Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Sub-nanomolar Affinity for a Salivary Biomarker

Abstract

We have attained a chemically modified DNA aptamer against salivary α-amylase (sAA), which attracts researchers’ attention as a useful biomarker for assessing human psychobiological and social behavioural processes, although high affinity aptamers have not been isolated from a random natural DNA library to date. For the selection, we used the base-appended base (BAB) modification, that is, a modified-base DNA library containing (E)-5-(2-(N-(2-(N6-adeninyl)ethyl))carbamylvinyl)-uracil in place of thymine. After eight rounds of selection, a 75 mer aptamer, AMYm1, which binds to sAA with extremely high affinity (Kd < 1 nM), was isolated. Furthermore, we have successfully determined the 36-mer minimum fragment, AMYm1-3, which retains target binding activity comparable to the full-length AMYm1, by surface plasmon resonance assays. Nuclear magnetic resonance spectral analysis indicated that the minimum fragment forms a specific stable conformation, whereas the predicted secondary structures were suggested to be disordered forms. Thus, DNA libraries with BAB-modifications can achieve more diverse conformations for fitness to various targets compared with natural DNA libraries, which is an important advantage for aptamer development. Furthermore, using AMYm1, a capillary gel electrophoresis assay and lateral flow assay with human saliva were conducted, and its feasibility was demonstrated.

Chemical Modifications of Nucleic Acid Aptamers for Therapeutic Purposes

“…structure determination using NMR spectroscopy in solution [99]. This and other reports indicated that NMR guided aptamer optimization could be an optional strategy for aptamer improving binding affinity [100][101][102].…”

Selection and characterization of novel DNA aptamer against colorectal carcinoma Caco‐2 cells

“…Recently, aptamers have also been proven as hopeful candidates of affinity molecules for target‐specific drug delivery . To date, many aptamers have been successfully selected against a variety of targets and applied on diverse fields . As compared with antibodies, aptamers are showing some unique advantages .…”

“…Aptamers are usually screened from a large (10 13 –10 16 ) oligonucleotide (ssDNA or RNA) pool by a technique called systematic evolution of ligands by exponential enrichment (SELEX) in vitro , which contains iterative rounds of selection with amplification steps (Fig. ).…”

“…It has been widely used for screening of cancer cell‐specific aptamers as well as identification of novel biomarkers . To date, many target‐specific aptamers have been successfully selected and applied on diverse fields . For instance, Macugen is the first United States Food and Drug Administration‐approved aptamer drug for the treatment of age‐related macular degeneration .…”

Targeted siRNA delivery using aptamer‐siRNA chimeras and aptamer‐conjugated nanoparticles

“…The detailed steps of the SELEX method are described in Figure . Since SELEX technology was established in 1990 (Ellington & Szostak, ), more than 1,500 aptamers have been identified and exploited for target validation and targeted therapeutics (Minagawa et al, ; Zhou & Rossi, ). Since the first use of aptamers as targeted therapeutics was demonstrated in 1990 by Sullenger, Gallardo, Ungers, and Gilboa (), a number of therapeutic aptamers have made their way into the clinic (Keefe et al, ; Sundaram, Kurniawan, Byrne, & Wower, ).…”