1. DNA Computing and Robotics Based on Deoxyribozymes

2. DAO-E Sierpinski triangle experiments Paul Rothemund, Nick Papadakis, Erik Winfree, PLoS Biology 2: e424 (2004) 340nm

3. Part 1: How do we program mixtures of molecules in solution? Part 2: How do we program behavior of an individual molecule?

4. Introduction to deoxyribozymes: Complementary Single stranded DNA (or RNA) Joyce 1995, 1997

5. Introduction to deoxyribozymes II: Joyce 1995, 1997

6. …and we can combine them with stem loops: S

7. …into a two-state switch: Detector gate or sensor or YES gate or …

8. Complete set of switches: AND Gate: NOT Gate: ANDANDNOT: i3i3

9. Before we give examples of gates, a reminder: 1 is an increase in fluorescence, 0 no such increase!

10. A bit more about FRET: Cleavage

11. Stojanovic et al., ChemBioChem 2001 Stojanovic et al., J. Am. Chem. Soc. 2002 Catalytic Molecular Beacons as Sensor Gates Joyce 1995, Breaker 1999, Tyagi, Kramer 1996

12. NOT Gates – Inverters: Stojanovic et al., J. Am. Chem. Soc. 2002

13. Dual Input Molecular Computation Elements: AND Gate FU t(min)

14. Triple Input Elements: ANDANDNOT (INHIBIT)

15. Triple Input Elements: ANDAND Harvey Lederman

16. Before we give examples of gates, a reminder: 1 is an increase in fluorescence, 0 no such increase!

17. Let’s add A (0 or 1) and X (0 or 1) and get O (0 or 1, or 2): A + X = O 1 1 01 00 00 01 2 100100 i1i1 i2i2 GRGRGRGRGR

18. Stojanovic & Stefanovic, J. Am. Chem. Soc. 2003 Implement addition with gates: 10 01 21

19. Now, a twist: 1 is an increase in fluorescence, 0 no such increase! 1

20. d6 d4 d9 d5 d1 d2 d3 d8 d7 ABCDEFG 1  2   3  4   5   6  7  8 9  Segments required J. Macdonald, Columbia University Here is 7-segment display:

21. Test by constructing simple 2+2-bit adder 10102+2:+= A1A1 A0A0 X1X1 X0X0 + 01011+1:+= Binary inputs 01101+2:+= 10012+1:+= Non general Display output Expected display Adder result “hard-wired” into decoder Designate 4 oligonucleotides as binary inputs J. Macdonald, Columbia University More arithmetical operations:

22. Determine logic gates required, and layout in wells (via Microsoft Excel) Very simple logic gate arrangement required: 4 YES gates 2 AND gates Fully constructible from reagents in freezer Tested with all input combinations…. J. Macdonald, Columbia University Adding 2+2 with visual display:

23. Perfect digital behavior Constructed & tested in a single day (A 1 A 0 + X 1 X 0  display) 01+01=01+10=10+01=10+10= No inputs (1+1=2) (1+2=3) (2+1=3) (2+2=4)Background Binary inputs: 7-segment Display Arithmetic B&W imager within ~1hr Color photograph ~5hrs Fluorescence reader ~20-30mins J. Macdonald, Columbia University; DNA 13, 2007

24. 2x2 multiplier with visual display: Gate arrangement 19 gates required J. Macdonald, Columbia University (A 1 A 0 × X 1 X 0  display)

25. 01 × 01 = (1×1=1) Arithmetic Binary inputs: 7-segment Display 01 × 10 =01 × 11 =10 × 01 =10 × 10 = 10 × 11 =11 × 01 =11 × 10 =11 × 11 =(00 × 00) (1×2=2)(1×3=3)(2×1=2)(2×2=4) (2×3=6)Arithmetic(3×1=3)(3×2=6)(3×3=9)no input control Binary inputs: 7-segment Display Color photography J. Macdonald, Columbia University 2x2 multiplier with visual display: (A 1 A 0 × X 1 X 0  display)

26. Silicomimetic automata project (MAYA): Macdonald et al. Nano Lett. 2006 Stojanovic&Stefanovic. Nat.Biotech. 2003 MAYA:MAYA-II:MAYA-III: Pei, Matamoros, et al. in testing Human designed Goal: Demonstrate power of molecular computing! Computer designedHuman designed Goal: What does it take to coordinate large number of gates? Goal: Field programmable arrays of gates! -Teaching “by example” -Selecting strategy (“carving”)

27. Implementation of tic-tac-toe playing algorithm with deoxiribozymes: Molecular Array of YES and ANDANDNOT Gates (MAYA) Stojanovic, Stefanovic, Nat. Biotech. 2003

28. MAYA vs. Milan: losing game Mg 2+ From Sci. Am. 2008

29. Is it possible to have serial circuits? (Upstream enzyme)(downstream enzyme)

30. Intergate Communication : Ligase-Cleavase Stanka Semova, Dmitry Kolpashchikov

31. Ligase-cleaves XOR circuit: JACS 2005 O IAIA IBIB XOR

32. What about cleavase-cleavase? a. Substrate as inhibitor c. Substrate as activator b. product as activator d. product as inhibitor Cf. Uri Alon, Nat. Rev. Genetics 2007, 450 “Network Motifs: Theory and Experimental Approaches”

33. Ben-Tov, Tamburi +inhibitor Connection type I: Substrate as inhibitor

34. Connection type II: Product as activator 50nM d-8-17, 500nM d-sub, 500nM stem- loop, 50nM up-8-17, 500nM input +MB cont +U +i,p MB Renjun Pei

35. Connection type III: Substrate as activator 100nM E6, 500nM d-sub, 500nM activator, 50nM 8-17 +act +U cont Renjun Pei, Aihua Shen

36. Connection type IV: Product as inhibitor 100nM E6, 500nM d-sub, 250nM stem-loop, 50nM 8-17 P +MB +U Renjun Pei, Aihua Shen

37. AND cascade: 100nM E6, 500nM d-sub, 350nM inh-1, 350nM inh-2, 50nM 8-17-1, 50nM 8-17-2 p E-1 2 Es 2 inhs E-2

38. Now we can do signal threshold: 100nM D, 500nM d-sub, 20nM U, 200nM inh at time 180 minutes. one eq.

39. In Circuits by Winfree’s group: Inputs Input Translation Computational subcircuit Signal restoration