Enhancing Colorimetric Detection of Nucleic Acids on Nitrocellulose Membranes: Cutting-Edge Applications in Diagnostics and Forensics.

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology.

Markedly divergent effects of Ouabain on a Temozolomide-resistant (T98G) vs. a Temozolomide-sensitive (LN229) Glioblastoma cell line.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor prognosis. GMB are highly recurrent mainly because of radio- and chemoresistance. Radiotherapy with Temozolomide (TMZ) is until today the golden standard adjuvant therapy, however, the optimal treatment of recurrent glioblastoma remains controversial. Ouabain belongs to the Cardiotonic Steroids (CTS) the natural ligands of the Na/K-ATPase (NKA). It is established that the NKA represents a signal transducer with either stimulating or inhibiting cell growth, apoptosis, migration and angiogenesis. Over the last decade evidence grew that CTS have anti-tumor properties especially in GBM. AIM: Proceeding from recent studies we wanted to further demonstrate a divergent effect of Ouabain on a TMZ-resistant (T98G) as compared to a TMZ-sensitive (LN229) GBM cell line. METHODS: We analyzed the effect of Ouabain on cell migration and plasma cell membrane potential (PCMP) in the LN229 and T98G GBM cell line as well as underlying mechanisms (Bcl-2 and p-Akt/pan-Akt expression). Moreover, we analyzed the anti-angiogenic effect of Ouabain on human umbilical vein endothelial cells (HUVECs). RESULTS: T98G cells showed a significant inhibition of cell migration and a significant depolarization of the PCMP at similar Ouabain concentrations (IC50 = 1.67 × 10-7 M) resp. (IC50 = 2.72 × 10-7 M) with a strong inverse correlation (R2 = 0.95). In contrast, LN229 cells did not respond to Ouabain in these assays at all. Similarly, only T98G but not LN229 cells revealed Bcl-2 down-regulation at nanomolar Ouabain concentrations. This unique response to Ouabain is associated with a down-regulation of pan-Akt in T98G cells 24 h after Ouabain (1.0 × 10-6 M) treatment. For the first time, the anti-angiogenic effect of Ouabain on HUVEC cells (IC50 = 5.49 × 10-8 M) was demonstrated which correlated strongly with the anti-migratory effect (R2 = 0.85). CONCLUSION: The TMZ-resistant T98G cell line as compared to the TMZ-sensitive LN229 cell line shows a high sensitivity towards Ouabain. We consider it as a promising new compound especially in recurrent GBM to overcome the resistance to TMZ and irradiation.

Determination of Aptamer Structure Using Circular Dichroism Spectroscopy.

G-quadruplexes can be important as three-dimensional structures for aptamers as they improve the properties of the nucleic acids like higher nuclease degradation resistance. For the characterization of the quadruplex type and its formation, circular dichroism (CD) spectroscopy is one of the most common used identification methods. It is possible to differentiate the parallel and antiparallel G-quadruplex forms as well as the different number of nucleic acid strands by very specific CD spectral patterns at distinct absorption wavelengths. In this chapter, the protocol describes the model characterization of the anthracycline aptamer DRN-10 by CD spectroscopy, in order to show the usefulness and simple handling of this method for analyzing three-dimensional nucleic acid structures.

Immobilization-Free Determination of Dissociation Constants Independent of Ligand Size Using MicroScale Thermophoresis.

The quantitative characterization of aptamer-ligand interactions is an important step in the aptamer development process. However, certain pitfalls impede KD determination, especially when working with small molecule ligands. These include altered binding behavior caused by ligand immobilization. Further, the compulsory requirement for major differences in size between the bound and unbound state makes small molecule ligands ineligible for separation-based methods. MicroScale Thermophoresis circumvents such limitations as binding is accurately quantified with both binding partners free in solution and independent of ligand size. In this chapter, we present a protocol for the characterization of a DNA aptamer binding to its small molecule ligand daunorubicin.

