The Design of Autonomous DNAThe Design of Autonomous DNANanomechanical Devices: Nanomechanical Devices:

Walking and RollingWalking and Rolling

John H. ReifJohn H. ReifDuke UniversityDuke University

Prior Nanomechanical Devices built oPrior Nanomechanical Devices built of DNAf DNA

SeemanSeeman used rotational transitions of ds DNA conformations between theused rotational transitions of ds DNA conformations between the B-form (right handed) to the Z-form (left-handed) controlled by ionicB-form (right handed) to the Z-form (left-handed) controlled by ionic effector molecules andeffector molecules and

Yurke and TurberfieldYurke and Turberfield used a fuel DNA strands acting as a hybridization catalyst toused a fuel DNA strands acting as a hybridization catalyst to generate a sequence of motions in another tweezers strand of DNAgenerate a sequence of motions in another tweezers strand of DNA extended this technique to be DNA sequence dependantextended this technique to be DNA sequence dependant the two strands of DNA bind and unbind with the overhangs tothe two strands of DNA bind and unbind with the overhangs to alternately open and shut the tweezersalternately open and shut the tweezers..

Other Related Work:Other Related Work: Shapiro’s recent autonomous 2 state DNA computing machineShapiro’s recent autonomous 2 state DNA computing machine uses DNA ligase and two restriction enzymeuses DNA ligase and two restriction enzyme

Bernard Yurke’s Molecular Tweezers (Bell Lab):Bernard Yurke’s Molecular Tweezers (Bell Lab): ComComposed of DNA and powered by DNA hybridizationposed of DNA and powered by DNA hybridization..

Two ds DNA arms are connected by a ssDNA hinge Two ds DNA arms are connected by a ssDNA hinge Two ssDNA “handles ” at the ends of the arms. Two ssDNA “handles ” at the ends of the arms. To close tweezers: To close tweezers: Add a special “fuel ” strand of ssDNA..Add a special “fuel ” strand of ssDNA.. The “fuel ” strand attaches to the handles and draws the two strand The “fuel ” strand attaches to the handles and draws the two strand arms together .arms together .

B-Z DNA Nanomechanical DeviceB-Z DNA Nanomechanical Device[Seeman, 1999][Seeman, 1999]

DNA Nanomechanical Device (Hao, Duke)DNA Nanomechanical Device (Hao, Duke)

Walking Triangles: Walking Triangles: By binding the short red strand (top figure) versus the long redBy binding the short red strand (top figure) versus the long redstrand (bottom figure) the orientation of and distance between the triangular tilestrand (bottom figure) the orientation of and distance between the triangular tiles is altered.s is altered.

Applications: Applications: Programmable state control for nanomechanical devicesProgrammable state control for nanomechanical devices.

Key restrictions on the use of prior DNA nanoKey restrictions on the use of prior DNA nanomechanical devices:mechanical devices:

Minor Restriction:Minor Restriction: They can only execute one type of motionThey can only execute one type of motion (rotational or translational).(rotational or translational).

Major Restriction:Major Restriction: Prior DNA devices require environmental changesPrior DNA devices require environmental changes such as temperature cycling or bead treatment of such as temperature cycling or bead treatment of biotin-streptavidin beads to make repeated motions.biotin-streptavidin beads to make repeated motions.

Our Technical Challenge:Our Technical Challenge: To make an autonomous DNA nanomechanical deviceTo make an autonomous DNA nanomechanical device that executes cycles of motionthat executes cycles of motion (either rotational or translational or both)(either rotational or translational or both) without external environmental changes.without external environmental changes.

Designs for the first autonomous DNADesigns for the first autonomous DNAnanomechanical devices that execute cycles of motionanomechanical devices that execute cycles of motion without external environmental changes.n without external environmental changes.

Walking DNA deviceWalking DNA deviceUses ATP consumption by DNA ligase in conjunctionUses ATP consumption by DNA ligase in conjunctionwith restriction enzyme operations.with restriction enzyme operations.

Rolling DNA deviceRolling DNA deviceUses hybridization energyUses hybridization energyGenerate random bidirectional movements that acquire after n Generate random bidirectional movements that acquire after n steps an expected translational deviation of O(n1/2).steps an expected translational deviation of O(n1/2).

Energy sources that can fuel Energy sources that can fuel DNA movements:DNA movements:

(i) ATP consumption by DNA ligase in(i) ATP consumption by DNA ligase inconjunction with restriction enzymeconjunction with restriction enzymeoperationsoperations

(ii) DNA hybridization energy in trapped stat(ii) DNA hybridization energy in trapped stateses

(iii) kinetic (heat) energy(iii) kinetic (heat) energy

Walking DNA Autonomous NanomechWalking DNA Autonomous Nanomechanical Device:anical Device:

Energetic: Uses ATP consumption by DNA ligase inEnergetic: Uses ATP consumption by DNA ligase in conjunction withconjunction with

restriction enzyme operations : Achieves randomrestriction enzyme operations : Achieves random bidirectional translational and rotational motionbidirectional translational and rotational motion around a circular ssDNA strand.around a circular ssDNA strand.

