Supported Phospholipid Bilayers: Ellipsometry, the FCS Zscan, and Timeresolved FCS

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The compaction mechanism of intermediate-sized
DNA molecules elucidated by fluorescence lifetime
correlation spectroscopy Relevance of DNA
compaction to Non-Viral Gene Therapy
Martin Hof
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Outline

• What is not known regarding the mechanism of DNA
  compaction? Relevance to Non-Viral Gene Therapy
• Confocal Fluorescence Correlation Spectroscopy,
  Dual-Colour FCS and Fluorescence Lifetime CS
• Compaction mechanism of intermediate-sized DNA
  elucidated by FLCS

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1a. What is known on the mechanism of DNA
compaction?
The term DNA compaction or DNA folding is
reserved to describe changes in a conformational
state of the DNA chain at the level of single
molecule.
Makita, Yoshikawa Biophys Chem 22 42 (2002)
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In viruses, bacteria, prokaryotes, and sperm
cells, the DNA chain is packed into extremely
dense structures by polyamine molecules such as
spermine (4), spermidine (3), cationic polymers
(protamines), etc..
Such a tightly packed state is used in living
systems for storage of long DNA molecules and the
DNA chain in these condensates is not accessible
for biological operations as transcription or
replication.
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Shape of condensed forms is characterized
Yoshikawa The shape of condensed forms depends
on DNA, condenser, and conditions Forms
characterized by Cryo-EM
(A) toroid T4 DNA spermine (B) rod - Lambda
DNA spermidine (C) racket - Pig liver DNA
spermidine (D) globule - T4 DNA condensed by
chiral dication (E) giant toroid - T4 DNA
spermidine at high monovalent salt concentration
(F) multitoroid - T4 DNA pteridinepolyamine
conjugate (G) D-shaped - T4 DNA spermine
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Compaction mechanism of a large-sized DNA
molecule linear bacteriophage T4 DNA (166 kbp)
was elucidated by single molecule fluorescence
microscopy
Yoshikawa
900 nm 90 nm (hydrodyn. radius)

• Compaction of DNA chain by multivalent cations is
  a
• non or all transition
• There are no intermediate states between unfolded
  and compact DNA conformations.
• Large discrete transition in a single DNA
  molecule appears continuous in the ensemble

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In contrast to such a significant change in
linear giant DNA, it is still unclear how
circular and/or intermediate size DNA folds into
a compact state! Most of theDNA in prokaryotes
has a circular structure. Even for eukaryotes,
the transcriptionally active part of giant DNA is
considered to in a circular Structure The
compaction of DNA is extensively studied in vitro
as it has a potential application in constructing
DNA carriers for a non-viral gene therapy

• Why is the mechanism still unclear?
• Even the unfolded DNA molecules are smaller or
  close to the resolution of an optical
  fluorescence microscope
• No single molecule data available
• Mechanism e.g. of a circular 10 kbp plasmid
  unkown.
• ?Non or all transition or Continuous?

Motivation for FLCS
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1b. Relevance of DNA compaction to Non-Viral Gene
Therapy
The compaction of DNA is extensively studied in
vitro as it has a potential application in
constructing DNA carriers for a non-viral gene
therapy
DNA as a drug
With gene vectors, DNA can be formulated to be
a medicine
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Introduction to gene therapy and its strategy
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Non-viral gene therapy formulation
Polyplex (cationic polymer-nucleic acid complex)
Poly-L-lysine
Lipoplex (cationic lipid-nucleic acid complex)
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DNA with negatively charged phosphate groups
-
Relevant DNA size is about 4,000 - 16,000 bp (2.6
to 10.4 x 106 Da)
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DNA with negative phosphate groups is neutralized
by polycationic lipopolyamines
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DNA with polycationic lipopolyamines
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Nanoparticles were formed by DNA and polycationic
lipopolyamines














diameter size 50 150 nm
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DNA nanoparticle enters the cell by endocytosis
process
Mitochondria
Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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Early endosome is formed by the bilayer cellular
membrane and carry DNA particles
Mitochondria
Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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Protons are pumped into the vesicles, to become
the late endosome with pH 5.5
Mitochondria
H
H
H
H
Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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Endosome is swollen due to water influx from the
proton pump process
Mitochondria














Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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Endosome membrane was ruptured due to the vesicle
swelling
Mitochondria














Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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DNA particle was released and escaped from
endosomes
Mitochondria


















Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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Delivered DNA is now in cytoplasm
Mitochondria
Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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DNA enters the nucleus through NPC with the
support from signaling protein
Mitochondria
Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
The nuclear envelope is perforated by thousands
of nuclear pore complexes (NPCs) that control the
passage of molecules in and out of the nucleus
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DNA dissociates from polyamines and expresses
the encoding protein
Mitochondria
Final protein product
Ribosome and Endoplasmic reticulum (ER)
Lysosome
Cytoplasm
Cell membrane
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It is still unclear how circular and/or
intermediate size DNA folds into a compact
state!

