Unravelling the lipocalin 2 interaction with aptamers: May rolling circle amplification improve their functional affinity?

Abstract

Anti-cancer biomarker aptamers are not always well characterized leading to unreliable performance in analytical assays  Fast isothermal rolling circle amplification (15 min) leads to a 10 3 -fold increase in the functional affinity of the aptamer  Most favorable interaction on the solid-surface due to increase in the local protein concentration  Different stability of aptamer-free protein and aptamer-bound protein complexes compromise the efficiency of competitive assays *Highlights (for review)No RCA With RCA

On the Electrochemical Detection of Alpha-Fetoprotein Using Aptamers: DNA Isothermal Amplification Strategies to Improve the Performance of Weak Aptamers

“…Previously we were able to observe an increase in the apparent affinity of a candidate tumor marker, NGAL, of three orders of magnitude using RCA as isothermal amplification technique [31]. In order to understand whether this improvement is general for any binding pair or it depends on the true affinity, we combined RCA with the short aptamer.…”

Improving the Analytical Performance of Weak Aptamers: DNA Isothermal Amplification Approaches

“…However, both values were above the previously described Kd (2.24 pM) [5]. Further binding measurements with other techniques (surface plasmon resonance spectroscopy and microscale thermophoresis) showed that the previously reported affinity of this aptamer might be overestimated [8].…”

“…The amplification protocol comprised the following steps: (1) hybridization of a circularizable single-stranded DNA template (padlock) to the 3′ end of the aptamer, which served as the primer; (2) in parallel. Optimization of reagent concentrations (padlock, T4 DNA ligase, phi29 DNA polymerase, detection probe) and reaction times (padlock hybridization and ligation, RCA, detection probe hybridization) was carried out, increasing both the quickness and cost-effectiveness of the assay [8].…”

Biosensors Made of Synthetic Functional Nucleic Acids Toward Better Human Health

“…A large proportion of aptasensors that were described in 2018 and 2019 were for general cancer biomarkers, meaning they were biomarkers that were useful for multiple cancer types. Biomarkers that were examined include PDGF-BB (LODs, 0.13 nM, 0.08 ng/mL, 0.52 nM, and 3.2 pM), − VEGF (LODs, 0.3 fM and 12 pM), − CD70 (LOD, 14 cells/mL), neutrophil gelatinase-associated lipocalin (NGAL), nucleolin, ,,, ATP (LOD, 0.01 pM), , epidermal growth factor receptor (EGFR, LODs, 5.64 fg/mL, 0.7 ng/mL, and 0.1 ng/mL), − MCF-7 cells (LOD, 61 cells/mL), telomerase (LOD, 100 cells), thymidine kinase 1 (LOD, 54 pg/mL), miRNA let-7a (LOD, 5.12 aM), cytochrome C (LOD, 25.90 nM), lysozyme (LOD, 0.94 nM), mucin 1 (LOD, 0.13 ng/mL), , epithelial cell adhesion molecule (EpCAM, LODs, 10 pM and 20 pg/mL), , N -glycolylneuraminic acid (Neu5Gc, LOD, 10 ng/mL), and CD63 exosome surface protein (LODs, 32 exosomes/μL, 73 exosomes/μL, 203 exosomes/μL). − The usefulness of many of these biosensors was evaluated in complex matrices, such as whole blood, serum, or cell lysate, or in the presence of whole cells. The strength of each of these biosensors is their diverse application to multiple cancers.…”