Evaluation of different conditions for ligating dumbbell-shaped oligonucleotides.
We tested three different standard ligation conditions (37 degrees C for 30 min, 16 degrees C for 24 h, and 4 degrees C for 48 h) to generate dumbbell-shaped oligonucleotides (ODNs) as transcription factor decoys for SOX9 and alphaA-crystallin binding protein 1 (CRYBP1), which are positive and negative transcriptional regulators for type II collagen expression in chondrocytes. Decoy ODN for CRYBP1 was successfully produced as a "dumbbell" by all three conditions. A small amount of decoy ODN for SOX9, however, remained unligated under all three ligation conditions. Ligation at 4 degrees C for 48 h appeared to be the least desirable for SOX9 ODN. Transfection experiments with the SOX9 ODN ligated in different conditions and a luciferase-based reporter system also supports this conclusion. In general, shorter incubation time produced more acceptable results for this ODN than incubation for a longer time. These data suggest that different ligation conditions should be tested prior to creating dumbbell-shaped ODNs for transfection experiments.
Yamagiwa
Hiroshi
H
Dept of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Bolander
Mark E
ME
Sarkar
Gobinda
G
D000818:Animals; D002460:Cell Line; D019902:Chondrocytes / Q000502:physiology*; D003459:Crystallins / Q000235:genetics*; D006609:High Mobility Group Proteins / Q000235:genetics*; D008024:Ligands; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics*; D055758:SOX9 Transcription Factor; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D003459:Crystallins; D006609:High Mobility Group Proteins; D008024:Ligands; D009841:Oligonucleotides; D055758:SOX9 Transcription Factor; D014157:Transcription Factors