Download Document

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
Methods
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 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.
the actual chemical reaction takes place
The stretching force
acting on DNA
fragments is thought
to originate from
highly
inhomogeneous flow
near the collapsing
cavitation bubbles
Image obtained
after gel
exposition and
scanning
The impulsive
shearing force
leads to
cleavage of the
phosphodiester
linkage of DNA
molecule
PAGE
A photograph and scheme of
sonication stage
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.
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.
Normalization
Results
Fig.1 Ultrasonic cleavage rates in 16 possible
dinucleotides
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
Figs. 2-5. Ultrasonic cleavage rates of the central phosphodiester bond in 256 possible tetranucleotides
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 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 sequencedependent 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
2
3
4
S.L. Grokhovsky, Molecular Biology (Russia, translated) 40, 276-283 (2006).
N. J. Pritchard, D. E. Hughes, A. R. Peacocke, Biopolymers, 4, 259–273 (1965).
R. Das, A. Laederach, S. M. Pearlman, D. Herschlag and R.B. Altman. RNA. 11, 344-354 (2005).
E. Duchardt, L. Nilsson and J. Schleucher, Nucleic Acids Research, 36, 4211-4219 (2008)