A Detailed Protein-SELEX Protocol Allowing Visual Assessments of Individual Steps for a High Success Rate
| Autor: | Pei-Zhuo Zhang, Yong Li, Changying Chen, Tao Wang, Tuong Ngoc-Gia Nguyen, Qingxia Zhao, Weihong Zhang, Wei Duan, Yingchun Hou, Miaomiao Sun, Phuong H.L. Tran, Kuisheng Chen, Wang Yin, Hadi AlShamaileh, Yumei Zhang |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Aptamer DNA Single-Stranded Enzyme-Linked Immunosorbent Assay Computational biology Biology 01 natural sciences Applied Microbiology and Biotechnology Polymerase Chain Reaction 03 medical and health sciences 010608 biotechnology Genetics Humans Genetics (clinical) 030304 developmental biology Gene Library Pharmacology Protocol (science) 0303 health sciences Ligand binding assay SELEX Aptamer Technique High-Throughput Nucleotide Sequencing Genetic Therapy Aptamers Nucleotide Small molecule Recombinant Proteins Complex materials Highly sensitive Drug delivery Molecular Medicine Systematic evolution of ligands by exponential enrichment |
| Zdroj: | Human gene therapy methods. 30(1) |
| ISSN: | 1946-6544 |
| Popis: | As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing, and laboratory medicine. Over the past decades, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers. Accordingly, the development of aptamers against new or novel targets is still a major obstacle for aptamer-based research and application. Here, an improved protein-SELEX procedure is presented for simplified and highly efficient isolation of aptamers against protein targets. Approaches are described that ensure a high success rate in aptamer selection by simplifying polymerase chain reaction procedures, introducing denature gel, utilizing an electro-elution-based single-stranded DNA separation strategy, as well as an enzyme-linked immunosorbent assay-based highly sensitive binding assay. In addition, a simplified sample preparation method for MiSeq-based next-generation sequencing is also introduced. While a recombinant protein as a bait protein for SELEX is discussed here, this protocol will also be invaluable for researchers wishing to develop aptamers against targets other than proteins such as small molecules, lipids, carbohydrates, cells, and micro-organisms for future gene therapy and/or diagnostics. |
| Databáze: | OpenAIRE |
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