Abstract

1. ULTRASONIC CLEAVAGE ANALYSIS: A NOVEL APPROACH TO STUDY THE SEQUENCE EFFECTS ON DNA STRUCTURAL DYNAMICS. Nechipurenko1 D.Yu., Il'icheva2 I.A., Golovkin2 M.V., Panchenko3 L.A., Polozov4 R.V., Nechipurenko2 Yu.D., Grokhovsky2 S.L. 1) Dep. of Physics, Moscow State University, Moscow 119992, Russia 2) Engelhardt Institute of Molecular Biology RAS, Vavilov str., 32 Moscow 119991, Russia 3) Dep. of Biology, Moscow State University, Moscow 119992, Russia 4) Institute of Theoretical and Experimental Biophysics, Puschino142290, Russia Abstract Recently we have developed a new method for studying sequence dependent structural dynamics of DNA fragments in solution. The approach is based on ultrasound - induced cleavage of DNA sugar phosphate backbone. Experiments demonstrate that ultrasonic cleavage of DNA observed in our case represents mechanochemical reaction induced by cavitational processes in irradiated solution. Sequence effects on DNA cleavage patterns obtained by PAGE were studied using consequent mono, di and tetranucleotide levels of description. Our analysis revealed the enhancement of cleavage rate in phosphodiester bond following deoxycitidine. At the dinucleotide level, cleavage rate of this bond diminishes in the following row of dinucleotides: dСрdG > dСрdА > dСрdТ >> dCpdC. Analysis at the tetranucleotide level has shown that for some dinucleotides the cleavage rate depends on the flanking base pairs to a greater extent than for the others. The average cleavage rates of central phosphodiester bond in 16 possible dinucleotides as well as in 256 possible tetranucleotides are reported. Distinctive features of obtained sequence-dependent ultrasonic cleavage rates correlate with data covering sequence effects on conformational mobility of DNA sugar phosphate backbone reported by NMR 13С spin relaxation, 31Р chemical shifts in solutions, molecular dynamics simulation studies and quantum chemistry computations. Thus, the increase in ultrasonic cleavage rate reflects enhanced conformational flexibility in particular sites of the sugar phosphate backbone. Hence, the obtained data on the cleavage rates in 256 possible tetranucleotides may provide information useful for studying structural peculiarities in functional genomic elements. Introduction Ultrasonic exposure of intact DNA fragments in solution results in sequence specific cleavage[1]. This cleavage is likely the result of hydrodynamic shearing forces which originate from high velocity gradients caused by the collapse of cavitation bubbles [2]. High velocity gradients in the streaming solution might exert the mechanical deformation of the molecule by friction forces. Thus, the observed cleavage of DNA fragments most likely represents complex mechanochemical process, which includes mechanical deformation of the molecule before the actual chemical reaction takes place Ultrasonic cleavage analysis covers cleavage properties of DNA fragments with lengths of several hundreds of base pairs. Thus, this method might elucidate structural properties of dsDNA fragments which become apparent on this scale and may not be accessed by NMR analysis or MD simulation. Methods The restriction fragments of dsDNA were 3’ end- radio-labeled and irradiated with ultrasound of 22 kHz frequency. Ultrasonic power subjected to the system was determined calorimetrically and exceeded the value of 60 Watts. The cleavage patterns were obtained by gel electrophoresis in PAAG. Gel images were analyzed using the SAFA software [3]: the cleavage profiles were built and normalized using values obtained by the linear filtration of the cleavage profiles. Image obtained after gel exposition and scanning The stretching force acting on DNA fragments is thought to originate from highly inhomogeneous flow near the collapsing cavitation bubbles The impulsive shearing force leads to cleavage of the phosphodiester linkage of DNA molecule PAGE A photograph and scheme of sonication stage Normalization Results We analyzed the cleavage patterns for 48 different DNA restriction fragments with lengths from 150 to 500 base pairs from λ – phage DNA and plasmids pBR322, pUC18, pGEM7(f+) (Promega), and their modified analogs which contained different insertions into polylinkers. The results of statistical analysis have shown that: 1) The cleavage rate just after deoxycitidine is considerably higher than in other positions and diminish in the order of dСрdG > dСрdА > dСрdТ >> dCpdC (see Fig.1 ). 2) The cleavage rates of the complementary dinucleotides are different (see Fig.1). 3) For the every dinucleotide the cleavage rate is dependent on the context. The extent of this dependence is modulated by the dinucleotide type (see Fig.2-5). Fig.1 Ultrasonic cleavage rates in 16 possible dinucleotides Figs. 2-5. Ultrasonic cleavage rates of the central phosphodiester bond in 256 possible tetranucleotides The increase of ultrasonic cleavage rate in phoshodiester linkage following deoxycytidine correlates with reported data on enhanced conformational flexibility of cytosine deoxyribose moiety obtained using combined NMR and MD simulation study [4]. Fig.6 Sugar phosphate backbone torsions The comparison of obtained sequence-dependent ultrasonic cleavage rates with various data covering reported sequence effects on conformational dynamics revealed that the conformational flexibility of deoxyribose is likely the dominant factor affecting ultrasonic cleavage rate of DNA. Sugar ring S↔N interconversion is coupled with trans↔gauche+ variations of δ (see Fig.6) , influencing the geometry of the C3’-O3’ bond involved in the ultrasonic cleavage of DNA. The enhancement of cleavage might be the result of such alteration: in North conformation of deoxyribose this bond tends to align with local helical axis of DNA fragment. When external stretching force acts on the molecule, the decrease of angle between helical axis and particular C3’-O3’ bond leads to increase in stretching component of its vibrational energy which in turn results in lowering the activation energy of hydrolysis reaction. Conclusions The analysis of sequence effects on ultrasonic cleavage rates of dsDNA revealed the unique properties of phosphodiester linkage following deoxycytidine. These results along with reported data on sequence-dependent structural dynamics of DNA allowed us to create the basis of interpretational framework for observed sequence-dependent ultrasonic cleavage of dsDNA in solution. References 1 S.L. Grokhovsky, Molecular Biology (Russia, translated)40, 276-283 (2006). 2 N. J. Pritchard, D. E. Hughes, A. R. Peacocke, Biopolymers,4, 259–273 (1965). 3 R. Das, A. Laederach, S. M. Pearlman, D. Herschlag and R.B.Altman. RNA. 11, 344-354(2005). 4E. Duchardt, L. Nilsson and J. Schleucher, Nucleic Acids Research, 36, 4211-4219 (2008)