|ZFIN ID: ZDB-PUB-031103-8|
Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics
Urtishak, K.A., Choob, M., Tian, X., Sternheim, N., Talbot, W.S., Wickstrom, E., and Farber, S.A.
|Source:||Developmental dynamics : an official publication of the American Association of Anatomists 228(3): 405-413 (Journal)|
|Registered Authors:||Farber, Steven, Sternheim, Nitzan, Talbot, William S., Urtishak, Karen|
|Keywords:||embryo, development, morpholino, digonucleotide, peptide nucleic acid|
|PubMed:||14579379 Full text @ Dev. Dyn.|
Urtishak, K.A., Choob, M., Tian, X., Sternheim, N., Talbot, W.S., Wickstrom, E., and Farber, S.A. (2003) Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics. Developmental dynamics : an official publication of the American Association of Anatomists. 228(3):405-413.
ABSTRACTNegatively charged homo-oligomers of alternating trans-4-hydroxy-L-proline/phosphonate polyamides with DNA bases (HypNA-pPNA) display excellent hybridization properties toward DNA and RNA, while preserving the mismatch discrimination, nuclease resistance, and protease resistance of peptide nucleic acids (PNAs). Similar properties are associated with morpholino phosphorodiamidate (MO) DNA mimics, which have been used in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. We evaluated mixed sequence HypNA-pPNAs as an alternative to MOs, and found that even a single central DNA mismatch lowered the HypNA-pPNA melting temperature by 16 degrees C. We then observed that the melting temperatures of HypNA-pPNA 18-mers hybridized to RNA 25-mers were comparable to the melting temperatures of MO 25-mers, and that two HypNA-pPNA mismatches lowered the melting temperature with RNA by 18 degrees C. In zebrafish embryos we observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail, and uroD, with greater mismatch stringency. Finally we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. HypNA-pPNAs designed to inhibit translation of specific zebrafish RNA targets thus demonstrated stringent hybridization properties, relative to DNA and MO oligomers, and present a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes.
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