Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulose-derived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.
- MeSH
- aptamerová technika SELEX metody MeSH
- aptamery nukleotidové chemie MeSH
- ELISA metody MeSH
- jednovláknová DNA chemie MeSH
- methylcelulosa chemie MeSH
- polymerázová řetězová reakce metody MeSH
- sodná sůl karboxymethylcelulosy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Systematic evolution of ligands by exponential enrichment (SELEX) is a well-established and efficient technology for the generation of oligonucleotides with a high target affinity. These SELEX-derived single stranded DNA and RNA molecules, called aptamers, were selected against various targets, such as proteins, cells, microorganisms, chemical compounds etc. They have a great potential in the use as novel antibodies, in cancer theragnostics and in biomedical research. Vast interest in aptamers stimulated continuous development of SELEX, which underwent numerous modifications since its first application in 1990. Novel modifications made the selection process more efficient, cost-effective and significantly less time-consuming. This article brings a comprehensive and up-to-date review of recent advances in SELEX methods and pinpoints advantages, main obstacles and limitations. The post-SELEX strategies and examples of application are also briefly outlined in this review.
- MeSH
- aptamerová technika SELEX MeSH
- biologická terapie MeSH
- imunoprecipitace MeSH
- infekce papilomavirem imunologie MeSH
- lidé MeSH
- malá interferující RNA MeSH
- mikro RNA MeSH
- oligonukleotidy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- recenze MeSH
DNA aptamers were developed against lipopolysaccharide (LPS) from E. coli O111:B4 and shown to bind both LPS and E. coli by a colorimetric enzyme-based microplate assay. The polyclonal aptamers were coupled to human C1qrs protein either directly using a bifunctional linker or indirectly using biotinylated aptamers and a streptavidin-C1qrs complex. Both systems significantly reduced colony counts when applied to E. coli O111:B4 and K12 strains across a series of 10x dilutions of the bacteria in the presence of human serum; it was diluted 1: 10(3) in order to avoid significant bacterial lysis by the competing alternate pathway of complement activation. A number of candidate DNA aptamer sequences were cloned and sequenced from the anti-LPS aptamer library for future screening of antibacterial or "antibiotic" potential and to aid in eventual development of an alternative therapy for antibiotic-resistant bacterial infections.
- MeSH
- antibakteriální látky farmakologie chemie imunologie MeSH
- aptamerová technika SELEX MeSH
- aptamery nukleotidové MeSH
- Escherichia coli imunologie účinky léků MeSH
- infekce vyvolané Escherichia coli farmakoterapie imunologie MeSH
- komplement C1 chemie imunologie MeSH
- lidé MeSH
- lipopolysacharidy genetika chemická syntéza MeSH
- molekulární sekvence - údaje MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
