When oligonucleotides are introduced into cells, they may prove to be toxic. This toxicity could be due to successful inhibition of the target gene function. However, toxicity could also be due to (1) binding to one or more nontarget proteins, (2) hybridization to one or more nontarget nucleic acid sequences, or (3) poisoning of the cells by small molecule impurities or endotoxins. The primary consideration is that the observed effects should be consistent with the biology of the system being examined. If cell death is observed, LNAs can be purified by desalting to remove small molecule contaminants. Alternatively, a fresh synthesis of LNA can be performed to determine if newly made material behaves similarly. Toxicity could also be caused by improper choice of transfection conditions, reagent concentrations, cell line, or lipids. The window between the conditions that produce optimal LNA delivery and those that cause cells to die is likely to be small. Use of fluorophore-labeled LNAs provides a convenient method for evaluating the success of a given protocol.
See invivo ready purification
- Baker, B.F., Lot, S.S., Condon, T.P., Cheng-Flournoy, S., Lesnik, E.A., Sasmor, H.M., and Bennett, C.F. 1997. 2'-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J. Biol. Chem. 272:11994-12000.
- Bondensgaard, K., Petersen, M., Singh, S.K., Rajwanshi, V.K., Kumar, R., Wengel, J., and Jacobsen, J.P. 2000. Structural studies of LNA:RNA duplexes by NMR: Conformations and RNase H activity. Chem. Eur. J. 6:2687-2695.
- Braasch, D.A. and Corey, D.R. 2001. Locked nucleic acids: Fine-tuning nucleic acid recognition. Chem. Biol. 8:1-7.