The free energy minimization algorithm includes a number of advanced options:
• Avoid isolated base pairs. The algorithm filters out isolated base pairs (i.e. stems of length 1).
• Apply different energy rules for Grossly Asymmetric Interior Loops (GAIL). Compute the minimum free energy applying different rules for Grossly Asymmetry Interior Loops (GAIL). A Grossly Asymmetry Interior Loop (GAIL) is an interior loop that is or where (see http://mfold.rna.albany.edu/doc/mfold-manual/node5.php).
• Include coaxial stacking energy rules. Include free energy increments of coaxial stacking for adjacent helices [Mathews et al., 2004].
• Apply base pairing constraints. With base pairing constraints, you can easily add experimental constraints to your folding algorithm. When you are computing suboptimal structures, it is not possible to apply base pair constraints. The possible base pairing constraints are:
• Force two equal length intervals to form a stem.
• Prohibit two equal length intervals to form a stem.
• Prohibit all nucleotides in a selected region to be a part of a base pair.
Base pairing constraints have to be added to the sequence before you can use this option - see below.
• Maximum distance between paired bases. Forces the algorithms to only consider RNA structures of a given upper length by setting a maximum distance between the base pair that opens a structure.

Specifying structure constraints

Structure constraints can serve two purposes in CLC Genomics Workbench: they can act as experimental constraints imposed on the MFE structure prediction algorithm or they can form a structure hypothesis to be evaluated using the partition function.

To force two regions to form a stem, open a normal sequence view and:

Select the two regions you want to force by pressing Ctrl while selecting - (use on Mac) | right-click the selection | Add Structure Prediction Constraints| Force Stem Here

This will add an annotation labeled "Forced Stem" to the sequence (see figure 21.5).

Figure 21.5: Force a stem of the selected bases.

Using this procedure to add base pairing constraints will force the algorithm to compute minimum free energy and structure with a stem in the selected region. The two regions must be of equal length.

To prohibit two regions to form a stem, open the sequence and:

Select the two regions you want to prohibit by pressing Ctrl while selecting - (use on Mac) | right-click the selection | Add Structure Prediction Constraints | Prohibit Stem Here

This will add an annotation labeled "Prohibited Stem" to the sequence (see figure 21.6).

Figure 21.6: Prohibit the selected bases from forming a stem.

Using this procedure to add base pairing constraints will force the algorithm to compute minimum free energy and structure without a stem in the selected region. Again, the two selected regions must be of equal length.

To prohibit a region to be part of any base pair, open the sequence and:

Select the bases you don't want to base pair | right-click the selection | Add Structure Prediction Constraints | Prohibit From Forming Base Pairs

This will add an annotation labeled "No base pairs" to the sequence, see 21.7.

Figure 21.7: Prohibiting any of the selected base from pairing with other bases.

Using this procedure to add base pairing constraints will force the algorithm to compute minimum free energy and structure without a base pair containing any residues in the selected region.

When you click Predict secondary structure () and click Next, check Apply base pairing constraints in order to force or prohibit stem regions or prohibit regions from forming base pairs.

You can add multiple base pairing constraints, e.g. simultaneously adding forced stem regions and prohibited stem regions and prohibit regions from forming base pairs.