Walking DNA Device Walking DNA Device ConstructionConstruction

The Road

A circular repeating strand R of ssDNA written in 5’ to 3’ direction frA circular repeating strand R of ssDNA written in 5’ to 3’ direction from left to right.om left to right.

consists of an even number n of subsequences, which we call stepconsists of an even number n of subsequences, which we call steppingstones, indexed from 0 to n-1 modulo n.pingstones, indexed from 0 to n-1 modulo n.

The iThe ithth steppingstone consists of a length L steppingstone consists of a length L (where L is between 15 to 20 base pairs) sequence (where L is between 15 to 20 base pairs) sequence AAii of ssDNA. the A of ssDNA. the Ai i repeat with a period of 2.repeat with a period of 2.

Walking DNA Device Walking DNA Device ConstructionConstruction

The iThe ithth Walker Walker

A unique a partial duplex DNA strand WA unique a partial duplex DNA strand W ii with 3’ ends i with 3’ ends i-1 -1 and i that arand i that are hybridized to consecutive ie hybridized to consecutive i -1-1

thth and i and ith th steppingstones Asteppingstones Ai-1i-1 and A and Aii

The Goal of the Device The Goal of the Device ConstructionConstruction

Bidirectional, translational movementBidirectional, translational movement both in the 5’ to 3’ direction (from left to right) andboth in the 5’ to 3’ direction (from left to right) and vise versa vise versa (in the 3’ to 5’ direction) on the road.(in the 3’ to 5’ direction) on the road.

The iThe ithth walker W walker Wii will reform to another partial duplex DNA strand will reform to another partial duplex DNA strand called the called the i+1i+1th walker Wth walker Wi+1i+1 which is which is shifted one unit over to the left shifted one unit over to the left or the right.or the right. Cycle back in 2 stages, so that WCycle back in 2 stages, so that W i+2i+2 = W = Wii for each stage i. for each stage i.

Use 2 distinct types of restriction enzymesUse 2 distinct types of restriction enzymes

Use DNA ligaseUse DNA ligase provides a source of energy (though ATP consumption) andprovides a source of energy (though ATP consumption) and a high degree of irreversibility.a high degree of irreversibility.

Simultaneous Translational and Rotational MovemSimultaneous Translational and Rotational Movementsents

Secondary structure of B-form dsDNA Rotates 2Secondary structure of B-form dsDNA Rotates 2∏ ∏ radians radians every approx 10.5 basesevery approx 10.5 bases So in each step of translational movement, the walker rotates 1/10.5 arSo in each step of translational movement, the walker rotates 1/10.5 ar

ound the axis of the road.ound the axis of the road.

Sequence designSequence design (i) use superscript R to denote the reverse of a sequence(i) use superscript R to denote the reverse of a sequence

(ii) use overbar to denote the complement of an ssDNA sequence.(ii) use overbar to denote the complement of an ssDNA sequence.

To ensure there is no interaction between a walkerTo ensure there is no interaction between a walker and more than one distinct road at a time:and more than one distinct road at a time: - - use a sufficiently low road concentration and solid support attachment use a sufficiently low road concentration and solid support attachment of the roads.of the roads.

To ensure there is no interaction between a road andTo ensure there is no interaction between a road and more than one walker:more than one walker: - - we use a sufficiently low walker concentration.we use a sufficiently low walker concentration.

Definition of the Walker WDefinition of the Walker W ii

walker Wwalker Wi i has:has:

the 3’ end the 3’ end i-1i-1 hybridized to steppingstone A hybridized to steppingstone A i-1i-1 on th on the road.e road.

the 3’ end i hybridized to steppingstone Athe 3’ end i hybridized to steppingstone A ii on the on the road.road.

Definition of the Stepper SDefinition of the Stepper S ii

Hybridization of the Walker Hybridization of the Walker to steppingstones of the to steppingstones of the

RoadRoad

Restriction Enzyme Cleavage of the Restriction Enzyme Cleavage of the WalkerWalker

Resulting Products of Resulting Products of CleavageCleavage

The Reformation of the WalkerThe Reformation of the Walker

Possible Movements of the Possible Movements of the WalkerWalker

Forward:Forward:

Stall:Stall:

The cleavage operation can be reversed by re-hybridizationThe cleavage operation can be reversed by re-hybridization

Reversal:Reversal:

The walker has two possible (dual) restriction The walker has two possible (dual) restriction enzymeenzymerecognition sites which can result in a reversal of recognition sites which can result in a reversal of movementmovement

Rolling DNA Autonomous NanomechaRolling DNA Autonomous Nanomechanical Devicenical Device

requires no temperature changesrequires no temperature changes makes no use of DNA ligase or any restriction enzymemakes no use of DNA ligase or any restriction enzyme it uses instead the hybridization energy of DNA in trapped statesit uses instead the hybridization energy of DNA in trapped states

Oglionucleotides used in the Rolling Oglionucleotides used in the Rolling DNA ConstructionDNA Construction

Let A0, A1, B0, B1 each be distinct oglionucleotides:Let A0, A1, B0, B1 each be distinct oglionucleotides: of low annealing cross-affinity,of low annealing cross-affinity, consisting of L (L can be between 15 to 20) bases pairs.consisting of L (L can be between 15 to 20) bases pairs.

