1 / 76

Recent Research in DNA Computing

Recent Research in DNA Computing. John Reif Dept Computer Science Duke University. Reif’s DNA Self-Assembly Group Current PhD Graduate Students Visiting Scholars: Abeer Eshra Reem Mokhtar Shalin Shah Xin Song Dan Fu Ming Yang. Prior Recent PhD Graduate Students

fawzia
Download Presentation

Recent Research in DNA Computing

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Recent Research in DNA Computing John Reif Dept Computer Science Duke University Reif’s DNA Self-Assembly Group Current PhD Graduate Students Visiting Scholars: Abeer Eshra ReemMokhtar Shalin Shah Xin Song Dan Fu Ming Yang Prior Recent PhD Graduate Students • Hieu Bui Nikhil Gopalkrishnan Peng Yin • Tianqi SongHarish Chandran Harish Chandran • Urmi Majumder Sudhanshu Garg

  2. Organization of Talk • Localized DNA Computation: Faster and Simpler Molecular-Scale Computing • Programming DNA-based Biomolecular Reaction Networks on Cancer Cell Membranes: localized circuits, molecular reaction networks • Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates: logic circuits, enzyme-based circuits • DNA-based Analog Computing: analog circuits

  3. Localized DNA ComputationHieu Bui and John Reif • Linear Cascade DNA Hybridization Chain Reactions (in solution, not localized) • Localized DNA Hybridization Chain Reactions on DNA Tracks • Localized DNA Hybridization Chain Reactions on DNA Origami

  4. Localized DNA Computation[Hieu Bui and John Reif] • Hieu Bui, SudhanshuGarg, ReemMokhtar, Harish Chandran, Vincent Miao and John Reif, Design and Analysis of Localized DNA Hybridization Chain Reactions, Small (2017), 1602983. DOI: 10.1002/smll.201602983  • Hieu Bui, SudhanshuGarg, Vincent Miao, Tianqi Song, ReemMokhtar, and John Reif, Design and Analysis of Linear Cascade DNA Hybridization Chain Reactions Using DNA Hairpins, Special Issue, Journal of New Physics, Vol. 19, (2017) 015006. doi:10.1088/1367-2630/aa53d0 • Hieu Bui, Shalin Shah, ReemMokhtar, Tianqi Song, SudhanshuGarg, John Reif, Localized DNA Hybridization Chain Reactions on DNA Origami, ACS Nano, Volume 12, Num. 2, pp 1146–1155(January 22, 2018). DOI: 10.1021/acsnano.7b06699259. • Hieu Bui and John H Reif, Localized DNA Computation, Chapter 19 in book: “From Parallel to Emergent Computing” (Edited by Andrew Adamatzky, SelimAkl, and Georgios Ch. Sirakoulis), CRC Press (February 18, 2019). Taylor & Francis Group of CRC Press. ISBN: 9781138054011   • Sudhanshu Garg, Hieu Bui, Abeer Eshra, Shalin Shah and John H Reif, Nucleic Acid Hairpins: A Robust and Powerful Motif for Molecular Devices, Chapter 7 in book: “Soft Nanomaterials” (Edited by Ye Zhang), World Scientific, (2019).

  5. Motivations • DNA – programmable, predictable, and self-assembly. • DNA circuits (AND, OR, NOT, etc.) – versatile, inexpensive, capable of parallel computing. • The locality effect may speedup the reactions. • Faster switching time compared to regular DNA hybridization reactions. • Minimal leakages due to the physical constraints. • Reusable sequences (gates) - scalable B-DNA (10.5bp, 0.34nm/bp, 2nm diameter)

  6. Linear Cascade DNA Hybridization Chain Reactions (in solution, not localized) • Autonomous reaction of controlled self-assembly. • Hairpins 1–9 are stable in the absence of initiator I. • The initiator binds at the sticky end of H1 and undergoes an unbiased strand displacement interaction to open the hairpin. • The newly exposed sequestered sticky end of H1 binds to the external sticky end of H2 and opens the hairpin to expose a sequestered sticky end on H2. • This cascade reaction continues until H9 is opened. • The designed product is a linear chain formed by staggered hybridization of nine hairpins. • Components of the linear cascade DNA hybridization chain reaction system consisting of 9 metastable DNA hairpins. Hieu Bui, Sudhanshu Garg, Vincent Miao, Tianqi Song, Reem Mokhtar, John Reif 2017 New J. Phys. 19 015006

