Abstract
To investigate the feasibility of using oligonucleotides in flow cytometry we describe a model system consisting of human neutrophil elastase (HNE) coated on 3.3 micro beads and a high affinity DNA ligand for HNE isolated by in vitro selection (SELEX). In this system the fluoresceinated DNA ligand was equally effective as an anti- HNE antibody in detecting HNE on beads. The location on and the chemistry of attachment of fluorescein to the DNA ligand is critical for the sensitivity of detection. DNA constructs in which fluorescein was conjugated via an ethylene glycol tether to either the 5'-end or near the 3'-end gave much higher signals than did probes with fluorescein directly conjugated to either end. Second-step staining with strepavidin-conjugated phycoerythrin was accomplished using a biotinylated DNA ligand in the initial staining of HNE beads. These data suggest that instead of, or in addition to, antibodies high affinity oligonucleotide probes can be useful in diagnostic applications based on flow cytometry.
Full Text
The Full Text of this article is available as a PDF (103.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Eaton B. E., Pieken W. A. Ribonucleosides and RNA. Annu Rev Biochem. 1995;64:837–863. doi: 10.1146/annurev.bi.64.070195.004201. [DOI] [PubMed] [Google Scholar]
- Ellington A. D., Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818–822. doi: 10.1038/346818a0. [DOI] [PubMed] [Google Scholar]
- Goetzman E. A. Flow cytometry: basic concepts and clinical applications in immunodiagnostics. Clin Lab Sci. 1993 May-Jun;6(3):177–182. [PubMed] [Google Scholar]
- Gold L., Polisky B., Uhlenbeck O., Yarus M. Diversity of oligonucleotide functions. Annu Rev Biochem. 1995;64:763–797. doi: 10.1146/annurev.bi.64.070195.003555. [DOI] [PubMed] [Google Scholar]
- Green L. S., Jellinek D., Bell C., Beebe L. A., Feistner B. D., Gill S. C., Jucker F. M., Janjić N. Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor. Chem Biol. 1995 Oct;2(10):683–695. doi: 10.1016/1074-5521(95)90032-2. [DOI] [PubMed] [Google Scholar]
- Haynes J. L. Principles of flow cytometry. Cytometry Suppl. 1988;3:7–17. doi: 10.1002/cyto.990090804. [DOI] [PubMed] [Google Scholar]
- Lin Y., Padmapriya A., Morden K. M., Jayasena S. D. Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11044–11048. doi: 10.1073/pnas.92.24.11044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin Y., Qiu Q., Gill S. C., Jayasena S. D. Modified RNA sequence pools for in vitro selection. Nucleic Acids Res. 1994 Dec 11;22(24):5229–5234. doi: 10.1093/nar/22.24.5229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]