventional diagnostic and therapeutic approaches in dealing with continuously changing infections highlight the necessity for innovative solutions. Aptamers, which are synthetic oligonucleotide ligands with a high degree of specificity and affinity, have demonstrated significant promise in the field of bacterial infection management. This review examines the use of aptamers in the diagnosis and therapy of bacterial infections. The scope of this study includes the utilization of aptasensors and imaging technologies, with a particular focus on their ability to detect conditions at an early stage. Aptamers have shown exceptional effectiveness in suppressing bacterial proliferation and halting the development of biofilms in therapeutic settings. In addition, they possess the capacity to regulate immune responses and serve as carriers in nanomaterial-based techniques, including radiation and photodynamic therapy. We also explore potential solutions to the challenges faced by aptamers, such as nuclease degradation and in vivo instability, to broaden the range of applications for aptamers to combat bacterial infections.

Optogenetic Tools for Regulating RNA Metabolism and Functions.

RNA molecules play a vital role in linking genetic information with various cellular processes. In recent years, a variety of optogenetic tools have been engineered for regulating cellular RNA metabolism and functions. These highly desirable tools can offer non-intrusive control with spatial precision, remote operation, and biocompatibility. Here, we would like to review these currently available approaches that can regulate RNAs with light: from non-genetically encodable chemically modified oligonucleotides to genetically encoded RNA aptamers that recognize photosensitive small-molecule or protein ligands. Some key applications of these optogenetic tools will also be highlighted to illustrate how they have been used for regulating all aspects of the RNA life cycle: from RNA synthesis, maturation, modification, and translation to their degradation, localization, and phase separation control. Some current challenges and potential practical utilizations of these RNA optogenetic tools will also be discussed.

Solution-based biophysical characterization of conformation change in structure-switching aptamers.

Structure-switching aptamers have become ubiquitous in several applications, notably in analytical devices such as biosensors, due to their ease of supporting strong signaling. Aside from their ability to bind specifically with their respective target, this class of aptamers also undergoes a conformational rearrangement upon target recognition. While several well-studied and early-developed aptamers (e.g., cocaine, ATP, and thrombin) have been found to have this structure-switching property, the vast majority do not. As a result, it is common to try to engineer aptamers into switches. This proves challenging in part because of the difficulty in obtaining structural and functional information about aptamers. In response, we review various readily available biophysical characterization tools that are capable of assessing structure switching of aptamers. In doing so, we delve into the fundamentals of these different techniques and detail how they have been utilized in characterizing structure-switching aptamers. While each of these biophysical techniques alone has utility, their real power to demonstrate the occurrence of structural change with ligand binding is when multiple techniques are used. We hope that through a deeper understanding of these techniques, researchers will be better able to acquire biophysical information about their aptamer-ligand systems and accelerate the translation of aptamers into biosensors.

Selection of DNA aptamers against Chikungunya virus Envelope 2 Protein and their application in sandwich ELASA.

Chikungunya fever, caused by Chikungunya virus (CHIKV) exhibits clinical features that mimic that of other arbovirus infections such as dengue. CHIKV Envelope 2 (E2) protein, an antigenic epitope of CHIKV, has been identified as an ideal marker for diagnostics. The current CHIKV antigen detection tests are largely based on antibodies but are beleaguered by issues such as sensitivity to high temperature, expensive and prone to batch-to-batch variations. Aptamers are suitable alternatives to antibodies as they are cheaper and have no batch-to-batch variations compared to antibodies. In this study, DNA aptamer selection against CHIKV E2 proteins was performed using two different randomized ssDNA libraries. Chik-2 (96-mer) and Chik-3 (76-mer) were isolated from these two libraries and were identified as the potential aptamers against CHIKV E2 protein. The binding affinity of Chik-2 and Chik-3 against CHIKV E2 protein was estimated at 177.5 ± 32.69 nM and 30.01 ± 3.60 nM, respectively. A sandwich ELASA was developed, and this assay showed a detection limit of 2.17 x 103 PFU/mL. The sensitivity and specificity of the assay were 80 % and 100 %, respectively. The assay showed no cross-reactivity with dengue-positive samples, demonstrating the enormous diagnostic potential of these aptamers for the detection of CHIKV.