  7. Linear Cascade DNA Hybridization Chain Reactions (in solution, not localized) • Simulation results of various linear cascade reaction lengths. The length depends on the number of hairpins involved in each reaction. • Simulation conditions are specified as the following: 5 nM concentration of hairpins, 6.5 nM concentration of reporter complex, 10 nM concentration of initiator, and the rate constant of displacing the reporter complex is common across all simulation experiments. • Linear cascade reactions when the rate constant is 105 M−1 s−1 (A) versus 1.3 × 106 M−1 s−1 (B). Simulation is performed based on the assumption that all hairpin–hairpin pairs follow the second order(bimolecular) chemical reaction. (A) (B) Hieu Bui, Sudhanshu Garg, Vincent Miao, Tianqi Song, Reem Mokhtar, John Reif 2017 New J. Phys. 19 015006

  8. Linear Cascade DNA Hybridization Chain Reactions (in solution, not localized) • Native PAGE gel of different linear cascade reactions involving 2 hairpins with and without addition of target DNA initiator. (A) • Native PAGE gel of different linear cascade reactions involving 4 hairpins with and without addition of target DNA initiator. (B) • Native PAGE gel of different linear cascade reactions involving 6 hairpins with and without addition of target DNA initiator. (C) A B C Hieu Bui, Sudhanshu Garg, Vincent Miao, Tianqi Song, Reem Mokhtar, John Reif 2017 New J. Phys. 19 015006

  9. Linear Cascade DNA Hybridization Chain Reactions (in solution, not localized) A • Kinetic characterization of the linear cascade DNA hybridization chain reactions. All samples were prepared at 5 nM of hairpin with 6.5 nM of reporter complex. • (A) Schematic of LCR system before and after interacting with the reporter complex. • (B) Effect of initiator concentration on the rate of linear cascade reaction. Hairpins do not hybridize in the absence of initiator(9 hairpins + 0× I, black). The linear cascade reaction occurs in the presence of 1× initiator concentration (9 hairpins + 1× I, red). The linear cascade reaction occurs at the same rate in the presence of excess initiator(9 hairpins + 8× I, green). • (C) Effect of hairpin concentration on the rate of crosstalk and leak reactions: 5 nM (black curve), 25 nM (red curve), and 50 nM (green curve)without the presence of initiator. B C Hieu Bui, Sudhanshu Garg, Vincent Miao, Tianqi Song, Reem Mokhtar, John Reif 2017 New J. Phys. 19 015006

  10. Linear Cascade DNA Hybridization Chain Reactions (in solution, not localized) • Effect of the number of hairpins on the rate of linear cascade reaction: 1 hairpin (black curve), 2 hairpins(red curve), 4 hairpins(green curve), 6 hairpins(blue curve), 9 hairpins(cyan curve). Note: the dotted horizontal line indicates the half-life of the linear cascade reaction (the time required for the reaction to reach 50% completion). Inset shows the real-time kinetic of all linear cascade reactions at equilibrium. • Empirical projection of the half-time completion as a function of hairpins. Note: each circle is an experimental result corresponding to the number of hairpins participating in the linear cascade reaction; a blue line is a fitted curve from those circles. Hieu Bui, Sudhanshu Garg, Vincent Miao, Tianqi Song, Reem Mokhtar, John Reif 2017 New J. Phys. 19 015006

  11. Localized DNA Hybridization Chain Reactions on DNA Tracks • Components of localized DNA hybridization chain reaction system: six metastable DNA hairpins anchored to a long DNA track via Ai domains. Prior to the addition of the initiator, DNA hairpins do not hybridize and are sequestered in the inactive states. • Triggered self-assembly of localized DNA hybridization chain reaction mechanism for the first and last steps upon sensing the presence of the catalyst / initator. Bui, H., Miao, V., Garg, S., Mokhtar, R., Song, T., Reif, J., Small 2017, 13, 1602983.

  12. Localized DNA Hybridization Chain Reactions on DNA Tracks • Fluorescence reporting mechanism to observe the kinetics of localized DNA hybridization reaction. A FRET pair consists of a fluorescence donor (green) and a fluorescence quencher (black). Upon sensing the completion, the FRET pair is disrupted by the last hairpin, giving rise to an increase in the fluorescence emission. Bui, H., Miao, V., Garg, S., Mokhtar, R., Song, T., Reif, J., Small 2017, 13, 1602983.

