using nanopores to sequence dna · the role of dna in biology - each human cell has two homologous...
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Using nanopores to sequence DNA
Aleksei Aksimentiev
Wednesday, November 27, 13
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phosphate
sugar ring
base
Double stranded DNA(persist. length ~50nm)
Single stranded DNA(persist. length ~1.5nm)
backboneDNA code is written in atomsHighly charged: 2 electron charges per 0.32nm
The sequence has direction:5’-AAGCTGGTTCAG-3’
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Double stranded DNA(persist. length ~50nm)
Single stranded DNA(persist. length ~1.5nm)
Highly charged: 2 electron charges per 0.32nm
The sequence has direction:5’-AAGCTGGTTCAG-3’
DNA code is written in atomsbackbone
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The role of DNA in biology
- Each human cell has two homologous copies of the genome (from father and mother), each containing ~3,100,000,000 (3.1x109) base pairs. Fits on a CD. - All cell types in a human body have the same genome, but appear different because different part of the genome are “ON” - Genes (protein coding part) occupy only 1.5% of the sequence. ~23,000 genes, twice as many as in a fruit fly. 50% of the genes have unknown function. - More than 50 percent are simple repeat sequences CGTCGTCGTCGT…, the “dark matter” of the genome. Difficult to characterize.- Among individuals, 99.9% of the sequence is similar. > 1,000,000 differences.
Central dogma of molecular biology
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The Human Genome Project
Duration: October 1990 - 2003
Discovered ALL 20,000-25,000 human genes
Determined complete sequence of the 3 billion DNA bases5’-ACCGGTGGGTGCATAGCTGTGCTGTAAGTGAAGTG AGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAG GTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTG GACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTA GTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT
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GTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGA TCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTATTGTGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGWednesday, November 27, 13
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GATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGA TCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTATTGTGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGAAACGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTG GCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTWednesday, November 27, 13
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CAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGATCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTATTGTGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGAAACGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTG GCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGCAGGTGTTGAAAGTCGATGTAGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCT CCTAGCTAGTCAGTGGTGTTCGTAGGTCAGTTGATGTCGATGTGAAATGCTGATGCTAGTGGACAGGGTGACTAGTGAATCGATGCTAGCCTAGCTAGTCAGTGGTGCTAGCTACGATCGATTTCAGGCTGCTGTGGGTGCATAGCTGTGCTGTAAGTGAAGTGAGGCGGWednesday, November 27, 13
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! and ~ 3,000,000 more pages!
(one month to show 24/7)
Just four letter:ACGT
~715 Mb
0 01 1
2 bits
8 bits = 1b
4/8*3*10^9DNA code is billion times moreefficient
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2006
2008
2010
$1,000,000
$100,000
Disruptive technologies emerged in: -2006 (Generation II) -2009 (Generation III)
$1,000 genomeexpected in 2013
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Conventional DNA sequencingNobel Prize in Chemistry 1980
As the DNA is synthesized, nucleotides are added on to the growing chain by the DNA polymerase.
The reactions start from the same nucleotide and end with a specific base
Fluorescence-based sequence gelhttp://bbrp.llnl.gov
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Electric readout of the DNA sequencing
A. Aksimentiev J.P. LeburtonK. SchultenS. SligarG. Timp
(2003)
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Coulter counter
patented in 1953 by W.H. Coulter
1/current (i.e. resistance) measurements when a particle enters the microscopic pore.
folk.uio.no/anderne/research.html
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The nanopore technology
Distributions of the electric field
A voltage bias is imposed across the membrane
Ionic current
time
I
+ + + + + +
- - - - - - - - -
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Ionic current through pore measured
current transients associated with
passage of dsDNA
- Isolates 1nm3 of volume- Automatic loading and reloading- Highly processive, single-file transport- Compatible with several detection schemes- No limit on the read length
The nanopore technology
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The nanopore technology
3 nm1000 times per minute
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The ionic current blockade reveals the sequence of the confined nucleotides
Sequencing DNA by measuring ionic current
Nature Reviews Drug Discovery 1, 77-‐84 (January 2002)
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Nature Biotech. 30: 349 - 353 (2012)
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Sequencing DNA using MspA
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Nature Biotechnology (2012)doi:10.1038/nbt.2171
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All-Atom Molecular Dynamics Simulations of Nanopore Systems
Massive parallel computerBlue Waters (UIUC): ~200,000 CPUs
Atoms move according to classical mechanics (F= ma)
Interaction between atoms is defined by molecular force field
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Time scale: ~ 0.1-100 µsLength scale: 10K - 100M atoms or (< 50 nm)3
Time resolution: 2 fsSpacial resolution: 0.1 A
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Setting up an simulation is like cooking
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-‐ protein
-‐ DNA
-‐ lipid
-‐ ions
-‐ water
Components
F = ma @ 300 K
Time step = 1 fs
Setting up an simulation is like cooking
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CompuEng conductance of α-‐hemolysin with molecular dynamics
Protein + lipid bilayer membrane + 1M water solution of KCl = ~300,000 atoms
Elec
tric
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Average electrostatic potential map
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Instantaneous current
Current-voltage curve of !-hemolysin
Scope is the current
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Instantaneous current
Ionic current at 120mV: Experiment: 120 pA Simulations: 130 pA
Current-voltage curve of !-hemolysin!"#$%&'()*("<<P`:=a">ABBaC
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MD simulations of current blockades in MspA
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`A
0 2000 4000 6000 8000 10000time (ns)
0
0.2
0.4
0.6
0.8
I (nA
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poly(dA)
0 20 40 60 80I/I0 (%)
0
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0.1
0.15
0.2
p(I/I
0)
poly(dT)I/I0 : 19.6±4.2%
I: 179 ±32 pA
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0.2
0.4
0.6
0.8
I (nA
)
poly(dT)
0 20 40 60 80I/I0 (%)
0
0.05
0.1
0.15
0.2
p(I/I
0)
poly(dA) I/I0 : 30.7±6%I: 281 ±48 pA
Simulated on D. E. Shaw Research computer system Anton
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Electric readout of the DNA sequencing
A. Aksimentiev J.P. LeburtonK. SchultenS. SligarG. Timp
(2003)
Wednesday, November 27, 13
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Eduardo Cruz-Chu(J. Phys. Chem. B, 2006)
Dangling Oxygen Non-Dangling Oxygen
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Wednesday, November 27, 13
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• DNA + nanopore
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User-Defined Forces in NAMD
TclBC TclForcesGridForces
Medium Medium Advanced
Estimated completion time: ~ 2 hours/sectionTclBC and TclForces: basic knowledge of Tcl
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The best of the two worlds
Solid-state pore Biological pore Hybrid pore?
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Wednesday, November 27, 13
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Using nanopores to trap DNA
2.5 nmText
Jeff Comer
Wednesday, November 27, 13
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Andre Geim Konstantin Novoselov
Wednesday, November 27, 13
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Graphene nanopores
46
Wednesday, November 27, 13
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Wednesday, November 27, 13
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Wednesday, November 27, 13
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Plasmonics tweezers for nanopore sensing
54
Experiment: Cees Dekker, Magnus Jonsson (TU Delo)
Wednesday, November 27, 13
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