Technical Reference #289

289.

Linear 2' O Methyl RNA Probes for the Visualization of RNA in Living Cells C. Molenaar; S. A. Marras; J. C. M. Slats; J.-C. Truffert; M. Lemaitre; A.K. Raap; R.W. Dirks; H.J. Tanke, Public Health Research Institute, Nucleic Acids Research, 29(289), (2001)
Link To Paper

Abstract:
U1snRNA U3snRNA 28 S ribosomal RNA poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2′ O-Methyl oligoribonucleotides (2′ OMe RNA). Antisense 2′ OMe RNA probes showed fast hybridization kin

Materials & Methods:
Probes For the various RNA targets described probes were selected on the basis of sequences in the NCBI genome database. Single-stranded regions or partly single-stranded regions in the secondary RNA structure for U1 snRNA and U3 snRNA (28) were targeted. They were varied in length structure and nucleotide modification. Sequences and GenBank IDs are listed in Table Table1.1. DNA oligonucleotides were synthesized using standard phosphoramidite chemistry and purified by HPLC. 2′ OMe RNA probes were synthesized using standard 2′ OMe phosphoramidite monomers. For linear probes fluorescent dyes were covalently linked to the 5′-end via a succinimidyl ester derivative (Molecular Probes Eugene OR). Molecular beacons were synthesized automatically with the 3′-4-dimethylaminoazobenzene-4′-sulfonyl (dabcyl)-quencher linked to the solid phase support (DABCYL-CPG Glen Research Sterling VA). Oligonucleotide chain elongation was performed via standard cyanoethyl-phosphoramidite chemistry and fluorophores were covalently linked to the 5′-end via iodoacetamide (Molecular Probes) or phosphoramidite derivatives (Glen Research) following release from the solid support. All 2′ OMe RNA probes were purified twice by reverse phase HPLC with a Waters 600E instrument equipped with a Waters 996 Photodiode Array Detector for simultaneous detection at three different wavelengths. Ion-molecular weights of purified probes were determined by mass spectrometry using a Dynamo time-of-flight instrument. The initial molecular beacon probes were designed following the guidelines described by Tyagi and Kramer (26) (http:\\www.molecular-beacons.org). The intramolecular configuration of molecular beacons and their target sequences was modeled using the DNA mfold program (www.ibc.wustl.edu/~zuker/dna/form1.shtml) which uses thermodynamic parameters established by SantaLucia (26). The molecular beacons did not contain self-complementary sequences in the loop. Beacons were equipped with stem sequences of varying length.

Microscopic Technique
Fluorescence Microscopy, Laser

Cell Type(s)
U2OS, R9G Cells