Let a0, a1 be oglionucleotidesLet a0, a1 be oglionucleotides derived from A0, A1 by changing a small number of bases,derived from A0, A1 by changing a small number of bases, so their annealing affinity with 0so their annealing affinity with 0RR, 1, 1RR respectively is somewhat reduced, respectively is somewhat reduced, but still moderately high.but still moderately high.

Strong Hybridization:Strong Hybridization: Hybridization between A0 and reverse complementary sequence 0Hybridization between A0 and reverse complementary sequence 0RR (or between A1 and reverse complementary 1(or between A1 and reverse complementary 1RR))

Weak Hybridization:Weak Hybridization: Hybridization between a0 and 0R (or between a1 and 1Hybridization between a0 and 0R (or between a1 and 1RR))

Key Idea:Key Idea: A strong hybridization is able to displace a weak hybridization.A strong hybridization is able to displace a weak hybridization.

Rolling DNA DeviceRolling DNA Device

The Road: an ss DNAThe Road: an ss DNA

with a0, a1, a0, a1, a0, a1, … in direction from 5’ to 3’,with a0, a1, a0, a1, a0, a1, … in direction from 5’ to 3’,consisting of a large number of repetitions of the consisting of a large number of repetitions of the sequences a0, a1.sequences a0, a1.

The Wheel: a cyclic ss DNAThe Wheel: a cyclic ss DNA

of base length 4Lof base length 4L

with 0with 0RR, 1, 1RR, 0, 0RR, 1, 1RR in direction from 5’ to 3’ in direction from 5’ to 3’

this corresponds to 1, 0, 1, 0 in direction from 3’ to 5’.this corresponds to 1, 0, 1, 0 in direction from 3’ to 5’.

DNA Fuel Loop StrandsDNA Fuel Loop StrandsPrimary Fuel Primary Fuel StrandStrand

Loop Loop ConfigurationConfiguration

ComplementComplementary Fuel ary Fuel StrandStrand

Loop Loop ConfiguratiConfigurationon

The Sequence of Events of a The Sequence of Events of a Feasible Movement of the Feasible Movement of the

WheelWheel

(1)(1)Hybridizations of a 0Hybridizations of a 0th th primary fuel strand:primary fuel strand:

Initial Hybridization of the second segment AInitial Hybridization of the second segment A0 0 of the 0of the 0th th

primaryprimary fuel strand with the reverse complementary segment 0fuel strand with the reverse complementary segment 0RR of of

the wheel.the wheel. Extension of that initial hybridization to a hybridization of Extension of that initial hybridization to a hybridization of

two firsttwo first segments Asegments A11, A, A0 0 of the 0of the 0thth primary fuel strand with the primary fuel strand with the

consecutiveconsecutive reverse complementary segments 1reverse complementary segments 1RR 0 0RR of the wheel. of the wheel.

The Sequence of Events of a The Sequence of Events of a Feasible Movement of the Feasible Movement of the

WheelWheel

2) Hybridizations of a type 0 complementary fuel strand:Hybridizations of a type 0 complementary fuel strand:

Hybridization with reverse complementary subsequences Hybridization with reverse complementary subsequences of the type 0of the type 0primary fuel strand,primary fuel strand,first at that fuel strand’s newly exposed 3’ end segment Afirst at that fuel strand’s newly exposed 3’ end segment A11

RR

then at Bthen at B00..

Formation of a type 0 fuel strand duplex removes the type Formation of a type 0 fuel strand duplex removes the type 0 fuel0 fuelstrands from the wheel, completing the step.strands from the wheel, completing the step.

Potential ApplicationsPotential Applications

Array Automata: Array Automata:

The state information could be stored at each siteThe state information could be stored at each siteof a regular DNA lattices, and additional mechanisms for finite stateof a regular DNA lattices, and additional mechanisms for finite statetransiting would provide for the capability of a cellular arraytransiting would provide for the capability of a cellular arrayautomata.automata.

Nanofabrication: Nanofabrication:

These capabilities might be used to selectivelyThese capabilities might be used to selectivelycontrol nanofabrication stages. The size or shape of the lattice maycontrol nanofabrication stages. The size or shape of the lattice maybe programmed through the control of such sequence-dependentbe programmed through the control of such sequence-dependentdevices and this might be used to execute a series of foldings devices and this might be used to execute a series of foldings

DNA LatticesDNA Lattices

Key Application: Molecular robotic ComponentsKey Application: Molecular robotic Components

DNA tiles of size 14 x 7 nanometersDNA tiles of size 14 x 7 nanometersComposed of short DNA strands with HollidayComposed of short DNA strands with Holliday