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Patrick O'Neil patrick at corona
Mon Mar 13 20:16:43 EST 1995

 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	           A
	U   U G G  C C G G   A
	A   G C C  G G C C   U
          U     |  |       A
                5' 3'

  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:

		       G G	
   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?


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