Challenging Traditional Approaches to Computation A Biomolecular Transducer Employing Ternary Language and Rendering a Biological Output Mark Chaskes and

Challenging Traditional Challenging Traditional Approaches to Approaches to ComputationComputation

A Biomolecular Transducer A Biomolecular Transducer Employing Ternary Language Employing Ternary Language and Rendering a Biological and Rendering a Biological OutputOutput

Mark Chaskes and Paul LazarescuMark Chaskes and Paul Lazarescu

Mentor: Tamar RatnerMentor: Tamar Ratner

The Schulich Faculty of ChemistryThe Schulich Faculty of Chemistry

Technion, Haifa, Israel, 32000Technion, Haifa, Israel, 32000

ObjectiveObjective

Design a theoretical biomolecular Design a theoretical biomolecular transducer to solve consecutive transducer to solve consecutive mathematical equations in ternary.mathematical equations in ternary.

-First divide an input by three and -First divide an input by three and then divide the yeild of that by two then divide the yeild of that by two

What is biomolecular What is biomolecular computing?computing?

A biomolecular A biomolecular computer is a group computer is a group of molecules that of molecules that ‘read’ dsDNA and ‘read’ dsDNA and can ‘print’ an output.can ‘print’ an output.

What is a DNA based What is a DNA based transducertransducer??

A transducer is not a PC; it has unique capabilities that an ordinary computer

does not.

Advantages include:

•Direct interface with a biological system

•Can release a biological output

•Able to compute in parallel

•Store large amounts of data in a small volume

Design on the Design on the Molecular LevelMolecular Level

Symbols are dsDNA strands

Restriction enzymes cleave the sequence at recognition sites

States are determined by the location of cleavage within the symbol

ProcessProcess

Reading 2 from S0 prints 0 and goes to S2

Divide by three transducer reading the input 2-0-0

State 0 2 0 0

ProcessProcess



0 State 2 0 0

ProcessProcess


Reading 0 from S0 prints 0 and encodes the output 0-2-0

0 2 State 0 0

ProcessProcess

S0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

Start

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

StartS0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

Start

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S1


Divide by two transducer reading the input 0-2-0

State 0 0 2 0

ProcessProcess

S0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

Start

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

StartS0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

Start

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S1



0 State 0 2 0

ProcessProcess

S0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

Start

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

StartS0

r 1 p 0

r 1 p 2 r 2 p 2

r 0 p 1

r 0 p 0

r 2 p 1

A.

Start

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S0 r0p0 S0

S0r2p1 S0

S0r1p0 S1

S1r1p2 S0

S1r0p1 S1

S1r2p2 S1

B.

S1



0 1 State 0 0

AATTCGGCCGTT..8 base..CTCCTCGCAGC..8 base..CTCGTTAGTCTTAGTCTTTGCTGAAATTTTAAGCCGGCAA..pairs ..GAGGAGCGTCG..pairs ..GAGCAATCAGAATCAGAAACGACTTTAA

BseRI Recognition

Site

EagI Recognition Site

Spacers

BbvI Recognition Site

2 0 0

Plasmid

Terminator

Molecular Design of the InputEncoding 2-0-0 in Ternary (18 in base ten)

AATTCGGCCGTT..8 base..CTCCTCGCAGC..8 base..CTCGTTAGTCTTAGTCTTTGCTGAAATTTTAAGCCGGCAA..pairs ..GAGGAGCGTCG..pairs ..GAGCAATCAGAATCAGAAACGACTTTAA

AGTCTT...8 base...CTCCTCGCAGC...2 baseAATCAGAA...pairs ...GAGGAGCGTCG...pairs...TCAG

S0 to S0, read 0, print 0

AGTCTT...8 base...CTCCTCGCAGC...1 baseAATCAGAA...pairs ...GAGGAGCGTCG...pairs...CCAT


AGTCTT...8 base...CTCCTCGCAGCAATCAGAA...pairs ...GAGGAGCGTCGGAGC


GGTATT...8 base...CTCCTCGCAGC...3 baseAACCATAA...pairs ...GAGGAGCGTCG...pairs...CAGA


GGTATT...8 base...CTCCTCGCAGC...2 baseAACCATAA...pairs ...GAGGAGCGTCG...pairs...CATA


GGTATT...8 base...CTCCTCGCAGC...1 baseAACCATAA...pairs ...GAGGAGCGTCG...pairs...AGCA


CTCGTT...8 base...CTCCTCGCAGC...4 baseAAGAGCAA...pairs ...GAGGAGCGTCG...pairs...AGAA


CTCGTT...8 base...CTCCTCGCAGC...3 baseAAGAGCAA...pairs ...GAGGAGCGTCG...pairs...ATAA


CTCGTT...8 base...CTCCTCGCAGC...2 baseAAGAGCAA...pairs ...GAGGAGCGTCG...pairs...GCAA


