langmuir, 2009, doi:10.1021/la90222g dna diffusivity decreases with increase in the mole fraction of...
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Langmuir, 2009, DOI:10.1021/la90222gDNA diffusivity decreases with increase in the mole fraction of the gel-phase lipid.
Epifluorescence tracking of naked GUVs (top) and microsphere adsorbed GUVs (bottom) reveal binding induced charge separation and slaved motion of lipids.
5µm
a
b c
DMPC
DMTAP+
Before particle binding
After binding
Lipids diffusion slaved by particle
-
0 30 60 90 120 150
-20
-15
-10
-5
0
Bin
din
g e
nth
alp
y (
10
6 cal/m
ol)
cnanoparticle
/cliposome
DMPC 20C DMPC/DMTAP 20C DMPC/DMTAP 40C
Enhanced cooperative binding
Particle binding induces DMTAP segregation at
liquid phaseCDMTAP 15%→50%
Isothermal titration calorimetry measures the binding energy quantitatively and shows that binding induces charge separation and facilitates further binding
Advance made here: particle binding induces segregation of lipid in membrane and gathers their reins
In progress: coupled diffusion of lipids and particles
Random walkers on a fluctuating
lipid tube
For Corrugated Surfaces D~N-1
Model Chain Diffusion on Surfaces
For Smooth Surfaces D~N-3/4
Experiments Show D~N-3/2
Include Surface Defects to Explain Data
Transition to experimental data when defect spacing is less than chain size
DNA mobility on homogeneous bilayers and vesicles
Adsorption of ss-DNA on supported cationic lipid bilayer composed of DMTAP or DOTAP
0.001
0.01
0.1
1
10
0.001 0.01 0.1 1 10DLipid (µm2/s)
DD
NA (
µm
2 /s)
Diffusivity of the adsorbed DNA () plotted as a function of the lipid (DMTAP) diffusivity. Also shown is the diffusivity of bacteriorhodopsin in liposomes plotted as a function of lipid mobility in the presence of protein at different Lipid(L)/Protein(P) ratios. L/P = 140 () and L/P = 30 () (adapted from Peters et al ,PNAS, 79,4317(1982))
Mobility of ss DNA tracks lipid mobility
Diffusion of large DNA on giant unilamellar vesicles
Diffusion of linear DNA
Diffusion of circular DNA
0.23m2/s0.2m2/s
No D
D=0.44m2/s
48.5kbp linear DNA (3.2*107g/mol) trajectory (black) on DOPC DOTAP (10%) GUV. The three smaller red circles show trajectories from same DNA molecule. Scale of this image is 40 m x 40 m. It is exciting to see that the DNA diffusivity differs from spot to spot, and from time to time, even in the absence of external electric field for electrophoresis.
2D projection of 2.6*107 g/mol circular DNA trajectory on giant lipid vesicle surface. From a plot of mean-square displacement against time, the straight line implies a translational diffusion coefficient of D = 0.66 m2/sec.
X2 (m
2 )
ConclusionsLipid mobility controls the diffusivity of short biopolymer adsorbates.Formation of raft-inspired lipid bilayers enhances the effectiveness of polyvalent recognition as well as adsorbed enzyme stability.Domain formation in lipid bilayers and biomolecule recognition can be actively controlled by adding divalent cations.Domains in lipid bilayers provide control over the adsorption and transport of DNA.Nanoparticles stabilize phospholipid vesicles by preventing vesiclefusion even at high vesicle concentrations.Nanoparticles do not interfere with receptor binding or functionalization of bilayer lipids.Nanoparticle binding can locally induce phase transitions in lipid membranes.
Education and Outreach: The project has already contributed to the
training and development of four graduate students (Krishna Athmakuri, Jeffrey Litt, Chakradhar Padala, and Yan Yu), one undergraduate student (Andrew Devine) and a high school student (Kevin Crimmins). Students are introduced to an interdisciplinary research environment and gain expertise in topics ranging from soft materials to nanotechnology, biophysics, transport phenomena, and biomaterials. Further training is provided through outreach efforts, such as presentations to high school students in the New Visions High School Program and to a high school teacher, Ms. Tammy Borland.
Confocal Images of GUVs containingi)5% and ii-iv)20% cholesterol.
Characterization by FRET of peptide clustering due to cholesterol dependentphase separation
Controlling Biomolecule Stability and Recognition Using Raft-Mimetic Lipid Bilayers
Actively Induced Phase Separation
Enhanced efficiency of recognitionusing raft-mimetic liposomes
Phase separation leads to a 2 order of magnitude increase in polyvalent recognition
Phase separation provides a general mechanism to increase efficiency of polyvalent recognition
Confocal image of Ca2+
induced phase separation of a GUV
Peptide-functionalizedlipid
Gel-Phase lipids
Fluid-Phase lipids
Angew. Chem. 119, 2257 (2007)
No cholesterol
5% cholesterol20% cholesterol
0
0.1
0.2
0.3
0.4
21 42 54 63 84
Diff
usiv
ity(
m2 /s
ec)
# of bases of ssDNA (N)
Novelty:Actively reconfigurable, nanostructures to control biomolecule adsorption and transport. This biologically-inspired approach seeks to implement, in the bioseparations context, the concept of lipid rafts.
