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Institute
Anthracyclines like daunorubicin (DRN) and doxorubicin (DOX) play an undisputed key role in cancer treatment, but their chronic administration can cause severe side effects. For precise anthracycline analytical systems, aptamers are preferable recognition elements. Here, we describe the detailed characterisation of a single-stranded DNA aptamer DRN-10 and its truncated versions for DOX and DRN detection. Binding affinities were determined from surface plasmon resonance (SPR) and microscale thermophoresis (MST) and combined with conformational data from circular dichroism (CD). Both aptamers displayed similar nanomolar binding affinities to DRN and DOX, even though their rate constants differed as shown by SPR recordings. SPR kinetic data unravelled a two-state reaction model including a 1 : 1 binding and a subsequent conformational change of the binding complex. This model was supported by CD spectra. In addition, the dissociation constants determined with MST were always lower than that from SPR, and especially for the truncated aptamer they differed by two orders of magnitude. This most probably reflects the methodological difference, namely labelling for MST vs. immobilisation for SPR. From CD recordings, we suggested a specific G-quadruplex as structural basis for anthracycline binding. We concluded that the aptamer DRN-10 is a promising recognition element for anthracycline detection systems and further selected aptamers can be also characterised with the combined methodological approach presented here.
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.
Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either "evolution in the test tube" of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the "biological" degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.
Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either "evolution in the test tube" of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the "biological" degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.
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 K-D of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a K-D 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.
Simple Summary
Urokinase-type plasminogen activator (urokinase, uPA) is a widely discussed biomarker for cancer prognosis and diagnosis. The gold standard for the determination of protein biomarkers in physiological samples is the enzyme-linked immunosorbent assay (ELISA). Here, antibodies are used to detect the specific protein.
In our study, recently published urokinase aptamers were tested for their use in a sandwich assay format as alternative specific recognition elements. Different aptamer combinations were used for the detection of uPA in a sandwich-assay format and a combination of aptamers and antibodies additionally allowed the differentiation of human high and low molecular weight- (HMW- and LMW-) uPA. Hence, uPA aptamers offer a valuable alternative as specific recognition elements for analytical purposes. Since aptamers are easy to synthesize and modify, they can be used as a cost-effective alternative in sandwich assay formats for the detection of uPA in physiological samples.
Abstract
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.