In situ imaging and isolation of proteins using dsDNA oligonucleotides.
As proteomics initiatives mature, the need will arise for the multiple visualization of proteins and supramolecular complexes within their true context, in situ. Single-stranded DNA and RNA aptamers can be used for low resolution imaging of cellular receptors and cytoplasmic proteins by light microscopy (LM). These techniques, however, cannot be applied to the imaging of nuclear antigens as these single-stranded aptamers bind endogenous RNA and DNA with high affinity. To overcome this problem, we have developed a novel method for the in situ detection of proteins using double-stranded DNA oligonucleotides. To demonstrate this system we have utilized the prokaryotic DNA-binding proteins LacI and TetR as peptide tags to image fusion proteins in situ using dsDNA oligonucleotides encoding either the Lac or Tet operator. Using fluorescent and fluorogold dsDNA oligonucleotides, we localized within the nucleus a TetR-PML fusion protein within promyelocytic leukaemia protein (PML) bodies by LM and a LacI-SC35 fusion protein within nuclear speckles by correlative light and electron microscopy (LM/EM). Isolation of LacI-SC35 was also accomplished by using biotinylated dsDNA and streptavidin sepharose. The use of dsDNA oligonucleotides should complement existing aptamer in situ detection techniques by allowing the multiple detection and localization of nuclear proteins in situ and at high resolution.
Dellaire
Graham
G
Programme in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
Nisman
Rozalia
R
Eskiw
Christopher H
CH
Bazett-Jones
David P
DP
D000818:Animals; D045744:Cell Line, Tumor; D022003:Cell Nucleus Structures / Q000737:chemistry; D005456:Fluorescent Dyes; D008853:Microscopy; D046032:Microscopy, Energy-Filtering Transmission Electron; D009687:Nuclear Proteins / Q000032:analysis* / Q000302:isolation & purification*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics
D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012097:Repressor Proteins