Transformative potential: The field of bio-separations is limited by use of passive surfaces. Our goal here is to remove this limitation. To accomplish this task, fundamental underpinnings are under development, those needed to design reconfigurable nanostructured surfaces that enable the separation of biomolecules in a manner that is far more facile and efficient than conventional strategies.
Potential Impact on Industry and Society: The understanding of biomolecule recognition and transport provided by this work will impact the design of novel technologies for biosensing, bioseparation, drug delivery, as well as the design of novel therapeutics. The project will also contribute to the training of graduate, undergraduate, and high school students and expose them to a stimulating interdisciplinary research environment.
Novel materialsTransportRaft-mimetic bilayers
A + + +
DNA mobility on heterogeneous bilayersDNA adsorption and diffusion on heterogeneous bilayers
Fluorescence micrographs of DNA adsorbed on bilayers consisting of: (a) 10 mole% DSPC and (b) 30 mole% DSPC. (c) Diffusivity of DNA adsorbed on heterogeneous supported bilayers as a function of mole% of the gel-phase lipid DSPC in the bilayer.
DNA electrophoresis on heterogeneous bilayers
Fluorescence micrographs of 84mer ssDNA labeled with Texas Red dye in the presence of an electric field. (a) t = 0 min, (b) t =8 min and (c) t =16 min
DNA diffusivity (D) decreased with size (N) whereas drift velocity is nearly independent of N.
0
0.1
0.2
0.3
0.4
0.5
21 42 54 63 84
Dri
ft ve
loci
ty (
m/s
ec)
# of bases of ssDNA (N)
Isothermal titration calorimetry shows the affinity of nanoparticles to lipid membrane depends on the surface charge of particles. The binding can be divided into two categories: enthalpy driven, and entropy driven, accordingly. The binding strength is >>kT.
Positively charged particles
0 100 200 300 400 500 600 7000
200
400
600
800
1000
1200
H (
kcal/m
ol)
cnanoparticle
/cliposome
0 40 80 120
0.0
0.5
1.0
Heat
flo
w (
cal/sec)
Injection sequence (min)
0 100 200 300 400 500 600
-5000
-4000
-3000
-2000
-1000
0
1000
2000
H (
kcal/m
ol)
cnanoparticle
/cliposome
0 50 100 150 200 250 300-1.5
-1.0
-0.5
0.0
0.5
Heat
flo
w (
ccal/sec)
Injection sequence (min)
C
A
Negatively charged particles
Enthalpy driven
Entropy driven
0 300 600
0.48
0.52
0.56
0.60
GP
cnanoparticle
/cliposome
0 100 200 300
-0.3
0.0
0.3
0.6
cnanoparticle
/cliposome
Gen
era
l p
ola
rizati
on
400 450 500 550 6000.0
0.5
1.0
Em
issio
n (
a.u
.)
Wavelength (nm)
0100200600
B
A
Dielectric environment sensitive fluorescence demonstrates the ability of nanoparticles to locally induce liquid-gel phase transition in lipid membranes, which is the driven force for particle binding.
Negatively charged particles, Liquid-gel
Positively charged particles,Gel->liquid
Advance made here: particle binding modulate the membrane structure significantly In progress: particle packing and induced local curvature
gel
θ-+
liquid
+- θ- +
electrostatic
Particle binding induces local phase transition in lipid membranes
PNAS ,105, 18171-18175 (2008)
B
D
Advance made here: cationic nanoparticles stabilize phospholipid vesicles up to dense volume fractions without fusion, allowing ligand-receptor binding.
In progress: interactions with DNA, especially plasmid DNA.
Soft Matter 3, 551 (2007) J. Phys. Chem. C 111, 8233 (2007)
Fluorescence autocorrelation functions showing that streptavidin binds effectively to vesicle-attached biotin even when nanoparticles stabilize the liposomes to which biotin is attached.
0.1 1 101E-3
0.01
0.1
1
MS
D/ t
(m
2 / sec
)
Time (sec)
500 nm
Δπ=300 mM
Δπ=400 mM
t=0 min
t=20 min
Δπ=200 mM
Δπ=100 mM
0 100 200 300 400 500
0.0
0.2
0.4
0.6
0.8
1.0
Nor
mal
ized
Inte
nsity
Osmotic Pressure (mM)
Naked GUV Stabilized GUV
Epifluorescence images of naked GUVs (top)
and nanoparticle-stabilized GUVs (bottom) reveal enhanced osmotic stress tolerance for the latter.
Nanoparticle-stiffened phospholipid vesicles
DiI FITC-SBP Merged
Heterogeneous (rapid quench)Heterogeneous (slow quench)Homogeneous
Enzyme Adsorption onto Raft-Like Domains Increases Stability
JACS 131, 7107 (2009)
Adsorption of SBP onto small domains (5 nm) imparts greater stability than larger domains (13 nm)
NIRT: Actively Reconfigurable Nanostructured Surfaces for the Improved Separation of Biological Macromolecules
Ravi Kane, Steve Granick, and Sanat Kumar, CBET - 0608978
Particle binding induces charge separation and slaved motion
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