Special course in Computer Science: Molecular Computing

Transcription

1 Special course in Computer Science: Molecular Computing Lecture 9: Engineering Natural Computation by Autonomous DNA-Based Biomolecular Devices Vladimir Rogojin Department of IT, Åbo Akademi Fall 2013

2 DNA-based biomolecular devices Basic features: Autonomous Programmable Executing steps with no exterior mediation after start Tasks can be modified without major redesigning the nanostructure Functions: Computation, 2D patterning, amplified sensing, molecular/nanoscale transport

3 Nanoscience Core challenge: Construction of molecular-scale structures and devices Key deficiency: The need for robust, error-free methods for self-assembly of complex devices out of large numbers of molecular components

4 Botton-Up self-assembly Microelectronics industry is reaching the physical limit of miniaturization by top-down lithographic fabrication techniques New botton-up methods are needed for self-assembling complex, aperiodic structures for nanofabrication of molecular electronic circuits That are significantly smaller than conventional electronics

5 DNA for Assembly of Biomolecular Devices Why use DNA for assembly of biomolecular devices? DNA-based nanostructures have some unique advantages: They are relatively easy to design Predictable in their geometric structures Have been experimentaly implemented in a number of labs A key principle: Use of DNA self-assembly processes to actuate the molecular assembly.

6 Conventional vs self-assembly electronics fabrication Conventional electronics fabrication is reaching the limit of minimization possible by top-down techniques Self-assembly operates naturally at the molecular scale, It does not suffer from the limitation in scale reduction

7 Computations at molecular scale Computational patterns are essentially woven into molecular fabric (DNA lattices) via carefully controlled and designed self-assembly processes

8 Functions of molecular-scale autonomous devices Executing molecular-scale computations Use as scaffolds or templates for the further assembly of other materials Example: hybrid molecular electronic architectures Example: high-efficiency solar cells Robotic movement and molecular transport For instance, programmable versions of cellular transport mechanisms Highly sensitive molecular detection and amplification of single molecular events Transduction of molecular sensing for drug delivery

9 Molecular computation Distributed parallel molecular computations: A computational step is implemented by multiple distinct interacting molecules. Example: the tiling assembly computations Local molecular computations: A computational step is implemented within a single molecule. Multiple computing devices can run in parallel Example: Whiplash polymerase chain reaction

10 Autonomous molecular computation Adleman's and others follow-ups required many tedious laboratory steps to execute Adleman's experiment had a completely autonomous step: Self-assembly of paths in the di-graph Autonomous: Does not require any supervision while running

11 DNA nanostructures DNA nanostructure: A multimolecular complex consisting of a number of ssdna that have partially hybridized along their subsegments The field pioneered by Seeman Typical motifs: A stem-loop ssdna loops back to hybridize on itself Used to form patterns on DNA nanostructures A sticky end Unhybridized ssdna protrudes from the end of a double helix Used to combine two DNA nanostructures together via hybridization of their complementary ssdna

12 DNA nanostructure motifs, example DNA setm-loop DNA sticky end

13 Holliday junction A mobile junction between four strands of NDA By Robin Holliday, 1964 Account for a particular type of exchange of genetic information known as homologous recombination Used to tie together various parts of DNA nanostructure

14 2D tiling 2D tiling by Wang in 1961 Design 2D entities (tiles) equipped with pads on their sides that could selectively attach to each other so that desired 2D patterns could be built from those tiles Berger, 1966 Universal computation could be done via tiling assemblies

15 DNA tiles Winfree et al., 1998: Proposed the concept of computational tiling assemblies for assembling DNA molecular constructs Core idea: use tiles composed of DNA to perform computations during their self-assembly process A DNA tile: A DNA nanostructure that has a number of sticky ends on its sides A sticky end pads, used to hybridize with the other DNA tiles

16 DNA lattices DNA lattice: DNA nanostructure composed of a group of DNA tiles That are assembled together via hybridization of their pads Strands composing DNA tiles are designed to have a melting temperature above the melting temperature of the pads When the solution is cooling down: First DNA hybridize to form tiles, then Then tiles hybridize to each other

