The program will look for the file in a directory specified during the installation. However, if an environment variable NN_PATH is defined, melting will search in this one first. Be careful, the option -A changes the default parameter set defined by the option -H.
###beginning###
-Hdnadna
-Asug96a.nn
-SAGCTCGACTC
-CTCGAGGTGAG
-N0.2
-P0.0001
-v
-Ksan96a
###end###
The nearest-neighbor approach is based on the fact that the helix-coil transition works as a zipper. After an initial attachment, the hybridisation propagates laterally. Therefore, the process depends on the adjacent nucleotides on each strand (the Crick's pairs). Two duplexes with the same base pairs could have different stabilities, and on the contrary, two duplexes with different sequences but identical sets of Crick's pairs will have the same thermodynamics properties (see Sugimoto et al. 1994). This program first computes the hybridisation enthalpy and entropy from the elementary parameters of each Crick's pair.
DeltaH = deltaH(initiation) + SUM(deltaH(Crick's pair))
DeltaS = deltaS(initiation) + SUM(deltaS(Crick's pair))
See Wetmur J.G. (1991) and SantaLucia (1998) for deep reviews on the nucleic acid hybridisation and on the different set of nearest-neighbor parameters.
The mismatching pairs are also taken into account. However the thermodynamic parameters are still not available for every possible cases (notably when both positions are mismatched). In such a case, the program, unable to compute any relevant result, will quit with a warning.
The two first and positions cannot be mismatched. in such a case, the result is unpredictable, and all cases are possible. for instance (see Allawi and SanLucia 1997), the duplex
A T
GTGAGCTCAT
TACTCGAGTG
T A
is more stable than
AGTGAGCTCATT
TTACTCGAGTGA
The dangling ends, that is the umatched terminal nucleotides, can be taken into account.
DeltaH(
AGCGATGAA-
-CGCTGCTTT
) = DeltaH(AG/-C)+DeltaH(A-/TT)
+DeltaH(initG/C)+DeltaH(initA/T)
+DeltaH(GC/CG)+DeltaH(CG/GC)+2xDeltaH(GA/CT)+DeltaH(AA/TT)
+Delta(AT/TG mismatch) +DeltaG(TC/GG mismatch)
(The same computation is performed for DeltaS)
Then the melting temperature is computed by the following formula:
Tm = DeltaH / (DeltaS + Rx ln ([nucleic acid]/F))
Tm in K
(for [Na+] = 1 M )
+ f([Na+]) - 273.15
correction
for the salt concentration (if there are only sodium cations in the solution)and to get the temperature in degree Celsius.
(In fact some corrections are directly included in the DeltaS see that of SanLucia 1998)
If the concentration of the two strands are similar, F is 1 in case of self-complementary oligonucleotides, 4 otherwise. If one strand is in excess (for instance in PCR experiment), F is 2 (Actually the formula would have to use the difference of concentrations rather than the total concentration, but if the excess is sufficient, the total concentration can be assumed to be identical to the concentration of the strand in excess).
Note however, MELTING makes the assumption of no self-assembly, i.e. the computation does not take any entropic term to correct for self-complementarity.
If there are only sodium ions in the solution, we can use the following corrections:
The correction can be chosen between
wet91a,
presented in Wetmur 1991
i.e.
16.6 x log([Na+] / (1 + 0.7 x [Na+])) + 3.85
san96a
presented in SantaLucia et al. 1996
i.e.
12.5 x log[Na+]
and
san98a
presented in SantaLucia 1998
i.e.
a correction of the entropic term without modification of enthalpy
DeltaS = DeltaS([Na+]=1M) + 0.368 x (N-1) x ln[Na+]
Where N is the length of the duplex (SantaLucia 1998 actually used 'N' the number of non-terminal phosphates, that is effectively equal to our N-1). CAUTION, this correction is meant to correct entropy values expressed in cal.mol-1.K-1!!!
[Mon+] = [Na+] + [K+] + [Tris+]
Where [Tris+] = [Tris buffer]/2. (in the option -t, it is the Tris buffer concentration which is entered).
