Comprehensive viral oligonucleotide probe design using conserved protein regions

Jabado, Omar J.; Liu, Yang; Conlan, Sean; Quan, P. Lan; Hegyi, Hédi; Lussier, Yves; Briese, Thomas; Palacios, Gustavo; Lipkin, W. I.

We sought to validate the probe design method by generating a simple thermodynamic model to predict hybridization signal based on sequence composition. We computed the change in Gibbs free energy (ÁG) for all expected probe-viral nucleic acid pairs in the West Nile virus hybridization experiments described above. The calculation method employed finds the most thermodynamically stable structure (minimum free energy) (28) based on empirically established nearest neighbor energies (29) . Strong signal was observed from probe-virus hybrids with ÁG of À32.5 kJ or less. Thus, this value was chosen as the threshold to classify a probe as likely to generate high signal when the cognate viral target is present (Figure 6 ). Probes will be designed in the area of short motifs of 20aa or 60nt Figure 3 . Comprehensive motif-based probe design. The EMBL viral database is clustered with a threshold of 98% nucleotide identity to create a non-redundant sequence database. Coding sequences are subjected to an amino acid motif search, and then probes are made from the underlying nucleic acid sequences. Similarly, nucleic acid motifs are found in non-coding sequences and used to make probes. Database coverage is checked; supplementary probes for highly divergent sequences are designed as necessary. Acronyms: Pfam-Protein Families database, MEME-Multiple Expectation maximization for Motif Elicitation, UTR-untranslated region, LTR-long terminal repeat.