Graphene oxide and Lambda exonuclease assisted screening of L-carnitine aptamers and the site-directed mutagenesis design of C-rich structure aptamer
Autor: | Yanmei Zhao, Mingzhu Gong, Zefeng He, Xia Liu, Yuhui Zhang, Jidong Yang, Dan Li, Ligang Xing, Ping Yan |
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Rok vydání: | 2021 |
Předmět: |
Exonucleases
0301 basic medicine Aptamer Electrospray ionization Biophysics DNA Single-Stranded Metal Nanoparticles Sequence alignment Sequence (biology) Biochemistry 03 medical and health sciences 0302 clinical medicine Carnitine Binding site Site-directed mutagenesis Molecular Biology Base Sequence Chemistry Circular Dichroism SELEX Aptamer Technique Mutagenesis High-Throughput Nucleotide Sequencing Stereoisomerism Sequence Analysis DNA Cell Biology Aptamers Nucleotide Bacteriophage lambda Combinatorial chemistry Spectrometry Fluorescence 030104 developmental biology Colloidal gold 030220 oncology & carcinogenesis Mutagenesis Site-Directed Nucleic Acid Conformation Graphite Gold |
Zdroj: | Biochemical and Biophysical Research Communications. 545:171-176 |
ISSN: | 0006-291X |
Popis: | In this study, Graphene Oxide (GO) was used to screen the binding with the aptamers of L-carnitine chiral enantiomers. The ssDNA library was prepared by the method of Lambda exonuclease. In addition, a simple casing device was designed to improve the purification and recovery efficiency of the small ssDNA fragments in the process of screening. Finally, more than 160,000 aptamer sequences were obtained by high-throughput sequencing. We determined the strongest affinity aptamer sequence, CA04, by the Resonance Rayleigh scattering (RRS) technology. We also analyzed the key binding sites (in the 16th position case) of the truncated aptamer sequence CAD10. Interestingly, we found that aptamer CA10 and CA06 were both C-rich bases through sequence alignment and analysis, and the aptamer CA10 was confirmed that the CA10 and CA06 were formed under acidic conditions (pH 4.5) by CD spectrum and ESI-MS analysis. The interaction between gold nanoparticles (AuNPs) and functionalized aptamer CA10 was analyzed. We used Site-directed mutagenesis design and QGRS Mapper to optimize aptamer CA10, where an optimal aptamer CA10-03 were obtained after affinity analysis. It is also proved to be an effective method to obtain stronger affinity aptamer. Meanwhile, Native-PAGE and UV spectrum analysis were performed on the mutation sequences, and the interaction with ThT was analyzed. Finally, it is hoped that my study can provide help for later identification and detection of L-carnitine. |
Databáze: | OpenAIRE |
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