1. A nanoscale programmable computing machine with input, output, software and hardware made of biomolecules Nature 414, 430-434 (2001) Kobi Benenson supervisor: Ehud Shapiro, Dept of Computer Science & Applied Math Acknowledgements: Ehud Keinan (Technion), Zvi Livneh (WIS), Tami Paz-Elizur (WIS), Rivka Adar (WIS), Aviv Regev (WIS), Irith Sagi (WIS), Ada Yonath (WIS)

2. “Medicine in 2050: Doctor in a Cell” Programmable Computer Molecular Input Molecular Output

3. Research goal: Design a simplest non-trivial molecular computing machine (two-state two-symbol finite automaton) that works on engineered inputs

4. Finite automaton: an example An even number of b ’ s S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 S1 S0 b a b a Two-states, two-symbols automaton

5. Automaton 1 bab S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S0

6. Automaton 1 bab S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S0 S0, b  S1

7. Automaton 1 ab S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S1

8. Automaton 1 ab S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S1 S1, a  S1

9. Automaton 1 b S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S1

10. Automaton 1 b S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S1 S1, b  S0

11. Automaton 1 S0, a  S0 S0, b  S1 S1, a  S1 S1, b  S0 An even number of b ’ s S0 The output

12. Rationale for the molecular design

13. b CGCAGC GCGTCG a CTGGCT GACCGA Rationale for the molecular design

14. b CGCAGC GCGTCG a CTGGCT GACCGA CAGC GGCT S0, a Rationale for the molecular design S0, b

15. b CGCAGC GCGTCG a CTGGCT GACCGA CAGC GGCT S0, aS0, b CGCAGC CG CTGGCT GA S1, aS1, b Rationale for the molecular design

16. Transitions abt CAGCCTGGCTCGCAGCTGTCGC GACCGAGCGTCGACAGCG S0, b Rationale for the molecular design

17. S0, b  S1 Transitions abt CAGCCTGGCTCGCAGCTGTCGC GACCGAGCGTCGACAGCG S0, b Rationale for the molecular design

18. Transitions bt CTGGCTCGCAGCTGTCGC GAGCGTCGACAGCG S1, a Rationale for the molecular design S0, b  S1

19. Transitions bt CTGGCTCGCAGCTGTCGC GAGCGTCGACAGCG S1, a Rationale for the molecular design S1, a  S1

20. Transitions t CGCAGCTGTCGC CGACAGCG S1, b Rationale for the molecular design

21. S1, b  S0 Transitions t CGCAGCTGTCGC CGACAGCG S1, b Rationale for the molecular design

22. S1, b  S0 Transitions TCGC S0, t Rationale for the molecular design

23. Output: S0 Transitions TCGC S0, t Rationale for the molecular design

24. Transition procedure: a concept abt CAGCCTGGCTCGCAGCTGTCGC GACCGAGCGTCGACAGCG S0, b Rationale for the molecular design

25. Transition procedure: a concept abt CAGCCTGGCTCGCAGCTGTCGC GACCGAGCGTCGACAGCG S0, b GTCG 4 nt 8 nt S0, b -> S1 Rationale for the molecular design

26. Transition procedure: a concept bt CAGCCTGGCTCGCAGCTGTCGC GACCGAGCGTCGACAGCG GTCG 4 nt 8 nt S0, b -> S1 Rationale for the molecular design

27. Transition procedure: a concept bt CTGGCTCGCAGCTGTCGC GAGCGTCGACAGCG S0, b -> S1 S1, a Rationale for the molecular design

28. Transition procedure: a concept bt CTGGCTCGCAGCTGTCGC GAGCGTCGACAGCG S1, a -> S1 S1, a Rationale for the molecular design

29. Transition procedure: a concept bt CTGGCTCGCAGCTGTCGC GAGCGTCGACAGCG S1, a -> S1 S1, a GACC 6 nt 10 nt Rationale for the molecular design

30. Transition procedure: a concept t CTGGCTCGCAGCTGTCGC GAGCGTCGACAGCG S1, a -> S1 GACC 6 nt 10 nt Rationale for the molecular design

31. Transition procedure: a concept t CGCAGCTGTCGC CGACAGCG S1, a -> S1 S1, b Rationale for the molecular design

32. Transition procedure: a concept t CGCAGCTGTCGC CGACAGCG S1, b -> S0 S1, b GCGT 8 nt 12 nt Rationale for the molecular design

33. Transition procedure: a concept CGCAGCTGTCGC CGACAGCG S1, b -> S0 GCGT 8 nt 12 nt Rationale for the molecular design

34. Transition procedure: a concept TCGC Output: S0 S0, t Rationale for the molecular design

35. In situ detection TCGC Output: S0 S0, t AGCG Detection molecule for S0 output Rationale for the molecular design

36. In situ detection TCGC Output: S0 AGCG Reporter molecule for S0 output Rationale for the molecular design

37. Inside the transition molecule S0,b -> S1 GTCG 4 nt 8 nt

38. Inside the transition molecule S0,b -> S1 GTCG 4 nt 8 nt GGATGACGAC CCTACTGCTG FokI

39. Inside the transition molecule S0,b -> S1 GTCG 4 nt 8 nt GGATGACGAC CCTACTGCTG 9 nt 13 nt FokI

40. Inside the transition molecule S0,b -> S1 GTCG GGATGACGAC CCTACTGCTG 9 nt 13 nt FokI

42. Inside the transition molecule S1,a -> S1 GACC 6 nt 10 nt

43. Inside the transition molecule S1,a -> S1 GACC 6 nt 10 nt GGATGACG CCTACTGC 9 nt 13 nt FokI

44. Inside the transition molecule S1,a -> S1 GACC GGATGACG CCTACTGC 9 nt 13 nt FokI

45. Inside the transition molecule S1,b -> S0 GCGT 8 nt 12 nt

46. Inside the transition molecule S1,b -> S0 GCGT 8 nt 12 nt GGATGG CCTACC 9 nt 13 nt FokI

47. Inside the transition molecule S1,b -> S0 GCGT GGATGG CCTACC 9 nt 13 nt FokI

48. Inside the transition molecule GACC GGATGACG CCTACTGC GTCG GGATGACGAC CCTACTGCTG GCGT GGATGG CCTACC S0 -> S1 S0 -> S0 S1 -> S1 S1 -> S0

49. Transition rules: complete list

50. Automata programs used to test the molecular implementation

51. Transition molecules: complete list

52. Input and detection molecules

53. Experimental testing of automaton programs A1 – A6

54. Computations over 6-symbol long input molecules

55. Parallel computation

56. Identification of the essential components

57. Close inspection of the reaction intermediates

58. An estimation of system fidelity

59. Summary 10 12 automata run independently and in parallel on potentially distinct inputs in 120  l at room temperature at combined rate of 10 9 transitions per second with accuracy greater than 99.8% per transition, consuming less than 10 -10 Watt.