Application of DNA aptamers to block enterotoxigenic Escherichia coli toxicity in a Galleria mellonella larval model.

Enterotoxigenic Escherichia coli (ETEC) is the major bacterial cause of diarrheal diseases in pigs, particularly at young ages, resulting in significant costs to swine farming. The pathogenicity of ETEC is largely dependent on the presence of fimbriae and the ability to produce toxins. Fimbriae are responsible for their initial adhesion to the intestinal epithelial cells, leading to the onset of infection. In particular, the F4 type (K88) fimbriae are often attributed to neonatal infections and have also been associated with post-weaning diarrheal infections. This disease is traditionally prevented or treated with antibiotics, but their use is being severely restricted due to the emergence of resistant bacteria and their impact on human health. Emerging approaches such as aptamers that target the F4-type fimbriae and block the initial ETEC adhesion are a promising alternative. The aim of this study is to assess the effectiveness of two aptamers, Apt31 and Apt37, in controlling ETEC infection in the G. mellonella in vivo model. Initially, the dissociation constant (KD) of each aptamer against ETEC was established using real-time quantitative PCR methodology. Subsequently, different concentrations of the aptamers were injected into Galleria mellonella to study their toxicity. Afterwards, the anti-ETEC potential of Apt31 and Apt37 was assessed in the larvae model. The determined KD was 81.79 nM (95% CI: 31.21-199.4 nM) and 50.71 nM (95% CI: 26.52-96.15 nM) for the Apt31 and Apt37, respectively, showing no statistical difference. No toxicity was observed in G. mellonella following injection with both aptamers at any concentration. However, the administration of Apt31 together with ETEC-F4+ in G. mellonella resulted in a significant improvement of approximately 30% in both larvae survival and health index compared to ETEC-F4+ alone. These findings suggest that aptamers have promising inhibitory effect against ETEC infections and pave the way for additional in vivo studies.

A novel peptide pair-based rapid fluorescent diagnostic system for malaria Plasmodium falciparum detection.

Advanced diagnostic materials, such as aptamers, are required due to the scarcity of efficient diagnostic antibodies and the low sensitivity of rapid diagnostic kits at detecting the malaria parasite, Plasmodium falciparum. METHODS: Two peptides M2.9 [(KPTAEQTESPELQSAPEN) and M2.17 (KILFNVYSPLGCTCECWV)] were designed using simple epitope prediction tools and modified against the merozoite surface antigen 2 of P. falciparum (Pf.MSP2) by 3-dimensional modeling based on binding affinity. Based on five prediction tools for hydropathy, M2.17 was selected as an appropriate capture peptide. A peptide-based fluorescence-linked immunosorbent assay (FLISA) and a peptide pair-based fluorescent immunochromatographic test strip (FICT) were developed to detect P. falciparum 3D7 (drug-sensitive) and P. falciparum K1 (multi drugs-resistant) strains. RESULTS: Bioinformatic analysis of two peptides demonstrated the potential binding affinity with the merozoite surface protein 2 of P. falciparum (Pf.MSP2) with a positive hydropathy value. The limit of detection (LOD) of FLISA was 10 parasites/µL and of a peptide pair-linked rapid FICT system was 5 and 200 parasites/µL for P. falciparum 3D7 and K1, respectively. Compared to commercial rapid detection systems (RDTs), a peptide pair-linked FICT system exhibited a 20-fold greater efficiency in detecting P. falciparum 3D7 and specifically discriminated another protozoan spp. CONCLUSION: A peptide pair-linked rapid diagnostic strip could be an alternative to conventional RDTs for monitoring wild-type and drug-resistant malaria parasites.

A DNA Aptamer for 2-Aminopurine: Binding-induced Fluorescence Quenching.