Label-Free Determination of the Kinetic Parameters of Protein-Aptamer Interaction by Surface Plasmon Resonance.

Due to their high specificity and affinity to target molecules, aptamers can be used as powerful tools in diagnostics, therapeutics, and environmental or food analytics. For the use in various applications, the detailed characterization of their binding behavior is an important step after selection to determine the interaction strength between the aptamer and its target and to find the best kinetics depending on the field of application. The surface plasmon resonance (SPR) spectroscopy is a powerful technology to investigate important parameters in molecular interaction, for example, kinetics, affinity, and specificity. The most-used system is the Biacore™ SPR system which comprises an optical biosensor for label-free monitoring of binding events in real time based on SPR. This biophysical phenomenon describes the changes in refractive index on a sensor surface which can be used to measure binding events and to determine kinetic constants. In this chapter, a detailed protocol for the determination of kinetic constants for protein-aptamer interaction is provided. An 82-nt long ssDNA aptamer which are targeted against human urokinase is used as a model system for determination of binding and dissociation constants using Biacore™ SPR technology. A detailed note section provides useful tips and pitfalls at the end of this chapter.

Aptamer-Based Sandwich Assay Formats for Detection and Discrimination of Human High- and Low-Molecular-Weight uPA for Cancer Prognosis and Diagnosis.

Urokinase-type plasminogen activator (urokinase, uPA) is a frequently discussed biomarker for prognosis, diagnosis, and recurrence of cancer. In a previous study, we developed ssDNA aptamers that bind to different forms of human urokinase, which are therefore assumed to have different binding regions. In this study, we demonstrate the development of aptamer-based sandwich assays that use different combinations of these aptamers to detect high molecular weight- (HMW-) uPA in a micro titer plate format. By combining aptamers and antibodies, it was possible to distinguish between HMW-uPA and low molecular weight- (LMW-) uPA. For the best performing aptamer combination, we calculated the limit of detection (LOD) and limit of quantification (LOQ) in spiked buffer and urine samples with an LOD up to 50 ng/mL and 138 ng/mL, respectively. To show the specificity and sequence dependence of the reporter aptamer uPAapt-02-FR, we have identified key nucleotides within the sequence that are important for specific folding and binding to uPA using a fluorescent dye-linked aptamer assay (FLAA). Since uPA is a much-discussed marker for prognosis and diagnosis in various types of cancers, these aptamers and their use in a micro titer plate assay format represent a novel, promising tool for the detection of uPA and for possible diagnostic applications.

Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

The use of high-affinity polyhistidine binders as masking probes for the selection of an NDM-1 specific aptamer.

The emergence of carbapenemase-producing multi-drug resistant Enterobacteriaceae poses a dramatic, world-wide health risk. Limited treatment options and a lack of easy-to-use methods for the detection of infections with multi-drug resistant bacteria leave the health-care system with a fast-growing challenge. Aptamers are single stranded DNA or RNA molecules that bind to their targets with high affinity and specificity and can therefore serve as outstanding detection probes. However, an effective aptamer selection process is often hampered by non-specific binding. When selections are carried out against recombinant proteins, purification tags (e.g. polyhistidine) serve as attractive side targets, which may impede protein target binding. In this study, aptamer selection was carried out against N-terminally hexa-histidine tagged New Delhi metallo-ß-lactamase 1. After 14 selection rounds binding to polyhistidine was detected rather than to New Delhi metallo-ß-lactamase 1. Hence, the selection strategy was changed. As one aptamer candidate showed remarkable binding affinity to polyhistidine, it was used as a masking probe and selection was restarted from selection round 10. Finally, after three consecutive selection rounds, an aptamer with specific binding properties to New Delhi metallo-ß-lactamase 1 was identified. This aptamer may serve as a much-needed detection probe for New Delhi metallo-ß-lactamase 1 expressing Enterobacteriaceae.