  13. Localized DNA Hybridization Chain Reactions on DNA Tracks • Kinetic characterization of triggered self-assembly hybridization chain reactions. • A) Localized DNA hybridization chain reactions with and without the presence of the long DNA track in two times excess initiators. Note: inset shows the fluorescence responses of the same systems but in the absence of the initiator. • B) Effect of localized DNA hybridization chain reaction rate as a function of initiator concentration. • C) Control kinetic analysis of reactions as a function of hairpins in the absence of long DNA track. C B A Bui, H., Miao, V., Garg, S., Mokhtar, R., Song, T., Reif, J., Small 2017, 13, 1602983.

  14. Localized DNA Hybridization Chain Reactions on DNA Tracks • Fluorescence experimental results of the two systems which are then used to quantify the speed-up from tethering DNA hairpins on the DNA track: • A) six DNA hairpins (no anchor domain, no track) in the presence of 2× initiators • B) six DNA hairpins bound to the long DNA track in the presence of 2× initiators. • Note: Dashed lines indicate half-time completion—the time required for the reaction to reach 50% completion. • C) Model to describe cascade reactions with and without the locality effect. • Note: AND symbol indicates bimolecular reaction; TRIANGLE symbol indicates unimolecular reaction Bui, H., Miao, V., Garg, S., Mokhtar, R., Song, T., Reif, J., Small 2017, 13, 1602983.

  15. Localized DNA Hybridization Chain Reactions on DNA Origami • DNA origami rectangle with modified staple strands (green circles). Metastable DNA hairpins tethering to DNA origami rectangle’s surface via modified staple strands through the anchor domains. • Mechanism of DNA hybridization reactions on DNA origami surface. Metastable DNA hairpins tethering to DNA origami rectangles via modified staple strands. Upon sensing the presence of the target initiator, the chain reactions occur among the hairpins, releasing the encoded information in the last hairpin. Hieu Bui, Shalin Shah, Reem Mokhtar, Tianqi Song, Sudhanshu Garg, and John Reif ACS Nano201812 (2), 1146-1155

  16. Localized DNA Hybridization Chain Reactions on DNA Origami • AFM images of DNA origami rectangle labeled with DNA hairpins. • Six metastable DNA hairpins self-assembled on DNA origami rectangle (a) • Six metastable DNA hairpins underwent cascade hybridization reactions after adding the initiator (b) • Six metastable DNA hairpins underwent cascade hybridization reactions after adding the biotin-labeled output to detect the reaction completion via binding to the streptavidin molecule (c) Hieu Bui, Shalin Shah, Reem Mokhtar, Tianqi Song, Sudhanshu Garg, and John Reif ACS Nano201812 (2), 1146-1155

  17. Localized DNA Hybridization Chain Reactions on DNA Origami • (a) Illustration of DNA origami used single-molecule TIRF experiment. Blue tokens indicate hairpins while red token indicates marker strand. • (b, c) TIRF images for DNA origami labeled with ATTO 647N marker and TAMRA initiator. The green channel is for 561 nm laser line (TAMRA label). Similarly, the red channel is for 635 nm laser line (ATTO 647N label). The approximate Gaussian localized images for both the channels. These images are analyzed for co-localization independent of the dynamic range values and noise. • (d) Co-localized image where yellow spots (highlighted in green boxes) representing DNA origami with hairpins. • Scale bars are 5 μm. Hieu Bui, Shalin Shah, Reem Mokhtar, Tianqi Song, Sudhanshu Garg, and John Reif ACS Nano201812 (2), 1146-1155

  18. Localized DNA Hybridization Chain Reactions on DNA Origami • a) Illustration of DNA origami used single-molecule TIRF experiment. Blue tokens indicate hairpins while red token indicates marker strand. • (b, c) TIRF images for DNA origami labeled with ATTO 647N marker dye and ATTO 488 output. The blue channel is for 488 nm laser line (ATTO 488 label). Similarly, the red channel is for 635 nm laser line (ATTO 647N label). The approximate Gaussian localized images for both the channels. • (d) Co-localized image where yellow spots (highlighted in green boxes) indicates DNA origami with reaction completion. • (e) Co-localization analysis for different reaction incubation times to observe kinetics. • Scale bars are 5 μm. Hieu Bui, Shalin Shah, Reem Mokhtar, Tianqi Song, Sudhanshu Garg, and John Reif ACS Nano201812 (2), 1146-1155

