title: Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection.
abstract: Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5' and 3' termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.
authors.affiliation: Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, Taichung, Taiwan.|Sections of Infectious Diseases, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan.
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Tsao
authors.affiliation: Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei, Taiwan.|Research Assistant Center, Chang Hua Show Chwan Health Care System, Changhua, Taiwan.
authors.forename: Ji-Ching
authors.initials: JC
authors.lastname: Lai
authors.affiliation: Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei, Taiwan.
authors.forename: Horng-Er
authors.initials: HE
authors.lastname: Horng
authors.affiliation: Department of Chest Medicine, Cheng-Ching General Hospital, Taichung, Taiwan.
authors.forename: Tu-Chen
authors.initials: TC
authors.lastname: Liu
authors.affiliation: Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung, Taiwan.
authors.forename: Chin-Yih
authors.initials: CY
authors.lastname: Hong
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002097:C-Reactive Protein / Q000032:analysis / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry*; D015723:Gene Library; D008280:Magnetics; D058185:Magnetite Nanoparticles / Q000737:chemistry; D009391:Nephelometry and Turbidimetry; D052156:SELEX Aptamer Technique / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D052157:Aptamers, Nucleotide; D017931:DNA Primers; D004277:DNA, Single-Stranded; D058185:Magnetite Nanoparticles; D002097:C-Reactive Protein