dna computation and circuit construction isabel vogt 2012
TRANSCRIPT
![Page 1: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/1.jpg)
DNA Computation and Circuit Construction
Isabel Vogt2012
![Page 2: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/2.jpg)
What is computation?
• 2+2=4• RULE: 1 if and only if A=1 and B=1, else 0
A B Output
0 0 0
1 0 0
0 1 0
1 1 1
Computation
![Page 3: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/3.jpg)
Computer
Inputs
Output
![Page 4: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/4.jpg)
DeoxyriboNucleic Acid (DNA)
![Page 5: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/5.jpg)
How can we engineer DNA to computesolutions to problems?
![Page 6: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/6.jpg)
DNA Replication = Information Transfer
![Page 7: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/7.jpg)
![Page 8: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/8.jpg)
The Hamiltonian Path Problem
A directed graph G with vertices vin and vout has a directed Hamiltonian path iff there exists a sequence of one-way edges e1…ei that begins at vin and ends at vout, and passes through every vertex exactly once.
Vin
Vout
2
3
1
4
05
![Page 9: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/9.jpg)
1. Generate random paths through the graph
2. Keep only those paths that begin with vin and end with vout
3. If G has n vertices, keep only those paths that enter exactly n vertices
4. Keep only those paths that enter each vertex at least once
5. If any paths remain, say YES, if not NO
150234
15150234
243
024315
4501
05
02315
051515
0234315
![Page 10: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/10.jpg)
Parallel Computing With DNA
€
O i
€
Oi
€
O 1
€
O 2
€
O1
€
O2
€
O1
€
O2€
O 1
€
O 2
![Page 11: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/11.jpg)
1. Generate random paths through the graph
€
Oi
Unique 20mer for each vertex
€
Oi→ j
Unique 20mer for every existing edge
Last 10mer of Oi and first 10mer of Oj
Mix together for all vertices vi in Gand for all edges eij
€
O i
€
Oi→ j
€
O0→2
€
O2→4€
O 0
€
O 2
€
O 4
Splints for G-specific ligation
Random Path through G
![Page 12: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/12.jpg)
2. Keep only those paths that begin with vin and end with vout
€
O 0
€
O 2
€
O 6… …
€
O0
€
O 6
PCR copy region between (inclusive) and
€
O 0
€
O 6
![Page 13: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/13.jpg)
3. If G has n vertices, keep only those paths that enter exactly n vertices
MW
120mer
Separate oligomers based upon size and keep only those of n(20) bases
![Page 14: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/14.jpg)
4. Keep only those paths that enter each vertex at least once
€
O i
€
Oi
Pull down for every vertex
![Page 15: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/15.jpg)
1. Generate random paths through the graph
2. Keep only those paths that begin with vin and end with vout
3. If G has n vertices, keep only those paths that enter exactly n vertices
4. Keep only those paths that enter each vertex at least once
5. If any paths remain, say YES, if not NO
1. Ligate G-specific paths through DNA hybridization
2. Run PCR with primers for and .
3. Separate oligomers on a gel and keep only those with length n(20)
4. Affinity chromatography for each vertex sequence
5. Amplify and run on a gel for a band
€
Oin
€
Oout
![Page 16: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/16.jpg)
• Truly parallel computation• Applicability:
– # oligomeric sequences grows linearly with # edges
– Amount of oligomer scales exponentially
• Efficiency:– Approximately 1020 ligation reaction per second– ΔG ≈ -8 kcal mol-1
– 2 x 1019 reactions for 1 J– 2nd Law of Thermodynamics: 34 x 1019
irreversible rxns per J
The future of computation?
![Page 17: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/17.jpg)
![Page 18: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/18.jpg)
Branch Migration
No Reaction
Irreversible Reaction
Reversible Reaction(see-sawing)
Chen and Ellington. Curr Opin Biotech, 21: 2010
![Page 19: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/19.jpg)
See-sawingReporting
Thresholding
![Page 20: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/20.jpg)
S6*
S6
T*
T*T* S5*
S5 T
S6S5T
S2Input
Gate
Reporter
![Page 21: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/21.jpg)
T*T* S5*
S5 T
S6S5
T
S2
S6*
S6
T*
Reporter
![Page 22: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/22.jpg)
T*T* S5*
S5
S6S5T
S2
T
S6*
S6
T*
Reporter
Output
![Page 23: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/23.jpg)
S6*T*
T*T* S5*
S5
S6S5T
S2
Reporter
T
“Reporting”
![Page 24: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/24.jpg)
T*T* S5*
S5
S6S5T
S2
T
S6*
S6
T*
Reporter
Output
![Page 25: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/25.jpg)
S6*
S6
T*
T*T* S5*
S5 T
S6S5T
S2Input
Reporter
“See-Sawing”
![Page 26: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/26.jpg)
T*T* S5*
S5 T
S6
S5T
S2Input
Fueled see-sawing: catalytic output release
S5 T
S7
Gate:Output
Fuel
XS
![Page 27: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/27.jpg)
Entropically Driven – back of the envelope calculation
€
W0 ≈M!
N!(M −N)!
W1 ≈M!
(N − k)!k!(M −N)!
For
€
N!> (N − k)!k!
⇒ W1 >W0€
k <N
2
Fuel strands catalyze complete release of output
![Page 28: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/28.jpg)
T*T* S5*
S5 T
S6
S5T
S2Input
Thresholding: Limited output release
Gate:Output
Threshold
0.5 eq
S2* T* S5*
S5
Longer Toehold No Toehold
![Page 29: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/29.jpg)
Threshold
0.5 eq
S2* T* S5*
S5
Longer Toehold No Toehold
Irreversiblepreferential binding
€
ΔGbinding(kcal /mol)•Rate increases exponentially with length of toehold sequence
•No toehold on the opposite side makes the reverse reaction negligible
Zhang and Winfree. JACS,131: 2009
![Page 30: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/30.jpg)
![Page 31: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/31.jpg)
FAN OUT
•Single input
•If above threshold – catalytically releases all output
FAN IN
•Many inputs
•Stoichiometrically releases single output
![Page 32: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/32.jpg)
![Page 33: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/33.jpg)
Dual-Rail Logic
•Makes use of two different sequences, one for ON and one for OFF
•Each OR, AND, ANDNOT, NAND, NOR gate is constructed by two gates
•Prevents computation before sequences are added
![Page 34: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/34.jpg)
OR Gate
OFF
ON
•Add either x0 or x1 to indicate OFF or ON
•OR Gate: OR for ON (output = 1) or AND for OFF (output =0)
![Page 35: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/35.jpg)
![Page 36: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/36.jpg)
Why did this work?
•Simplicity•Abstraction•Tolerance
Clamps
Toehold length
Temperature
•A lot of careful troubleshooting!
![Page 37: DNA Computation and Circuit Construction Isabel Vogt 2012](https://reader030.vdocuments.us/reader030/viewer/2022020106/56649c7d5503460f94931ecd/html5/thumbnails/37.jpg)
Why do we care?
•Functional, useful computers?
•Computation + DNA nanostructures
•See-sawing in RNAi and miRNAs?
•Regulation in an “RNA world”