A nanoscale programmable computing machine with input, output, software and hardware made of biomolecules Nature 414, (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)

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

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

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

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

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

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

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

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

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

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

Rationale for the molecular design

b CGCAGC GCGTCG a CTGGCT GACCGA Rationale for the molecular design

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Transition rules: complete list

Automata programs used to test the molecular implementation

Transition molecules: complete list

Input and detection molecules

Experimental testing of automaton programs A1 – A6

Computations over 6-symbol long input molecules

Parallel computation

Identification of the essential components

Close inspection of the reaction intermediates

An estimation of system fidelity

Summary 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 Watt.