Single-strand DNA aptamers as probes for protein localization in cells.
The accurate localization of proteins in fixed cells is important for many studies in cell biology, but good fixation is often antagonistic to good immunolabeling, given the density of well-preserved cells and the size of most labeled antibody probes. We therefore explored the use of single-stranded oligonucleotides (aptamers), which can bind to proteins with very high affinity and specificity but which are only approximately 10 kD. To evaluate these probes for general protein localization, we sought an aptamer that binds to a widely used protein tag, the green fluorescent protein (GFP). Although this quest was not successful, we were able to solve several practical problems that will confront any such labeling effort, e.g., the rates at which oligonucleotides enter fixed cells of different kinds and the extent of nonspecific oligonucleotide binding to both mammalian and yeast cell structures. Because such localization methods would be of particular value for electron microscopy of optimally fixed material, we also explored the solubility of aptamers under conditions suitable for freeze-substitution fixation. We found that aptamers are sufficiently soluble in cold organic solvents to encourage the view that this approach may be useful for the localization of specific proteins in context of cellular fine structure.
Stanlis
Kristi K H
KK
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA.
McIntosh
J Richard
JR
D000818:Animals; D002478:Cells, Cultured; D004277:DNA, Single-Stranded / Q000737:chemistry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D007614:Macropodidae; D008854:Microscopy, Electron; D009154:Mutation; D015345:Oligonucleotide Probes / Q000737:chemistry; D011506:Proteins / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000166:cytology; D012995:Solubility
D004277:DNA, Single-Stranded; D008164:Luminescent Proteins; D015345:Oligonucleotide Probes; D011506:Proteins; D049452:Green Fluorescent Proteins