Nonequilibrium effects in DNA microarrays: a multiplatform study - - PowerPoint PPT Presentation

▶
nonequilibrium effects in dna microarrays a multiplatform
SMART_READER_LITE
LIVE PREVIEW

Nonequilibrium effects in DNA microarrays: a multiplatform study - - PowerPoint PPT Presentation

Sep 04, 2023 167 likes •394 views

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data Nonequilibrium effects in DNA microarrays: a multiplatform study Jean-Charles Walter KU Leuven, Belgium Collaborators : Myriam Kroll , Jef Hooyberghs

slide-1
SLIDE 1

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Nonequilibrium effects in DNA microarrays: a multiplatform study

Jean-Charles Walter KU Leuven, Belgium Collaborators : Myriam Kroll, Jef Hooyberghs and Enrico Carlon LAFNES11, Dresden July 14

DNA Microarrays

slide-2
SLIDE 2

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Outline

1

Introduction : DNA and microarrays

2

Two- and Three-state models

3

Analysis of experimental data

DNA Microarrays

slide-3
SLIDE 3

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

DNA double helix

DNA Microarrays

slide-4
SLIDE 4

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Information in the cell DNA

transcription

RNA

translation

Proteine

replication

Antisense strand RNA polymerase mRNA Transcript Sense Strand A G C G U AC G C G C G U AT A C GU A C GC G C G C G C G U A U A T T T T TT TT T T TT A A AA A A A A A A A TT T C G GG GG G G G G G G G G G C C C C C C C C C C C C C C G

3' 5' 3' 5'

5' 3'

DNA Microarrays

slide-5
SLIDE 5

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

DNA microarrays : principle

Strands on the surface : probes Strands labelled in solution : targets Gene expression, detection of mutations...

DNA Microarrays

slide-6
SLIDE 6

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Two-state model

dθ dt = ck1(1 − θ) − k−1θ k1/k−1 = e−∆G/RT

DNA Microarrays

slide-7
SLIDE 7

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Two-state model

θeq = ce−∆G/RT 1 + ce−∆G/RT ≈

c,−∆G≪1 ce−∆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 = 1 α(c + e∆G/RT)

DNA Microarrays

slide-8
SLIDE 8

Introduction : DNA and microarrays Two- and Three-state models Analysis 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

10

θ

dynamical saturation Langmuir chemical saturation isotherm t=4h t=17h t=86h

~1/RT T = 337K, c = 5pM and k1 = 104s−1M−1.

DNA Microarrays

slide-9
SLIDE 9

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Three-state model

G ∆ G ∆ G ∆ = γ ’

θ1 θ 2

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

slide-10
SLIDE 10

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Three-state model

  

dθ1 dt = ck1(1 − θ1 − θ2) + k−2θ2 − (k−1 + k2)θ1, dθ2 dt = 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

slide-11
SLIDE 11

Introduction : DNA and microarrays Two- and Three-state models Analysis 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

10

θ

regime Langmuir isotherm ~ 1/RTexp intermediate dynamical saturation chemical saturation ~1/RTeff t=86h t=17h t=4h

T = 337K, c = 5pM, k1 = 105s−1M−1, k2 = 1s−1 and γ = 1/3.

DNA Microarrays

slide-12
SLIDE 12

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Agilent microarrays

(a) (c) (d) (b) DNA Microarrays

slide-13
SLIDE 13

Introduction : DNA and microarrays Two- and Three-state models Analysis 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

slide-14
SLIDE 14

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Agilent microarrays

t

  • 8
  • 6
  • 4
  • 2
  • ∆∆Gµarray (kcal/mol)

10 10

2

10

4

10

6

I

L=30 nt, 50 pM, 65°C, 17h (a)

L

  • 8
  • 6
  • 4
  • 2
  • ∆∆Gµarray (kcal/mol)

10 10

2

10

4

10

6

I

L=30 nt, 50 pM, 65°C, 86h

(b)

  • 8
  • 6
  • 4
  • 2
  • ∆∆Gµarray (kcal/mol)

10 10

2

10

4

10

6

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

slide-15
SLIDE 15

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Affymetrix microarrays

Data from Suzuki et al (BMC Genomics, 2007) Design : 150 = 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

slide-16
SLIDE 16

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Affymetrix microarrays

15 20 25 30

  • ∆G (kcal/mol)

10 10

3

10

5

I

1.4fM 14fM 140fM 1.4pM 14pM 140pM 1.4nM

~1/RTexp ~1/RTeff

t = 16h, Texp = 318K, Teff = 850K= 2.7Texp

DNA Microarrays

slide-17
SLIDE 17

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Affymetrix microarrays

10 15 20 25 30 35

  • ∆G (kcal/mol)

10 10

3

10

5

I

14fM 140fM 1.4pM

k1 = 105s−1M−1, k2 = 1s−1 and γ = 0.374

DNA Microarrays

slide-18
SLIDE 18

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Conclusion

Thermodynamics of DNA µarray still poorly understood. Discrepancy between experiments and the two-state model : 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

slide-19
SLIDE 19

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Agilent microarrays

  • 8
  • 6
  • 4
  • 2
  • ∆∆Gµarray (kcal/mol)

10 10

2

10

4

10

6

I

c=2pM c=10pM c=50pM c=250pM L=30, 65°C, 17h

Intensity proportional to the concentration : far from saturation.

DNA Microarrays

slide-20
SLIDE 20

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Agilent microarrays

14 16 18 20 22 24 26 28 30

  • ∆Gsol (kcal/mol)

10 10

2

10

4

10

6

I c=50pM c=500pM c=5000pM

equilibrium region non-equilibrium region

L=30, T=55°C, 17h

T

DNA Microarrays

slide-21
SLIDE 21

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

DNA double helix

Estimation of the Gibbs free energy : ∆G = ∆H − T∆S with the nearest-neighbor model : ∆G T A C A T G

  • = ∆G

T A A T

  • + ∆G

A C T G

  • ,

where the values are estimated in solution.

DNA Microarrays

slide-22
SLIDE 22

Introduction : DNA and microarrays Two- and Three-state models Analysis of experimental data

Affymetrix microarrays

10 20 30 40

  • ∆G (kcal/mol)

10

  • 14

10

  • 12

10

  • 10

10

  • 8

10

  • 6

10

  • 4

10

  • 2

10

θ

5.10

5pM

5.10

3pM

5.10

1pM

5.10

  • 1pM

5.10

  • 3pM

T = 337K, t = 17h, k1 = 105s−1M−1, k2 = 1s−1 and γ = 1/3.

DNA Microarrays