17 Types of DNA tiles DX tile TX tile Cross tile

18 Types of DNA tiles DX tiles (Seeman and Winfree, 1998): Two parallel DNA helices linked by immobile Holliday junctions. This tiles formed large 2D lattices Pads on two sides Rectangular shape TX tiles Three DNA helices Have topological properties allowing for strands to propagate in useful ways through tile latticies Pads on two sides Rectangular shape

19 Types of DNA tiles Cross tiles (Yan et al.): Roughly square Have pads on all four sides Allows binding with neighbors in all four directions

20 Programming tiling assembly Programming tiling assembly: Design pads of tiles in a smart way, so that tiles assemble together as intended (positive design problem) Proper design ensure that only the adjacent pads of neighboring tiles are complementary, so only those pads hybridize together (negative design problem, avoid unintended hybridization)

21 Software for the Design of DNA Tiles A number of prototype computer software systems have been developed for the design of the DNA sequences composing DNA tiles, And for optimizing their stability Examples: TileSoft (Yin et al., 2004), developed jointly by Duke and Caltech Provides a graphically interfaced sequence optimization system for designing DNA secondary structures NanoEngineer (by Nanorex, Inc.) Enhanced capabilities for DNA design

22 TileSoft: sequence optimization software for designing DNA secondary structures.

23 Autonomous Computations Using Self-assembly of DNA Nanostructures Two-layer linear assembly of TX tiles executing bit-wise cumulative XOR computation Cumulative XOR computation: Input: n bit binary vector Output: n bit binary vector The i-th bit ecuals XOR of the first i input bits Example: Input: Output:

24 Implementing cumulative XOR by DNA self-assembly How to provide data input to a molecular computation using DNA tiles? Input sequence of n bits implemented as a specific series of input tiles With the input bits 0's and 1's encoded by distinct short subsequences Two different tile types were assembled according to specific sticky-end associations

25 Implementing cumulative XOR by DNA self-assembly

26 Implementing cumulative XOR by DNA self-assembly Blue input tiles Through paddings encode 0/1 bit value Green corner starting tiles Red computation/output tiles

27 Implementing cumulative XOR by DNA self-assembly Red computation/output tiles Through paddings encode 4 input conditions for each i'th bit: Result of i-1'th XOR (0/1) and i'th input bit (0/1) And two input depended possible outputs: 0 XOR 0 = 1 XOR 1 = 0 0 XOR 1 = 1 XOR 0 = 1

28 Implementing cumulative XOR by DNA self-assembly Computing cumulative XOR: Input: 1110 Output:

29 Implementing cumulative XOR by DNA self-assembly Computing cumulative XOR: Input: 1010 Output:

30 Implementing cumulative XOR by DNA self-assembly, reading results Using endonuclease to recognize sites corresponding to 0/1 at the tile assembly Another way: Use AFM observable patterning Designing the tiles computing bit 1 to have stem loop protruding from the top of the tile AFM: Atomic force microscopy A very high-resolution type of scanning probe microscopy Resolution: order of fractions of a nanometer

31 Finite-State Computations via Disassembly of DNA Nanostructures Reverse to the assembly-based computation techniques Input: A linear double-stranded DNA nanostructure Computation: Digesting part of the nanostructure each step At each step: a sticky end at one end of the nanostructure encodes the current state The finite transition is determined by hybridization of the current sticky end with a small rule nanostructure Which encodes the finite-state transition rule

32 Finite-State Computations via Disassembly of DNA Nanostructures Digesting part of the nanostructure each step At each step: a sticky end at one end of the nanostructure encodes the current state The finite transition is determined by hybridization of the current sticky end with a small rule nanostructure Which encodes the finite-state transition rule A restriction enzyme recognizes the sequence encoding the current input as well as the current state, cuts the appended end of the linear DNA and exposes a new sticky end encoding the next state

33 Finite-State Computations via Disassembly of DNA Nanostructures

38 Finite-State Computations via Disassembly of DNA Nanostructures Hardware-software complex DsDNA with ssdna overhang and protein restriction enzyme Potential application (Adar et al., 2004): Medical diagnosis and therapeutics

