W508 Nucleic Acids Research, 2005, Vol. 33, Web Server issue Figure 2. Target sequence evaluation phase. (A) The 10 highest scoring alignments for a gene sequence are presented in a BLAST report and alignment summary. (B) Putative start sites identified by either method can be selected, and the TIS position highlighted in gray text in a display of the target gene sequence. binding is estimated by adding the number of hydrogen bonds occurring in all possible self-to-self, ungapped alignments. For example, Figure 3 illustrates a morpholino self-to-self align- ment with an intra-strand binding score of 30, 18 from six G–C bonds and 12 from six A–T bonds. All intra-strand binding alignments are reported, and the alignments yielding the maximum global intra-strand binding and the maximum intra-strand binding constrained to a four-base window are noted (Figure 4A). The list of putative morpholino oligonuc- leotides can be ﬁltered using base composition, or using the intra-strand binding score to allow users to identify sequences with the least intra-strand binding. Default thresholds for base composition and intra-strand binding properties are set to existing morpholino design guidelines; however, users may modify these values to ﬁt custom design criteria. A critical aspect of antisense reagent design is the evalu- ation of the reagent’s target speciﬁcity (11,13). AMOD pro- vides a second set of BLAST comparisons between select morpholinos and genomic sequences or the transcriptome to identify all sequences complementary to the selected mor- pholino oligonucleotides (Figure 4B). These comparisons enable a user to select a morpholino that maximizes binding to the target gene with high speciﬁcity while minimizing bind- ing to other gene targets that may induce effects unrelated to the speciﬁc knockdown of the target gene. User interaction A design session on the AMOD web server begins with the entry of a user-selected target nucleotide sequence in the