Delivered by Publishing Technology to: Sankar Basu IP: 116.193.141.16 On: Thu, 19 Dec 2013 19:57:45 Copyright: American Scientific Publishers COMMUNICATION Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Bioinformatics and Intelligent Control Vol. 2, 316–320, 2013 polDNAmelt: Local Melting Within Polymeric DNA—An Improved Method and Its Applications Sankar Basu 1 and Dhananjay Bhattacharyya 2 ∗ 1 Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India 2 Computer Science Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India Local pseudo-unimolecular partial melting in polymeric cellular DNA is very much different from bimolecular comprehensive melting of short oligonucleotides in solutions. Several softwares esti- mate the melting temperature pretty accurately for the latter case and are used routinely for primer designing in PCR. However, the local partial melting in polymeric DNA involves the formation of a short-lived interior bubble constrained by double helical DNA at both the ends and thus (i) the melting-annealing transition becomes concentration independent and (ii) the fraying effect due to dangling ends are ruled out. Based on these features, the current study appropriately modifies the thermodynamic equations for calculation of local melting within polymeric DNA and incorpo- rates them in a web-server (http://www.saha.ac.in/biop/www/db/local/nsdnamelt.html). Applications of the method in validating annotated promoter sequences and origin of replication have also been surveyed. Keywords: T m , Local Pseudo-Unimolecular Partial Melting, Short-Lived Interior Bubble, Concentration Independent Dna Melting, Fraying Effect. We report the availability of a web-server, polDNAmelt (http://www.saha.ac.in/biop/www/db/local/nsdnamelt.html) to analyze local melting within polymeric DNA. Theoreti- cal localization of melting nucleation sites distributed over chromosomal double stranded DNA (ds-DNA) is crucial to study since only the converted single stranded DNA is active for most of the biochemical processes (e.g., repli- cation, transcription, recombination, etc.). Local melting within polymeric DNA with both the ends being held in double helical conformation is significantly different from melting of short oligonucleotides in solution. In either case, the main forces stabilizing a ds-DNA are base pair- ing and stacking interactions. 1 While there are two types of Watson-Crick base pairs (bp) present in ds-DNA with different hydrogen bonding strength, there are ten unique stacking arrangements between any two consecutive base pairs leading to ten different stacking energy increments. Thus, different base pairing and stacking interaction ener- gies across diverge sequences (of say, identical length) require different amount of kinetic energies to transfer to unstructured melted out conformations. These evolution- arily conserved melting nucleation sites inhomogeneously distributed throughout the genome, map to local minima of stability with respect to their immediate neighboring ∗ Author to whom correspondence should be addressed. sequences. Although one can determine T m considering only Watson-Crick base pairing energy (i.e., GC con- tent) in a sequence, inclusion of heterogeneous stacking considerably improves agreements between theory and experiment for long polymeric DNA. 2 Experimental char- acterizations of duplex stability by (calorimetric and/or spectroscopic) DNA-melting studies have worked out several libraries of stacking-based melting thermodynamic parameters. 2–11 Quite a few softwares and servers 12–16 are available for prediction of melting temperature (T m  of short oligonucleotides using these libraries but their use to determine sequence dependent local melting can be erro- neous. The present study attempts to modify appropriate thermodynamic equations for calculation of local melting within long polymeric DNA and incorporates them in polDNAmelt. In a ds-DNA (devoid of mismatches), stacking occurs between every two consecutive base pairs (Nearest Neigh- bors: NN) once the Watson-Crick hydrogen bonding donor–acceptor pairs are stereo-specifically positioned. In the available libraries of stacking-based NN-step param- eters (H 0 , S 0 , G 0 , the contribution of hydrogen bond- ing is implicitly included. PolDNAmelt utilizes the library derived by Santalucia 3 and adds these individual step parameters (NN propagation energies) for each consecutive NN-step along a polymeric DNA. This library is ‘a single 316 J. Bioinf. Intell. Control 2013, Vol. 2, No. 4 2326-7496/2013/2/316/005 doi:10.1166/jbic.2013.1058