39 Addressable 2D DNA Lattices Tiling computations to form patterned nanostructures to which other materials can selectively bound An addressable 2D DNA lattice has a number of sites with distinct ssdna This provides a superstructure for selectively attaching other molecules at addressable locations

40 Attaching molecules to DNA lattice There are many types of molecules to which one can attach DNA Each of these DNA-tagged molecules can then be assembled by hybridization of their DNA tags to a complementary sequence of ssdna Located within an addressable 2D DNA lattice Hereby, we can program the assembly of each DNA-tagged molecule onto a particular site of the addressable 2D DNA lattice

41 Attaching materials to DNA Materials that directly bind to specific DNA segments: DNA, RNA Proteins Peptides, Etc. Materials that bind indirectly: Some metals (e.g., gold) Carbon nanotubes This provides techniques for attaching heterogenous materials at molecular-scale level Example: it could be potentially used for attaching molecular electronic devices

42 Assembly of Patterned 2D DNA Lattices by Scaffold Strands A scaffold strand A long ssdna around which shorter ssdna assemble To form structures larger than individual tiles This method may be used to form an arbitrary 2D pixel image by designing the scaffold strand in a smart way Example: DNA origami: Scaffold ssdna has weak secondary structure and few long complementary segments Many Short stapple ssdna hybridize to the scaffold ssdna and keep the structure together in a designated shape

43 DNA origami

44 2D computational assembly by DNA tile's pads Any computable 2D pattern can be assembled by tiles Winfree and others: Pascal's (Sierpinski) Triangle: Each tile determines and outputs to neighborhood pads the XOR of two of the tile pads Self-assembled binary counter The resulting 2D counting lattice may have major applications to patterning molecular electronic circuits

45 2D computational assembly by DNA tile's pads Sierpinski triangle

46 2D computational assembly by DNA tile's pads Binary counter

47 Hierarchical assembly of 2D DNA Lattices Assemble DNA lattices in multiple stages Example: Tiles assembled prior mixing with other tiles Unique ssdna pads direct tiles to designed locations White pixels are turned on by binding avidin protein at programmed sites

48 The Need for Error Correction at the Molecular-Scale Significant level of errors in self-assembled molecular devices due to: Errors in synthesizing underlying DNA strands Errors in biomolecular operations of used to assemble and modify DNA nanostructures Biochemistry addresses the problem in terms of a number of purification and optimization procedures However, this is not enough: Need to design methods for decreasing errors of self-assembly and to enable self-repair of DNA tiling lattices

49 Winfree's Proofreading Scheme for Error-Resilient Tilings Replace each tile with a subarray of tiles That provide sufficient redundancy to quadratically reduce errors Drawbacks: increases the area of the assembly by a factor of 4 Quadratic reduction of errors

50 Reif's Compact Scheme for Error-Resilient Tilings More compact method: Replace original pads so that: Each tile executes the original computation at its location as well as The computation of a particular neighbor Quadratic reduction of errors without increasing the assembly size Error-correcting codes may also be used

51 Activatable Tiles for Reducing Errors Potentially major source of errors: Needed: The uncontrolled assembly of tilin assemblies in reverse directions Methods for controlled directional assembly of tiling assemblies Activatable tiles (by Majumder et al., 2007) Equip tiles with states (active/inactive) Implemented by a powerful DNA polymerase enzyme that allows tiles to transition between active (allow assembly) and inactive (forbid assembly) states

52 Three-Dimensional DNA Lattices So far we have considered 2D structures It seems much more challenging to assemble 3D DNA lattices of high regularity There are a number of very important applications to nanoelectronics and biology: Conventional lithographic techniques allow for very small number of layers. 3D nanoelectronic devices such as memory would provide much improvement in density 3D DNA lattice that assembled with sufficient regularity and regular interstices may help considerably in protein crystallography: A protein might be captured within each of the lattice interstices, allowing it to be in a fixed orientation

