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Dresden University of Technology Department of Computer Science

An Object Oriented Simulation of Real Occurring Molecular Biological Processes for DNA Computing and its Experimental Verification

• T. Hinze, U. Hatnik, M. Sturm

Dresden DNA Computation Group email: dnacomp@tcs.inf.tu-dresden.de www: http://wwwtcs.inf.tu-dresden.de/dnacomp

• T. Hinze, U. Hatnik, M. Sturm

1/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

Contents

1. State of the Art in DNA Computing 2. Side Effects of DNA Operations 3. A Probabilistic Approach to DNA Computing 4. An Object Oriented Simulation Tool 5. Selected DNA Operations 6. A PCR Example 7. Conclusions

• T. Hinze, U. Hatnik, M. Sturm

2/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

State of the Art in DNA Computing

vision

• establish a universal biocomputer in theory and laboratory
• biocomputer based on a formal model should feature by

– computational completeness (universality), reliability – high operational speed using massive data parallelism – high storage capacity and density, persistence of stored data – DNA reusability, energy efficient processing without mech. wear challenges

• making DNA operations error resistent reducing side effects
• bridging the gap between formal models of DNA computing and

lab-reality

• ur approach
• T. Hinze, U. Hatnik, M. Sturm

3/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

Gap between Models and Lab-Reality

side effects of DNA operations

• not controllable, unreproducible, stochastically occurring effects
• f molecular biological processes used as DNA operations
• can sum up in sequences of DNA operations
• lead to unexpected, unprecise, unreproducible or even unusable

final results of experimental DNA computations frequently used abstractions of formal models of DNA computing

• nly linear DNA used as data carrier (words of formal languages)
• unrestricted approach; arbitrary (
• ) number of strand copies
• unique result strands detectable absolutely reliable
• all DNA operations performed completely and reproducible

idea to bridge the gap

• specification of DNA operations on molecular level
• include side effects specified by statistical parameters into the

description of DNA operations

probabilistic approach

• T. Hinze, U. Hatnik, M. Sturm

4/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

Side Effects of DNA Operations

(min. # copies) artifacts (diff. from structure) loss of linear DNA strands by forming hairpins, insertion deletion (% deletion rate, max. length of deletion) point mutation (% mutation rate) sequence) in DNA (differences mutations classification of side effects synthesis

• perations performed with

state of the art laboratory techniques bulges, loops, junctions, and compositions of them (% loss rate of tube contents)

• lin. DNA

(differences from incomplete reaction (% unprocessed strands) failures in reaction procedure unspecificity (% error rate, maximum difference) supercoils strand instabilities caused by temperature or pH impurities by rests of reagences loss of DNA strands (% loss rate of tube contents) perfect specification

• f reaction)

undetectable low DNA concentration in brackets: statistical parameters : supported in simulation tool : significant side effect caused by the operation annealing melting union ligation digestion labeling polymerisation PCR gel electrophoresis affinity purification

• T. Hinze, U. Hatnik, M. Sturm

5/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

A Probabilistic Approach to DNA Computing

properties

• multiset based, nondeterministic, restricted model
• description of DNA operations on level of single nucleotides and

strand end labels

• peration param., side effect param.
• recently supported: synthesis, annealing, melting, union, ligation,

digestion, labeling, polymerisation, PCR, affinity purification, gel electrophoresis; formal description by prog. language

• peration control
• iteration of molecular events, probability-controlled
• probabilities of molecular events depend on: DNA pool, number
• f strand copies, operation parameters, side effect parameters
• iteration terminates iff empty list (matrix) of possible mol. events
• T. Hinze, U. Hatnik, M. Sturm

6/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

A Probabilistic Approach to DNA Computing

iteration exemplified by annealing (simplified)

C G G A

6 strands

T A A G C T G C C A T C G G A

6 strands

A A G C T A C G C G G C A T T A G C G C A T G C C G A G T T A A A T G C C G T A C G G A T A G C G C A T G C C G A C G G T loop T A A A G C T C C G A T G G A G C T T G A A G C T A C C A T C G G A A A G C T A C G C G G C A T T A G C G C A T G C C G A G T T A A A G C T C C G A T G G A G C T T G A A G C T C C A T C G G A A A G C T A C G C G G C A T T A G C G C A T G C C G A G T T A G G T A G C C T C G A A

10 strands

T C G A G G T A G C C T C G A A T A C C T C G A A G T

10 strands

T C G

possible complete molecular reactions: probability for collision: possible complete molecular reactions:

* 10

2

p 6 = * 6

2

p 10 =

no new product possible complete molecular reactions: probability for collision:

* 6

2

p 6 =

modified DNA pool 9 x 5 x 1 x possible complete molecular reactions: probability for collision:

* 10

2

p 10 =

0.15 0.23 0.39 0.23 number of DNA strands: p = 10 + 6 = 16 minimum nucleotide bonding rate for stable hybridized DNA double strands: 50% molecular event: strand hybridization DNA pool 10 x 6 x

Select one molecular event randomly with respect to the probability distribution Determine all possible reaction products from this molecular event and select one of them Modify DNA pool 1. 2. 3. 4. Create list (matrix) of molecular events and their probabilities including side effects 4. 3. 2. 1.

probability for collision:

• T. Hinze, U. Hatnik, M. Sturm

7/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

An Object Oriented Simulation Tool

main features

• specification of DNA operations on the level of single nucleotides

and strand end labels using probabilistic approach

• number of strand copies considered

concentrations of different DNA strands and their influence to the behaviour in op. process

• each DNA operation processed inside a virtual test tube collecting

a multiset of DNA strands, several test tubes supported

• each DNA operation characterized by a set of specific

parameters and side effect parameters

• arbitrary sequences of DNA operations including propagation of

side effects can be visualized and logged

• Java, simulation tool requires at least Java Development Kit 2.0

algorithms for molecular interactions algorithms for sets of DNA strands (DNA strand, reactants) algorithms for process control sequences tubes frame sequences sequences tubes sequences model interface

• bject
• ther objects

simulation algorithm a) b)

• T. Hinze, U. Hatnik, M. Sturm

8/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

Selected DNA Operations – Synthesis

• peration parameters:

tube name: nucleotide sequence (5’-3’): number of strand copies:

side effect parameters:

point mutation rate: deletion rate maximum deletion length: tube1 AGGCACTGAGGTGATTGGC 8 000 0.06% 0.06% 11% of strand length

• utput of

test tube contents

• T. Hinze, U. Hatnik, M. Sturm

9/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

Selected DNA Operations – Digestion

contents test tube

• utput of

tube2 5% 5% wildcarded recognition sequence: rate of star activity (unspecificity): rate of not executed molecular cuts:

side effect parameters:

and restriction site: recognition sequence tube name:

• peration parameters:

5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ T A C C G G C G G C N N N N G G C A T C C C G G

• T. Hinze, U. Hatnik, M. Sturm

10/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

A PCR Example (I)

correct synthesized strands incorrect synthesized strands carrying point mutations and deletions 941 copies of template2) 59 strands of template2) 91 strands of primer1, (totally 90 strands of primer2, 942 copies of template1,

5

7909 copies of primer1,

5 6 7 941

(7910 copies of primer2,

942

58 strands of template1,

7910 7909

1000 copies template1 Synthesis Synthesis Synthesis 1000 copies template2 Synthesis Union Union Union 8000 copies primer1 8000 copies primer2

• T. Hinze, U. Hatnik, M. Sturm

11/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

A PCR Example (II)

3rd PCR cycle 1st PCR cycle 2nd PCR cycle lane 1: PCR negative control lanes 2, 3, 4: PCR product (simulation screenshot) lane 5: 50bp marker 100 50 4 3 2 1 short double stranded PCR fragments) strands with deletions resulting in too unwanted strands (including amplified primer rests template (5965 copies) correct amplified double stranded 5

1 1 2 2 6 2713 2739 5965

• min. bonding rate: 50%
• max. length: 100bp

Melting Polymerisation Annealing

• min. bonding rate: 50%

Melting Polymerisation

• max. length: 100bp

Annealing

• min. bonding rate: 50%
• max. length: 100bp

Annealing Polymerisation Melting

• T. Hinze, U. Hatnik, M. Sturm

12/13 Simulation of Molecular Biological Processes

Dresden University of Technology Department of Computer Science

Conclusions

results

• first formal model of DNA computing considering side effects of

DNA operations using a probabilistic, restricted, and multiset based approach

• contribution to bridge the gap between experimental and

theoretical DNA computing

• supports experimental setup of DNA algorithms as well as

implementations of models for DNA computation

• prediction of experimental results and cost effective optimization
• f error reducing and error compensating operation sequences
• bject oriented simulation tool based on this approach enables a

flexible, interoperable, and ergonomic model handling further work

• extension to additional side effects concerning nonlinear DNA
• application to the implementation of distributed splicing systems
• T. Hinze, U. Hatnik, M. Sturm

13/13 Simulation of Molecular Biological Processes