If [Mon+] = 0, the divalent ions are the only ions present
and the melting temperature is :
1/Tm(Mg2+) = 1/Tm(1M Na+) + a - b x ln([Mg2+]) + Fgc x (c + d x ln([Mg2+]) + 1/(2 x (Nbp - 1)) x (- e +f x ln([Mg2+]) + g x ln([Mg2+]) x ln([Mg2+]))
where :
a = 3.92/100000.
b = 9.11/1000000.
c = 6.26/100000.
d = 1.42/100000.
e = 4.82/10000.
f = 5.25/10000.
g = 8.31/100000.
Fgc is the fraction of GC base pairs in the sequence and Nbp is the length of the sequence (Number of base pairs).
If [Mon+] > 0, there are several cases because we can have a competitive DNA binding between monovalent and divalent
cations :
If the ratio [Mg2+]^(0.5)/[Mon+] is inferior to 0.22, monovalent ion influence is dominant, divalent cations can be disregarded and the melting temperature is :
1/Tm(Mg2+) = 1/Tm(1M Na+) + (4.29 x Fgc - 3.95) x 1/100000 x ln([mon+]) + 9.40 x
1/1000000 x ln([Mon+]) x ln([Mon+])
where : Fgc is the fraction of GC base pairs in the sequence.
If the ratio [Mg2+]^(0.5)/[Mon+] is included in [0.22, 6[,
we must take in account both Mg2+ and monovalent cations
concentrations. The melting temperature is :
1/Tm(Mg2+) = 1/Tm(1M Na+) + a - b x ln([Mg2+]) + Fgc x (c + d x ln([Mg2+]) +
1/(2 x (Nbp - 1)) x (- e + f x ln([Mg2+]) + g x ln([Mg2+]) x ln([Mg2+]))
where : a = 3.92/100000 x (0.843 - 0.352 x [Mon+]0.5 x ln([Mon+])).
b = 9.11/1000000.
c = 6.26/100000.
d = 1.42/100000 x (1.279 - 4.03/1000 x ln([mon+]) - 8.03/1000 x
ln([mon+] x ln([mon+]).
e = 4.82/10000.
f = 5.25/10000.
g = 8.31/100000 x (0.486 - 0.258 x ln([mon+]) + 5.25/1000 x ln([mon+] x ln([mon+] x ln([mon+]).
Fgc is the fraction of GC base pairs in the sequence and Nbp is the length of the sequence (Number of base pairs).
Finally, if the ratio [Mg2+]^(0.5)/[Mon+] is superior to 6, divalent ion influence is dominant, monovalent cations can be disregarded and the melting temperature is :
1/Tm(Mg2+) = 1/Tm(1M Na+) + a - b x ln([Mg2+]) + Fgc x (c + d x ln([Mg2+]) +
1/(2 x (Nbp - 1)) x (- e + f x ln([Mg2+]) + g x ln([Mg2+]) x ln([Mg2+]))
where :
a = 3.92/100000.
b = 9.11/1000000.
c = 6.26/100000.
d = 1.42/100000.
e = 4.82/10000.
f = 5.25/10000.
g = 8.31/100000.
Fgc is the fraction of GC base pairs in the sequence and
Nbp is the length of the sequence (Number of base pairs).
It is important to realise that the nearest-neighbor approach has been established on small oligonucleotides. Therefore the use of melting in the non-approximative mode is really accurate only for relatively short sequences (Although if the sequences are two short, let's say < 6 bp, the influence of extremities becomes too important and the reliability decreases a lot). For long sequences an approximative mode has been designed. This mode is launched if the sequence length is higher than the value given by the option -T (the default threshold is 60 bp).
The melting temperature is computed by the following formulas:
DNA/DNA:
Tm = 81.5+16.6*log10([Na+]/(1+0.7[Na+]))+0.41%GC-500/size
DNA/RNA:
Tm = 67+16.6*log10([Na+]/(1.0+0.7[Na+]))+0.8%GC-500/size
RNA/RNA:
Tm = 78+16.6*log10([Na+]/(1.0+0.7[Na+]))+0.7%GC-500/size
This mode is nevertheless strongly disencouraged.
