Evaluation of aptamer specificity with or without primers using clinical samples for C-reactive protein by magnetic-assisted rapid aptamer selection

Abstract

Aptamers with primer binding sites are necessary for the SELEX (Systematic Evolution of Ligands byEXponential enrichment) process. Such primer sequences cause non-specific binding by their nature and raise the incidence of false positives. Thus, the use of aptamers without primer or with minimum primer, to shorten the length of selected aptamers, has become an interesting research topic. Recently, a primer-free aptamer selection protocol based on the magnetic-assisted rapid aptamer selection (MARAS) technology has been reported to generate primer-free aptamers with high affinity and specificity. Adversely, the yield of a suitable aptamer was low and the advantage of MARAS was deteriorated. In this study, multiple negative selection runs were used in the MARAS procedure to remove aptamers with non-specific binding to unwanted biomolecules. Smart aptamers with predetermined affinity for C-reactive protein were isolated by window-MARAS. The specificity of aptamers was validated by using blind serum samples and compared with those using monoclonal antibody-based nephelometry analysis. The relative specificity of aptamers with or without primers was evaluated, revealing that the specificity of the aptamer with primer is similar to that of the aptamer without primer. By using a randomized library with primers, the simplicity and rapidity of MARAS in generating aptamers are preserved, and by implementing multiple negative selection, MARAS could efficiently select aptamers with high binding affinity and specificity for clinical applications.

Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

“…However, most of these methods are labor intensive or time consuming with immense dependency on expensive resources. As an alternative, introduction of multiple negative selection steps in SELEX ensures removal of primer-binding domain associated non-specificity to a major extent (Li et al, 2017 ). Tsao et al ( 2017 ) employed three strategies to enhance the specificity of their aptamers: (i) multiple rounds of negative selection to remove non-target oligonucleotides; (ii) use of primer-free library to minimize interference from non-participating sequences and (iii) enhanced stringency of selection i.e., use of externally applied magnetic field to suppress non-specific events in MARAS aptamer selection.…”

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts

“…Thus, the AT1 sequence from the vegetable Arabdopsis thaliana was inserted in binding assays according to the method proposed by Graziani and colleagues [17]. Other groups and we have also demonstrated the binding of DNA aptamers and the internalization of RNA aptamers in various types of cells using qPCR [18,24,28,29] opening up the possibility of future clinical application of this technique as an e cient alternative to the use of ow cytometry. The results of the binding assays are shown in Fig.…”

Unravelling the binding mode of a methamphetamine aptamer: a spectroscopic and calorimetric investigation

“…The primers are necessary to amplify target-bound sequences by PCR during the SELEX process. They do not belong to the randomized part of the aptamer and thus can be removed without significant effect on ligand binding (43). Additionally, they can convolute results by interacting non-specifically with the ligand and so their removal is desirable for our subsequent studies.…”