2-Aminopurine (2AP) is a fluorescent analog of adenine, and its unique properties make it valuable in various scientific and biotechnological applications. Its fluorescence property probes local dynamics in DNA and RNA because the surrounding bases quench its fluorescence. 2AP-labeled probes that can bind to specific DNA or RNA sequences, enabling the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases. In this study, we isolated aptamers for 2AP using the library immobilization capture-SELEX technique. Two major aptamer families were isolated after 15 rounds of screening. The Kd values for the 2AP1 aptamer from family 1 are 209 nM in a fluorescence assay and 72 nM in an isothermal titration calorimetry test. The 32 nM 2AP limit of detection was tested. Additionally, we conducted some mutation analysis. Furthermore, we tested the selectivity of our aptamer using various molecules with similar structures and discovered that it can bind adenine and adenosine as well.

Molecular Modeling Methods in the Development of Affine and Specific Protein-Binding Agents.

High-affinity and specific agents are widely applied in various areas, including diagnostics, scientific research, and disease therapy (as drugs and drug delivery systems). It takes significant time to develop them. For this reason, development of high-affinity agents extensively utilizes computer methods at various stages for the analysis and modeling of these molecules. The review describes the main affinity and specific agents, such as monoclonal antibodies and their fragments, antibody mimetics, aptamers, and molecularly imprinted polymers. The methods of their obtaining as well as their main advantages and disadvantages are briefly described, with special attention focused on the molecular modeling methods used for their analysis and development.

Sandwich enzyme-linked aptamer-based assay for the detection of Trichomonas vaginalis.

Trichomoniasis is the most prevalent curable, non-viral sexually transmitted infection (STI), with an estimated 156 million new infections in 2020. It can potentially result in adverse birth outcomes as well as infertility in men, whilst it also increases the risk of acquiring HIV and contracting other vaginal infections. It is mostly prevalent among women in low-income countries and especially in Africa and the Americas. This STI is caused by Trichomonas vaginalis (TV) and a robust, cost-effective, sensitive, specific and rapid diagnostic test is urgently required. We report the screening of 6 full-length and 4 truncated aptamers previously selected in our group for use in a microplate-based sandwich assay. The combination of dual aptamers comprising a short 14-mer truncated capture aptamer (termed A1_14mer) and a full-length non-truncated reporter aptamer (A6) was elucidated to be the optimum pair for a sensitive sandwich enzyme-linked aptamer assay (ELAA) for the detection of TV achieving a detection limit of 3.02 × 104 TV cells/mL. The results obtained with the A1_14mer-A6 ELAA correlate excellently with wet-mount microscopy for the detection of TV in clinical specimens, cervicovaginal lavages and vaginal swabs, highlighting the potential clinical application of this assay for cost-effective population screening and subsequent prevention of the onset of complications associated with undiagnosed and untreated TV.

Aptamer-quantum dots platform for SARS-CoV-2 viral particle detection by fluorescence microscopy.

The virus is the smallest known replicative unit, usually in nanometer-range sizes. The most simple and sensitive detection assay involves molecular amplification of nucleic acids. This work shows a novel, straightforward detection based on the interaction of viral particles with fluorescent nanoconstructs without using enzymatic amplification, washing or separation steps. Fluorescent nanoconstructs are prepared with individual quantum dots of different emitting green and red fluorescence as a core. They are decorated with aptamers developed to recognise the receptor-binding region of the SARS-CoV-2 spike protein. Nanoconstructs can recognise SARS-CoV-2 viral particles fixed onto a coverglass generating aggregates. Meanwhile, SARS-CoV-2 viral particles/nanoconstructs complexes in solution yield aggregates and complexes, which a fluorescence microscope can visualise. The multiple molecular recognition allowed the detection of SARS-CoV-2 viral particles from a few microliters of patient swabs. This specific SARS-CoV-2/nanoconstructs interaction generates insoluble and precipitating aggregates. By using a mixture of green and red fluorescent nanoconstructs, upon the viral particle interaction, they yield heterochromatic green, red and yellow spectral fluorescence, easily identifiable by a fluorescence microscope. Washing and separation steps are not required, and aggregates allow one to easily recognise them, offering a sensitive, simple, and cheap alternative for viral detection.