Nonequilibrium effects in DNA microarrays: a multiplatform...
Transcript of Nonequilibrium effects in DNA microarrays: a multiplatform...
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Nonequilibrium effects in DNA microarrays:a multiplatform study
Jean-Charles WalterKU Leuven, Belgium
Collaborators :Myriam Kroll, Jef Hooyberghs and Enrico Carlon
LAFNES11, DresdenJuly 14
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Outline
1 Introduction : DNA and microarrays
2 Two- and Three-state models
3 Analysis of experimental data
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
DNA double helix
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Information in the cell
DNAtranscription
RNAtranslation
Proteine
replication
Antisense strand RNA polymerase
mRNA Transcript
Sense Strand
AG CG
UAC
G CG
CG
UAT
ACGU
ACGC
GCG
CGC
GUA
UAT
T T T TT TT T T TTA A AA A
AAAAAA TT TC G GG GG G
GGGGGGGG
C C C
CCCCCCCCCCC G
3' 5'
3'5'
5'3'
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
DNA microarrays : principle
Strands on the surface : probes
Strands labelled in solution : targets
Gene expression, detection of mutations...
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Two-state model
dθdt
= ck1(1 − θ)− k−1θ
k1/k−1 = e−∆G/RT
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Two-state model
θeq =ce−∆G/RT
1 + ce−∆G/RT≈
c,−∆G≪1ce−∆G/RT
θ(t) = θeq(1 − e−t/τ ) (θ(0) = 0)
{
k1 = Cst = α
k−1 = α · e∆G/RT
τ =1
ck1 + k−1=
1α(c + e∆G/RT )
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Two-state model
5 10 15 20 25 30 35 40-∆G (kcal/mol)
10-8
10-6
10-4
10-2
100
θdynamical saturation
Langmuir chemical saturation
isotherm
t=4ht=17ht=86h
~1/RT
T = 337K, c = 5pM and k1 = 104s−1M−1.
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Three-state model
G∆G∆ G∆= γ’
θ1 θ2
k
k
k
−2
2
1
k−1
Hooyberghs, Baiesi, Ferrantini and Carlon (PRE, 2010)
JCW, Kroll, Hooyberghs and Carlon (J.Phys.Chem.B, 2011)
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Three-state model
dθ1dt = ck1(1 − θ1 − θ2) + k−2θ2 − (k−1 + k2)θ1,
dθ2dt = k2θ1 − k−2θ2,
k1/k−1 = e−∆G′/RT k2/k−2 = e−(∆G−∆G′)/RT ,
k1 = α; k−1 = αe∆G′/RT ,
k2 = ω; k−2 = ωe(∆G−∆G′)/RT .
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Three-state model
5 10 15 20 25 30 35 40-∆G (kcal/mol)
10-8
10-6
10-4
10-2
100
θ
regime
Langmuir isotherm ~ 1/RTexp
intermediate
dynamical saturation
chemical saturation
~1/RTeff
t=86ht=17ht=4h
T = 337K, c = 5pM, k1 = 105s−1M−1, k2 = 1s−1 andγ = 1/3.
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Agilent microarrays
(a)
(c)
(d)
(b)
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Agilent microarrays
Design :
1 single Target :3’ ATTCGCCTATTGGACTACGTATTGCTCAGC 5’
Perfect Match Probe :5’ TAAGCGGATAACCTGATGCATAACGAGTCG 3’
One Mismatch Probes :5’ TAACCGGATAACCTGATGCATAACGAGTCG 3’
Two Mismatches Probes :5’ TAAGCTGATAACCTGATGCATCACGAGTCG 3’
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Agilent microarrays
t
-8 -6 -4 -2 0-∆∆Gµarray
(kcal/mol)10
0
102
104
106
I
L=30 nt, 50 pM, 65°C, 17h
(a)L
-8 -6 -4 -2 0-∆∆Gµarray
(kcal/mol)10
0
102
104
106
I
L=30 nt, 50 pM, 65°C, 86h
(b)
-8 -6 -4 -2 0-∆∆Gµarray
(kcal/mol)10
0
102
104
106
I
L=25 nt, 500 pM, 65°C, 17h
(c)
Fit : k1 = 5 · 103s−1M−1, k2 = 1s−1 and γ = 1/3.
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Affymetrix microarrays
Data from Suzuki et al (BMC Genomics, 2007)Design :
150 6= Targets (L = 25nt) :3’ ATTCGCCTATTGGACTACGTATTGCTCAGC 5’
Probes with varying length :Perfect Match :
5’ TAAGCGGATAACCTGATGCATAACGAGTCG 3’ (L = 25nt)5’ AAGCGGATAACCTGATGCATAACGAGTCG 3’ (L = 24nt)
· · ·
5’ TGCATAACGAGTCG 3’(L = 14nt)One Mismatch :
5’ TAAGCGGATAACCTGATGCATAACGAGTCG 3’ (L = 25nt)5’ AAGCGGATAACCTGATGCATAACGAGTCG 3’ (L = 24nt)
· · ·
5’ CCTGATGCATAACGAGTCG 3’ (L = 14nt)
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Affymetrix microarrays
15 20 25 30-∆G (kcal/mol)
10
103
105
I
1.4fM14fM140fM1.4pM14pM140pM1.4nM
~1/RTexp
~1/RTeff
t = 16h, Texp = 318K, Teff = 850K= 2.7Texp
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Affymetrix microarrays
10 15 20 25 30 35-∆G (kcal/mol)
10
103
105
I
14fM140fM1.4pM
k1 = 105s−1M−1, k2 = 1s−1 and γ = 0.374
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Conclusion
Thermodynamics of DNA µarray still poorly understood.
Discrepancy between experiments and the two-statemodel : nonequilibrium effects.
Weaker slope : effective temperature.
Saturation of the signal at a lower value :dynamical saturation.
Consequences on the behavior of the devices.
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Agilent microarrays
-8 -6 -4 -2 0-∆∆Gµarray
(kcal/mol)10
0
102
104
106
I c=2pM
c=10pM
c=50pM
c=250pML=30, 65°C, 17h
Intensity proportional to the concentration :far from saturation.
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Agilent microarrays
14 16 18 20 22 24 26 28 30-∆G
sol (kcal/mol)
100
102
104
106
I
c=50pM
c=500pM
c=5000pMequilibrium
region
non-equilibrium region
L=30, T=55°C, 17h
T
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
DNA double helix
Estimation of the Gibbs free energy :
∆G = ∆H − T∆S
with the nearest-neighbor model :
∆G(
T A CA T G
)
= ∆G(
T AA T
)
+∆G(
A CT G
)
,
where the values are estimated in solution.
DNA Microarrays
Introduction : DNA and microarraysTwo- and Three-state modelsAnalysis of experimental data
Affymetrix microarrays
0 10 20 30 40-∆G (kcal/mol)
10-14
10-12
10-10
10-8
10-6
10-4
10-2
100
θ 5.105pM
5.103pM
5.101pM
5.10-1
pM
5.10-3
pM
T = 337K, t = 17h, k1 = 105s−1M−1, k2 = 1s−1 andγ = 1/3.
DNA Microarrays