I have downloaded the program and used it on a short RNA sequence but
got puzzling (to me) results.
My sequence is 22 bases long:
5'-C C G U A U U U G G C C G G A A U A C C G G-3'
Asking the program to calculate structure(s) at 37 degrees Celsius with no
constraints as to basepairing, etc, I get a single structure:
5'-C C G U A U U U G G C C G G A
3'-G G C C U
According to the program, this structure's free energy is -3.54 kJ or
about 0.8 kcal/mol (???!!!). When I calculate by hand using numbers from
Ann.Rev.Biophys.Chem. 1988. 17: 167-192, Turner and Sugimoto, I get a
deltaG of -2.1 kcal/mol or about -9 kJ. This is hardly the most stable
structure, by my calculations, either (I also have no idea how to treat
the unpaired part of the strand - what deltaH or deltaS value(s) to
assign, if any. Any help there?).
By entering basepairing constraints to allow for coaxial stacking, I
get one of the structures I was expecting (and wanted):
U U G G C C G G A
A G C C G G C C U
U | | A
For this structure, using Turner numbers, I get a deltaG of -4.9
kcal/mol - using a modified number for the coaxial stacking region
between the 5' and 3' ends (from a more recent Turner paper in PNAS from
1994). In any case, even if I use the normal Turner numbers from the 1988
paper to calculate deltaG, I still get a much larger free energy value
than the program, which gives this structure an energy of -3.31 kJ (vs my
roughly -16 kJ calculation) and only after I twist the program's arm by
requiring certain bases to pair with others.
Another (final) structure I have to force out of the program with
constraints on basepairing is the, to me, seemingly obvious structure:
5'- C C G U A U U U C
3'- G G C A U A A G
C G C
The program gives this structure an energy of -1.21 kJ (at 37 degrees)
while my hand calculation gives it a deltaG of -3.2 kcal/mol or around
-12 kJ. What's up? Am I miscalculating somewhere? Leaving something
obvious out? I am assigning deltaG values from the 1988 Turner paper for
nearest neighbor propagation, plus the penalty for the 5 member loop.
Does anyone else get the values I mention? Has anyone else used the program?
I realize that it likely doesn't take into account coaxial stacking
reinforcements of structural stability (my nucleic acid biochem
instructor doesn't know of a program that does as yet either) but still,
even ignoring that, the coaxial structure I depict above is more stable
than the first structure, which appears to be a transition state on the way
to or on the way from the coaxial structure. It is less stable, by the
deltaGs AND deltaHs I calculate for them. Am I stupid? Or is there an
apparent flaw in this program's calculations?