DNA Computing and Robotics Based on Deoxyribozymes

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

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

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

Introduction to deoxyribozymes II: Joyce 1995, 1997

…and we can combine them with stem loops: S

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

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

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

A bit more about FRET: Cleavage

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

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

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

Triple Input Elements: ANDANDNOT (INHIBIT)

Triple Input Elements: ANDAND Harvey Lederman

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

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

Stojanovic & Stefanovic, J. Am. Chem. Soc Implement addition with gates:

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

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:

Test by constructing simple 2+2-bit adder :+= A1A1 A0A0 X1X1 X0X :+= Binary inputs :+= :+= 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:

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:

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

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

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)

Silicomimetic automata project (MAYA): Macdonald et al. Nano Lett Stojanovic&Stefanovic. Nat.Biotech 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”)

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

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

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

Intergate Communication : Ligase-Cleavase Stanka Semova, Dmitry Kolpashchikov

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

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”

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

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

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

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

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

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

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