  19. Localized DNA Hybridization Chain Reactions on DNA Origami • (a) Schematic of localized cascade DNA hybridization chain reaction on the surface of DNA origami rectangle before and after the completion. • (b) Kinetic characterization of localized cascade DNA hybridization chain reaction on DNA origami rectangle (green curve). Kinetic characterization of cascade DNA hybridization chain reaction without DNA origami rectangle (purple curve). Each solution was kept at 5 nM concentration and the initiator was added in 2× excess. • (c) Effect of localized cascade DNA hybridization chain reaction rate as a function of initiator concentration. Hieu Bui, Shalin Shah, Reem Mokhtar, Tianqi Song, Sudhanshu Garg, and John Reif ACS Nano201812 (2), 1146-1155

  20. Summary of Localized Linear Cascade Results • Design, simulation, and synthesis of linear cascade DNA hybridization chain reactions. • 9 distinct hairpins underwent cascade hybridization chain reactions upon sensing the presence of the target initiator. • Design, simulation, and synthesis of linear cascade DNA hybridization chain reactions on DNA tracks. • 6 distinct hairpins underwent cascade hybridization chain reactions • The speedup was observed due to the locality effect. • Design, simulation, and synthesis of linear cascade DNA hybridization chain reactions on DNA origami. • 6 distinct hairpins underwent cascade hybridization chain reactions • The speedup was observed due to the locality effect, consisting with prior studies. • Proof-of-concept examples which can be applied to other surfaces to utilize the locality effect. • Combination with other DNA circuits to carry out surface DNA computation.

  21. Organization of Talk • Localized DNA Computing • Programming DNA-based Biomolecular Reaction Networks on Cancer Cell Membranes • Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates • DNA-based Analog Computing

  22. Programming DNA-based Biomolecular Reaction Networks on Cancer Cell Membranes Tianqi Song and John Reif Tianqi Song, Hieu Bui, SudhanshuGarg, AbeerEshra, Daniel Fu, Shalin Shah, Ming Yang, ReemMokhtar, and John Reif, Programming DNA-Based Biomolecular Reaction Networks on Cancer Cell Membranes, submitted for publication(2019)

  23. DNA-based Biomolecular Reaction Networks on Cancer Cell Membranes initiator output DNA strand displacement reactions D Song et al. (2019) In submission

  24. Goals, Principles and Solutions Song et al. (2019) In submission

  25. Node Design • Two modules: reaction module (DNA hairpin) and addressing module. • Two modules are paired by the hybridization between domains A and A*. DNA strand displacement reaction sgc4f is an aptamer to target a particular cell membrane receptor. Song et al. (2019) In submission

  26. Cascade Reaction Abstraction: output initiator sgc8c sgc4f Song et al. (2019) In submission

  27. Capability and Applications • Linear cascades • Complex reaction networks • Logic circuits • Cancer cell recognition and therapies Song et al. (2019) In submission

  28. Linear Cascades Ramos Cancer Cell Lines: CCRF-CEM TC01 TD08 TC01 TD08 TE13 sgc8c sgc4f TC01 Flow cytometry results: high ratios (in fluorescence intensity) between Cellwith circuit and Cellonly (data from three repeats). sgc8c sgc4f Song et al. (2019) In submission

  29. A Reaction Network on CCRF-CEM sgc8c sgc4f TE13 TC01 TE17 Path 1 sgc8c sgc4f TC01 Path 2 sgc8c TE13 TC01 Path 3 sgc8c sgc4f TE17 sgc8c TE13 Path 4 TE17 Song et al. (2019) In submission

  30. Control Experiments Low ratios (in fluorescence intensity) between Cellwith reporter and Cellonly (data from three repeats). Song et al. (2019) In submission

  31. Logic Computing and Cancer Cell Recognition sgc4f AND sgc8c sgc8c sgc4f Aptamers (Receptors) Cell Lines Low ratios are expected for HeLa, Ramos and K562. Song et al. (2019) In submission

  32. Further On-Going Work • Cancer therapies: output of a circuit opens a DNA-based capsule containing drug molecules. • Distributed computing among different cell types.