Melting is currently accurate only when the hybridisation is performed at pH 71.
The computation is valid only for the hybridisations performed in aqueous medium. Therefore the use of denaturing agents such as formamide completely invalidates the results.
Allawi H.T., SantaLucia J. (1998). Nearest Neighbor thermodynamics parameters for internal G.A mismatches in DNA. Biochemistry 37: 2170-2179
Allawi H.T., SantaLucia J. (1998). Thermodynamics of internal C.T mismatches in DNA. Nucleic Acids Res 26: 2694-2701.
Allawi H.T., SantaLucia J. (1998). Nearest Neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects. Biochemistry 37: 9435-9444.
Bommarito S., Peyret N., SantaLucia J. (2000). Thermodynamic parameters for DNA sequences with dangling ends. Nucleic Acids Res 28: 1929-1934
Breslauer K.J., Frank R., Blï¿½ker H., Marky L.A. (1986). Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA 83: 3746-3750
Freier S.M., Kierzek R., Jaeger J.A., Sugimoto N., Caruthers M.H., Neilson T., Turner D.H. (1986). Improved free-energy parameters for predictions of RNA duplex stability. Biochemistry 83:9373-9377
Owczarzy R., Moreira B.G., You Y., Behlke M.B., Walder J.A. (2008) Predicting stability of DNA duplexes in solutions containing Magnesium and Monovalent Cations. Biochemistry 47: 5336-5353.
Peyret N., Seneviratne P.A., Allawi H.T., SantaLucia J. (1999). Nearest Neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G and T.T mismatches. dependence and pH effects. Biochemistry 38: 3468-3477
SantaLucia J. Jr, Allawi H.T., Seneviratne P.A. (1996). Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35: 3555-3562
Sugimoto N., Katoh M., Nakano S., Ohmichi T., Sasaki M. (1994). RNA/DNA hybrid duplexes with identical nearest-neighbor base-pairs hve identical stability. FEBS Letters 354: 74-78
Sugimoto N., Nakano S., Katoh M., Matsumura A., Nakamuta H., Ohmichi T., Yoneyama M., Sasaki M. (1995). Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34: 11211-11216
Sugimoto N., Nakano S., Yoneyama M., Honda K. (1996). Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nuc Acids Res 24: 4501-4505
Watkins N.E., Santalucia J. Jr. (2005). Nearest-neighbor t- hermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Research 33: 6258-6267
Wright D.J., Rice J.L., Yanker D.M., Znosko B.M. (2007). Nearest neighbor parameters for inosine-uridine pairs in RNA duplexes. Biochemistry 46: 4625-4634
Xia T., SantaLucia J., Burkard M.E., Kierzek R., Schroeder S.J., Jiao X., Cox C., Turner D.H. (1998). Thermodynamics parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 37: 14719-14735
For review see:
SantaLucia J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 95: 1460-1465
SantaLucia J., Hicks Donald (2004) The Thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Struct. 33: 415 -440
Wetmur J.G. (1991) DNA probes: applications of the principles of nucleic acid hybridization. Crit Rev Biochem Mol Biol 26: 227-259
You can use MELTING through a web server at http://bioweb.pasteur.fr/seqanal/interfaces/melting.html
If an infile is called, containing the address of another input file, it does not care of this latter. If it is its own address, the program quit (is it a bug or a feature?).
In interactive mode, a sequence can be entered on several lines with a backslash
AGCGACGAGCTAGCCTA\
AGGACCTATACGAC
If by mistake it is entered as
AGCGACGAGCTAGCCTA\AGGACCTATACGAC
The backslash will be considered as an illegal character. Here again, I do not think it is actually a bug (even if it is unlikely, there is a small probability that the backslash could actually be a mistyped base).
This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.
You should have received a copy of the GNU General Public License along with this program; if not, write to the Free Software Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA 02111-1307 USA
See the file ChangeLog for the changes of the versions 4 and more recent.