adapter design and duplex energy values (comp)...

rhubner at molbiol.ox.ac.uk rhubner at molbiol.ox.ac.uk
Tue Mar 5 05:59:57 EST 1996


hello friends  ;-)

 I'm in the process of designing an oligonucleotide pair to get an adapter 
with 4bp-overhangs on each end... I have a designed a couple of oligos with the 
desired ends and got kcal/mol values for each 13-mer from primer analysis 
software... There is a  stable dimer of 6 bp  (plus one base further another 
single base match) with -10.3 kcal/mol and because one overhang is for NotI RE 
the GGCC 3'-dimer has also a high value of -9.3 kcal/mol...
 
 In order to get an idea about the value for the desired 9 bp dimer I used the 
program FoldRNA in GCG with the seq written from 5' -> 3'  and a stretch of 
"N" inserted in between... I had seen something like this in a Biotechniques 
article a few years ago where 10 NNNNNNNNNN had been used to make a circular 
[RNA] molecule to check out for primer dimers... 
 The obtained value was -12.3 kcal/mol... however, the value increases when I 
reduce the number of Ns and is for instance -13.6 kcal/mol if 6 are inserted 
instead of 10... if 50 Ns are inserted it can be about -9 kcal/mol... and if 
no Ns are inserted [ hence 4 base overhang loops directly back onto stem!] I
read -12.0 kcal/mol...

 Well, now I don't know which [if any] of those approximations I can accept... 
and I would be indeed very grateful, if somebody could let me know how I could 
calculate a suitable value... I suspect that during slow cooling of the heated oli
mixture the designed molecule should perform properly even if a slight risk 
remains for those +6 bp stretches versus the "full-length" 9 bp duplex with 
4-base overhangs...

 Thank you very much for any insights - I highly appreciate!

 Have a nice time,
 Roland

 




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