53 Nucleic Detection Protocols, Detection Problem A fundamental task of many biochemical protocols: Sense a particular molecule and amplify the response Detection of particular DNA/RNA very importat in medicine Ideally, the detection protocol should be sensitive enough to detect few copies of target molecules and amplify the respective signal

54 Autonomous Molecular Computation Derived from PCR The Polymerase Chain Reaction: Selectively amplifies DNA molecules Selection performed via primers oligonucleotides matching ends of the amplified molecules Whiplash PCR: A method for local molecular computation: ssdna encodes a program describing state transition rules of finite state computing machine Sequence of rule subsequences Separated by polymerase stopper sequences A rule encodes transition from a state to the other state

55 Whiplash PCR

56 Whiplash PCR

57 Isothermal Autonomous PCR Detection and Whiplash PCR Neither the original PCR nor Whiplash PCR execute autonomously Isothermal methods for PCR (by Walker et al., 1992) They require thermal cycling Via strand displacement amplification Isothermal reactivating Whiplash PCR (by Reif and Majumder, 2009) Makes use of a strand-displacing polymerization enzyme

58 Autonomous Molecular Cascades for DNA Detection Isothermal enzyme-free highly sensitive detection of a particular DNA strand (Dirks and Pierce, 2004) Triggered amplification by hybridization chain reaction Uses multiple copies of two distinct DNA nanostructures T and T' initially in a test tube When ssdna S added to the tube, S hybridize with T and releases S' part from T S' hybridizes with T' and releases S, The story repeats... The domino effect

59 Autonomous Molecular Cascades for DNA Detection

60 Hybridization Reactions for Autonomous DNA Computations Winfree, Zhang et al., 2007 A general powerful scheme for executing any Boolean circuit computation via cascades of DNA hybridization reactions The unique property: No use of any enzymes No temperature cycling Only hybridization

61 Molecular Transport Many molecular-scale tasks may require the transport of molecules A number of other tasks would be considerably aided by an ability to transport within nanostructures Example: Living cells using protein motors powered by ATP

62 Nonautonomous DNA Motor Devices DNA actuator powered by DNA hybridization (Yurke et al., 2000) Required temperature and/or reagent cycling Nonautonomous

63 Autonomous DNA Walkers Almost all man-made motors run without external mediation Almost all natural systems molecular motors run without external mediation It is essential to develop autonomous DNA devices that do not require external mediation while executing movements Reif in 2003 proposed two designs of autonomous DNA walkers that could traverse DNA nanostructures in both directions

64 Restriction Enzyme-Based Autonomous DNA Walkers First experimentally demonstrated autonomous DNA walker by Yin et al., 2004 Employed restriction enzymes and ligaze The road - a linear DNA nanostructure with a series of attached ssdna steps is self-assembled A fixed-length segment of DNA helix walker with short sticky ends feet are hybridized to the first two steps of the road The walker performs sequential movement: At the start of each step the feet are hybridized to two further consecutive steps The restriction enzyme cuts the DNA helix where the backward foot is attached, exposes a new sticky end foot, ready to hybridize with the next step. And so on...

65 DNAzyme-Based Autonomous DNA Walkers By Mao's group, 2005 The DNA walker from above with a DNAzyme replacing the restriction enzyme DNAzyme DNA nanostructure performing the function of an enzyme

66 Programmable Autonomous DNA Devices: Nanobots Important applications of the DNA walkers from above: Transport of molecules within large self-assembled DNA nanostructures. However, those are simple devices able to move only along predefined rail Potential applications may be vastly increase if those walkers could perform computations i.e., decide where to go basing on the current situation according to a given program

67 Challenges for Self-assembled DNA Nanostructures Complex, error-free DNA patterning to the scale of at least pixels Required for a functional molecular electronic circuit for a simple processor A programmable DNA nanobot autonomously executing a task critical to nano-assembly An application of self-assembled DNA nanostructures to medical diagnosis First implementations: Adar et al., 2004: finite-state computing DNA device for medical diagnosis detects certain RNA levels, computes diagnoses, provides and appropriate response ( nano-doctor ) Sahu and Reif, 2008: a DNAzyme autonomous DNA nanobot for medical diagnosis