Divide-by-three ComputationFirst Restriction

AATTCGGCCGTT CTCGTTAGTCTTAGTCTTTGCTGAAATTTTAAGCCGGC AATCAGAATCAGAAACGACTTTAA



















Divide-by-three ComputationFirst Restriction

AATTCGGCCGTT CTCGTTAGTCTTAGTCTTTGCTGAAATTTTAAGCCGGC AATCAGAATCAGAAACGACTTTAA


Transition Molecule S0 to S2, reading 2, printing 0

DNA Ligase



















Divide-by-three ComputationFirst Ligation

AATTCGGCCGTTAGTCTT...8 base...CTCCTCGCAGCCTCGTTAGTCTTAGTCTTTGCTGAAATTTTAAGCCGGCAATCAGAA...pairs ...GAGGAGCGTCGGAGCAATCAGAATCAGAAACGACTTTAA



















Divide-by-three ComputationFirst Ligation



Continue cycle of restriction, hybridization,

and ligation until terminator is cleaved


AATTCGGCCGTTAGTCTTCTCGTTAGTCTT TGCTGAAATTTTAAGCCGGCAATCAGAAGAGCAATCAG CTTTAA

Divide-by-three ComputationFinal Restriction

AATTCGGCCGTTAGTCTTCTCGTTAGTCTT TGCTGAAATTTTAAGCCGGCAATCAGAAGAGCAATCAG CTTTAA

TGCTGA...Reporter...AAACGACT....Gene 0....ACGA

Detection Molecule

Divide-by-three ComputationFinal Ligation

AATTCGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATTTTAAGCCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA

0 2 0

This transducer has printed 020, which is 6 in base ten (610) .

Check: 18/3 = 6? Yes.

Divide-by-three ComputationFinal Ligation


Biological Function 0

Biological Function 0 could be releasing a drug, changing the bacteria phenotype, etc .


Divide-by-two ComputationTransition Stage

A third restriction enzyme that cleaves within its recognition site is necessary only when consecutive computation (using two

separate transducers) occurs.

AATTC GGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATTTTAAGCCGG CAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA

GGCCTTTCTCCTCGCAGCT AAAGAGGAGCGTCGACCGG

Reinsertion Molecule


Reinsertion of the recognition sites is also required for consecutive computation.

AATTCGGCCTTTCTCCTCGCAGCTGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATTTTAAGCCGGAAAGAGGAGCGTCGACCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA


AATTCGGCCTTTCTCCTCGCAGCTGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATTTTAAGCCGGAAAGAGGAGCGTCGACCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA

Entire cycle repeats again until terminator is cleaved once more

Divide-by-two Computation

AATTAGTCTTGGTATTAGTCTT TGCTGA...Reporter...TGCTGAAATTTTAATCAGAACCATAATCAG CT....Gene 0....ACGACTTTAA

Divide-by-two ComputationFinal Restriction

AATTAGTCTTGGTATTAGTCTT TGCTGA...Reporter...TGCTGAAATTTTAATCAGAACCATAATCAG CT....Gene 0....ACGACTTTAA

TGCTGA...Reporter...AAACGACT....Gene 0....ACGA

Detection Molecule

Divide-by-two ComputationFinal Ligation

AATTAGTCTTGGTATTAGTCTTTGCTGA...Reporter...TGCTGA...Reporter...TGCTGAAATTTTAATCAGAACCATAATCAGAAACGACT....Gene 0....ACGACT....Gene 0....ACGACTTTAA

Divide-by-two ComputationFinal Ligation

0 1 0) 310(

This transducer has printed 010, which is 3 in base ten.

Check: (18/3)/2 = 3? Yes.

AATTAGTCTTGGTATTAGTCTTTGCTGA...Reporter...TGCTGA...Reporter...TGCTGAAATTTTAATCAGAACCATAATCAGAAACGACT....Gene 0....ACGACT....Gene 0....ACGACTTTAA

Biological Function 0

Discussion & Discussion & ConclusionsConclusions

This project worked as expected. This project worked as expected.

18 18 ÷ 3= ÷ 3= 66 ; 6 ; 6 ÷ 2= ÷ 2= 33

No molecule encoded the recognition site of an enzymeNo molecule encoded the recognition site of an enzyme

Proof of concept worked however not done in practicality.Proof of concept worked however not done in practicality.

Transducers engineered functioned as coded Transducers engineered functioned as coded

AcknowledgementsAcknowledgements

We would like to sincerely thank Mr. We would like to sincerely thank Mr. Russell N. Stern for his generosity and Russell N. Stern for his generosity and donation.donation.

Thank you to the Louis Herman Israel Thank you to the Louis Herman Israel Experience Fund for their contribution.Experience Fund for their contribution.

We would also like to thank our mentor We would also like to thank our mentor Tamar Ratner, for her continued Tamar Ratner, for her continued dedication and help.dedication and help.

Finally, we would like to thank Finally, we would like to thank Professor Ehud Keinan for allowing us Professor Ehud Keinan for allowing us to use his laboratory and his student.to use his laboratory and his student.

Documents

Challenging Traditional Approaches to Computation A Biomolecular Transducer Employing Ternary Language and Rendering a Biological Output Mark Chaskes and