BMC Genomics 2008, 9:339 http://www.biomedcentral.com/1471-2164/9/339 TFhigeudreeta3iled interface The detailed interface. This demonstrates that pknots was used to compute an MFE of -23.7 kcal/mol for the 100 bases fol- lowing the AAAAAU slippery site at position 1653 of the EST2 gene. When randomized 100 times using Fisher-Yates shuffling, a mean MFE of -17.4 kcal/mol was computed for a normal distribution of correlation coefficient 0.9627. The MFE distribution of the randomized sequences is on the right; with the idealized normal distribution in red. The black vertical line marks the mean MFE of the randomized sequences, and the green vertical line marks the MFE of the native sequence. This secondary structure is significantly more stable than random (z score = -2.10). The predicted mRNA secondary structure of this sequence is shown below using both bracket notation, and using a Feynman diagram. To avoid complications of untranslated intronic sequences, the PRFdb contains only mature mRNA sequences (cDNA sequences primarily). Sequences are imported into the PRFdb from a web inter- face using Genbank accession numbers, yeast genome accessions, or raw sequences. Each new sequence is first passed through a simple text filter that searches for slip- pery sites following the International Union of Pure and Applied Chemistry (IUPAC) pattern 'N NNW WWH' (or X XXY YYZ), where N (X) denotes any three identical bases, W (Y) denotes AAA or UUU, H (Z) ≠ G, and spaces indi- cate the incoming (zero) reading frame. Since the distance between the end of the slippery site and the downstream stimulatory sequence is important, a spacer of 1 to 8 nt was incorporated into the algorithm. Downstream sequences (100, 75 and 50 nt) are passed to RNAMotif with a descriptor looking for the potential to form an mRNA pseudoknot. Sequence windows passing this min- imal test are passed to multiple pseudoknot predicting mRNA secondary structure prediction algorithms, includ- ing Pknots [16], Nupack [17], and HotKnots [18]. Mfold [19] and the Iterative Loop Matching algorithm are not used because Mfold does not predict pseudoknots and ILM does not provide the minimum free energy values for predicted secondary structures. After folding, every sequence is ran- domized using one or more algorithms including: Fisher- Yates shuffling to maintain dinucleotide frequencies, codon frequencies, or nucleotide frequencies. The result- ing random sequence windows are then refolded without searching for pseudoknots. This process is repeated a fixed number of times (100 by default) to create a distribution of sequence specific randomized MFEs. These resulting distribution of randomized MFEs is then compared the MFE of the original sequence window. These values are used to compute a z score, thus providing a measurement of the significance of the native sequence. Page 5 of 7 (page number not for citation purposes)