Abstract

ULTRASONIC CLEAVAGE ANALYSIS: A NOVEL APPROACH TO STUDY THE

SEQUENCE EFFECTS ON DNA STRUCTURAL DYNAMICS. ULTRASONIC CLEAVAGE ANALYSIS: A NOVEL APPROACH TO STUDY THE

SEQUENCE EFFECTS ON DNA STRUCTURAL DYNAMICS.

AbstractRecently 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.

IntroductionUltrasonic 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.

The impulsive shearing force

leads to cleavage of the phosphodiester linkage of DNA

molecule

MethodsThe 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.

A photograph and scheme of sonication stage

Image obtained after gel

exposition and scanning

Results

ConclusionsThe 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.

ConclusionsThe 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.

References1 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). 4 E. Duchardt, L. Nilsson and J. Schleucher, Nucleic Acids Research, 36, 4211-4219 (2008)

References1 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). 4 E. Duchardt, L. Nilsson and J. Schleucher, Nucleic Acids Research, 36, 4211-4219 (2008)

Figs. 2-5. Ultrasonic cleavage rates of the central phosphodiester bond in 256 possible tetranucleotides

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, Russia3) Dep. of Biology, Moscow State University, Moscow 119992, Russia

4) Institute of Theoretical and Experimental Biophysics, Puschino142290, Russia

Fig.1 Ultrasonic cleavage rates in 16 possible dinucleotides

Normalization

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.

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.6 Sugar phosphate backbone torsions

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].

The stretching force acting on DNA

fragments is thought to originate from

highly inhomogeneous flow

near the collapsing cavitation bubbles

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