  33. Organization of Talk • Localized DNA Computing • DNA-based Analog Computing: analog circuits • Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates • Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates: logic circuits, enzyme-based circuits

  34. Localized DNA ComputationTianqi Song and John Reif • Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates • DNA-based Analog Computing • Programming DNA-based Biomolecular Reaction Networks on Cancer Cell Membranes

  35. Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates[Tianqi Song and John Reif] Tianqi Song, Abeer Eshra, Shalin Shah, Hieu Bui, Daniel Fu, Ming Yang, Reem Mokhtar, and John Reif, Fast and Compact DNA Logic Circuits Based on Single-Stranded Gates Using Strand-Displacing Polymerase, in submission (2019) Song et al. (2019) In submission

  36. Motivation Reduce complexity Improve speed news.mit.edu Song et al. (2019) In submission

  37. Goals, Principles and Solutions Song et al. (2019) In submission

  38. Single-Stranded Logic Gates: OR Gate Gate Design and Performance Gate Strands Prepared by Fuel F Two Reaction Paths to Produce Output in OR Gate Each gate strand is at 1x (1x = 100 nM). Each input strand is at 1x when encoding “1”, otherwise 0x. Each domain is 30 nt long. Reporting Reaction Song et al. (2019) In submission

  39. Single-Stranded Logic Gates: AND Gate Two Reaction Paths to Produce Output in AND Gate Gate Design and Performance Path 1 Path 2 Each gate strand is at 1x (1x = 100 nM). Each input strand is at 2x when encoding “1”, otherwise 0x. d Side Reaction in AND Gate waste Song et al. (2019) In submission waste

  40. 2-Layer Cascades 1x 2x 1x 1x Rule of cascading: a gate should always produce enough output for its downstream gates. Song et al. (2019) In submission

  41. 3-Layer Cascades 2x 2x 1x 2x 1x 1x Song et al. (2019) In submission

  42. Fan-in and Fan-out 1x 1x 1x 1x 1x Output O1 4x 1x 1x 1x 1x Song et al. (2019) In submission

  43. A Square-Root Circuit Dual-rail Logic Circuit 2x 1x 1x 1x 1x 1x 2x 2x 1x 2x Dual-rail logic (as in Qian et al. (2011) Science), each variable in the original circuit is represented by two corresponding variables in the circuit of dual-rail logic format. For example, variable “A” is represented by two variables “A-0” and “A-1” in the dual-rail logic circuit: A is logic “1” ̶> A-1 is logic “1”, A-0 is logic “0”; A is logic “0” ̶> A-1 is logic “0”, A-0 is logic “1”. Song et al. (2019) In submission

  44. A Square-Root Circuit Song et al. (2019) In submission

  45. Signal Amplification and Restoration Once every several layers For nonideal inputs to a circuit Song et al. (2019) In submission

  46. Discussion • Further improvements: more compact mechanism of signal restoration. • Future work: dynamical circuits (using only polymerase) like oscillators, controllers, etc.

  47. Organization of Talk • Localized DNA Computing • Compact and Fast DNA Logic Circuits Using Strand-Displacing Polymerase and Single-Stranded Logic Gates • Programming DNA-based Biomolecular Reaction Networks on Cancer Cell Membranes • DNA-based Analog Computing

  48. DNA-based Analog Computing Tianqi Song and John Reif Tianqi Song, SudhanshuGarg, Hieu Bui, ReemMokhtar, and John H Reif, Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers, ACS Synthetic Biology (Dec 2017). DOI: 10.1021/acssynbio.6b00390Daniel Fu, Shalin Shah, Tianqi Song and John H Reif, DNA-based Analog Computing, Chapter in book: Synthetic Biology: Methods and Protocols, Edited by ‪Jeffrey C. Braman), In Series Methods in Molecular Biology, published by Springer, pp. 411-417 (2018). ISBN 978-1-4939-7795-6Tianqi Song, SudhanshuGarg, Hieu Bui, ReemMokhtar, and John H. Reif, Analog Computation by DNA Strand Displacement Circuits, ACS Synthetic Biology, 5, 898−912  (July, 2016). DOI: 10.1021/acssynbio.6b00144 

  49. Motivation • Processing continuous signal directly • Compact circuits for cellular sensing

  50. Two Architectures • The first architecture: circuits for basic arithmetic operations (addition, subtraction and multiplication) and polynomials. • The second architecture: compact circuits to compute functions like sqrt(x), ln(x), and exp(x).

More Related