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      Modified DNA Aptamers for C-Reactive Protein and Lactate Dehydrogenase-5 with Sub-Nanomolar Affinities

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          Abstract

          Human C-reactive protein (CRP) and lactate dehydrogenase are important markers in clinical laboratory testing—the former is used to detect in vivo inflammation, and the latter is used to detect cell necrosis and tissue destruction. We developed aptamers that bind to human CRP and human lactate dehydrogenase-5 (LDH-5) with high affinities (dissociation constants of 6.2 pM and 235 pM, respectively), applying the systematic evolution of ligands by exponential enrichment (SELEX) method, and by using a modified DNA library containing the following base-appended base modifications: analog adenine derivative at the fifth position of uracil (U ad), analog guanine derivative at the fifth position of uracil (U gu), and analog adenine derivative at the seventh position of adenine (A ad). A potential application of these aptamers as sensor elements includes high-sensitivity target detection in point-of-care testing.

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          Most cited references 48

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          Generation of high-affinity DNA aptamers using an expanded genetic alphabet.

          DNA aptamers produced with natural or modified natural nucleotides often lack the desired binding affinity and specificity to target proteins. Here we describe a method for selecting DNA aptamers containing the four natural nucleotides and an unnatural nucleotide with the hydrophobic base 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds). We incorporated up to three Ds nucleotides in a random sequence library, which is expected to increase the chemical and structural diversity of the DNA molecules. Selection experiments against two human target proteins, vascular endothelial cell growth factor-165 (VEGF-165) and interferon-γ (IFN-γ), yielded DNA aptamers that bind with KD values of 0.65 pM and 0.038 nM, respectively, affinities that are >100-fold improved over those of aptamers containing only natural bases. These results show that incorporation of unnatural bases can yield aptamers with greatly augmented affinities, suggesting the potential of genetic alphabet expansion as a powerful tool for creating highly functional nucleic acids.
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            A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics

             J SantaLucia (1998)
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              Aptamers as Therapeutics.

              Aptamers are single-stranded nucleic acid molecules that bind to and inhibit proteins and are commonly produced by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers undergo extensive pharmacological revision, which alters affinity, specificity, and therapeutic half-life, tailoring each drug for a specific clinical need. The first therapeutic aptamer was described 25 years ago. Thus far, one aptamer has been approved for clinical use, and numerous others are in preclinical or clinical development. This review presents a short history of aptamers and SELEX, describes their pharmacological development and optimization, and reviews potential treatment of diseases including visual disorders, thrombosis, and cancer.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                13 April 2020
                April 2020
                : 21
                : 8
                Affiliations
                [1 ]NEC Solution Innovators, Ltd., 1-18-7, Shinkiba, Koto-ku, Tokyo 136-8627, Japan; minagawa-hir@ 123456nec.com (H.M.); k-horii@ 123456nec.com (K.H.); iwa-waga@ 123456nec.com (I.W.)
                [2 ]Graduate School of Integrated Basic Sciences, Nihon University, 3-25-40 Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan; kataoka.yuka@ 123456nihon-u.ac.jp (Y.K.); fujita.hiroto@ 123456nihon-u.ac.jp (H.F.)
                Author notes
                [* ]Correspondence: mkuwa@ 123456chs.nihon-u.ac.jp (M.K.); shiratori@ 123456nec.com (I.S.); Tel.: +81-3-5317-9398 (M.K.); +81-3-5534-2619 (I.S.)
                Article
                ijms-21-02683
                10.3390/ijms21082683
                7215426
                32294882
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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