W506–W511 Nucleic Acids Research, 2005, Vol. 33, Web Server issue doi:10.1093/nar/gki453 AMOD: a morpholino oligonucleotide selection tool Eric W. Klee1,4, Kyong Jin Shim3,4, Michael A. Pickart2,4, Stephen C. Ekker2,4 and Lynda B. M. Ellis1,4,* 1Department of Laboratory Medicine and Pathology, 2Department of Genetics, Cell Biology and Development, 3Department of Computer Science and Engineering and 4Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, Minneapolis, MN, USA Received February 11, 2005; Revised and Accepted April 1, 2005 ABSTRACT AMOD is a web-based program that aids in the func- tional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome data- bases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design 200 morpholinos for a func- tional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at http://www. secretomes.umn.edu/AMOD/. INTRODUCTION Vertebrate genomes contain an estimated 20–30K genes involved in diverse processes; many encoding proteins with unknown function. The annotation of these genes remains a major step in understanding the vertebrate genome. The development of morpholino-based gene ‘knockdown’ techno- logy provides a method for identifying function from primary sequence, on a genome-wide scale in many vertebrates (1–7). Sequence-driven screens for functional annotation of genomic data are being developed in systems that lack the high cost, signiﬁcant time and infrastructure commitments associated with traditional model vertebrates. RNAi-based screening in nematode (8) and ﬂy tissue culture cells (9) have applied ‘knockdown’ strategies to sequence-speciﬁc annotation, and siRNA has been effectively applied in mammalian tissue culture models (10). However, these approaches remain impractical for large-scale in vivo work. Phosphorodiamidate morpholino oligonucleotides (morpho- linos) are non-classical antisense reagents that modulate gene expression by inhibiting protein translation or inducing altern- ative splicing events. A synthetic DNA analog that contains a six-member morpholino ring and a neutral charge phosphoro- diamidate backbone, morpholinos are resistant to nuclease digestion (8) and are freely water-soluble (9). Morpholinos overcome many limitations associated with traditional anti- sense reagents (11) and have been effectively used in many eukaryotes (1–7,11,12). The effect morpholinos cause on the expression is determined by the position targeted within a nucleotide sequence. Morpholinos targeting the 50-untrans- lated regions (50-UTRs) in proximity to the translational initiation site (TIS) disrupt ribosomal complex formation and inhibit protein translation of mRNA, while morpholinos targeting pre-mRNA splice sites can induce alternative splicing events (12–14). Consequently, effective morpholino design requires a clear understanding of nucleotide sequence characteristics in addition to the biochemical properties of the morpholino oligonucleotides. Since signiﬁcant sequence analyses are required for informed morpholino design, the application of this technology to large-scale screens (5,15) necessitates a software tool capable of efﬁcient and accurate target sequence selection and morpholino design. Programs for siRNA design include some, but not all, of the necessary processes required for morpholino design (16–19). Both siRNA and morpholino design require computation of biochemical properties of short oligonucleotides, including base composition and homogeneous nucleotide run calcula- tions. However, siRNA does not require a detailed analysis of oligonucleotide binding position relative to target nucleotide *To whom correspondence should be addressed at Mayo Mail Code 609, 420 SE Delaware Street, Minneapolis, MN 55455, USA. Tel: +1 612 625 9122; Fax: +1 612 624 6404; Email: lynda@umn.edu Present address: Michael A. Pickart, Department of Biology, University of Wisconsin–Stout, Menomonie, Wisconsin, WI, USA ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org