• Mechanism e.g. of a circular 10 kbp plasmid
  unkown.
• ?Non or all transition or Continuous?

Motivation for FLCS
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2a. Principles of confocal Fluorescence
Correlation Spectroscopy

• signal fluorescence (auto or labeled)
• confocal microscope ?illuminated fl volume
  element
• concentration of dye or labeled molecule in nM
  range
• ? single molecule

• Under this single molecule condition diffusion
  in and out of the focus lead to large
  fluctuations in fluorescence signal
• Monitored time evolution of fluorescence
  fluctuations (in the case discussed in this talk
  due to diffusion)

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AutoCorrelation Function
Translational diffusion fitting experimental
ACF

• Single component Works very well
• Multiple components j Strong limitations

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2b. Dual-color FCS
Designed for multicomponent diffusional
analysis Components must have different spectral
properties (i.e. two dyes differing in excitation
and emission) Autocorrelation functions
Parallel monitoring of concentrations and
diffusion characteristics of differently
labeled species Cross-correlation function
Interaction of the components Dual-beam or
two-photon excitation setup with several detectors
Separation by optical filters!
Two dyes!
Schwille, P. MeyerAlmes, F. J. Rigler, R.,
Biophysical Journal 1997, 72, (4), 1878-1886.
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2c. Fluorescence Lifetime CS - FLCS
Designed for multicomponent diffusional
analysis Components must have different excited
state decays (e.g. two dyes differing in
fluorescence lifetime) ACFs Parallel
monitoring of concentrations and diffusion
characteristics of labels with different
lifetime Cross-correlation Fs Interaction of
the components One pulsed laser and Time
Correlated Single Photon Counting detection
Separation by statistical lifetime filters
calculated from individual and overall decay
patterns
Böhmer, M. Wahl, M. Rahn, H. J. Erdmann, R.
Enderlein, J., Chemical Physics Letters
2002,353,(5-6),439-445.
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Schematic setup for FLCS measurements
Sample
Confocal Microscope
Objective
Expansion lens
Excitation filtr
Dichroic mirror
Pico-second pulsed laser with MHz repetition
frequency
Emission filter
Data storing card in Time-Tagged Time-resolved
mode
Lens
Detection pinhole
Fast efficient single photon detector
Benda A, Hof M, Wahl M, Patting M, Erdmann R,
Kapusta P TCSPC upgrade of a confocal FCS
microscope REVIEW OF SCIENTIFIC INSTRUMENTS 76
(3) Art. No. 033106 MAR 2005
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Time-Tagged Time-Resolved (TTTR) measurement mode
Fluorescence photon
Laser pulse
Time-Resolved
2480
1240
3120
Nanosecond-Time ps
Time-Tagged
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Mathematical filters for a two component mixture
Measured histogram linear combination of two
lifetime components
W(1) and W(2) are total number of photons for
each lifetime component
? filters fj(i) coefficients for every lifetime
component i and every TCSPC channel j
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Separation of fluctuation traces by lifetime
filters
Statistical (mathematical) filters are applied on
the whole data file to reconstruct
virtual-intensity traces for every lifetime
component
Auto-correlation Cross- correlation Auto-cor
relation
2 ns comp
Mixture
5 ns comp
Kapusta P, Wahl M, Benda A, Hof M, Enderlein J.
 JOURNAL OF FLUORESCENCE 17 (1) 43-48 JAN 2007
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Dual-color FCS x FLCS
Designed for similar purpose Different principle
and realization
Immense advantage of FLCS SINGLE DYE
LABELING Microenvironmental change can
hardly induce a large spectral shift easily
change a lifetime
FLCS has the potential to use one dye for
monitoring two environments First example Benda
et al., Langmuir 2006 in SLBs
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3. Compaction mechanism of an intermediate-sized
DNA Spermine induced condensation of Picogreen
labeled DNA (10 kbp)
Makita, Yoshikawa Biophys Chem 22 42 (2002)
Is FLCS suitable for monitoring the
condensation? Does FLCS give us information on
the mechanism?
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Titration of PicoGreen labeled circular DNA
(10 kbp) by spermine
Spermine-DNA nanoparticle (folded state) is
diffusing much faster than DNA with a length of
several ?m Apparent PN before condensation much
larger than PN calculated from used DNA
concentration, after condensation apparent PN
close to concentration. Why??
Kral T, Hof M, Langner M BIOLOGICAL CHEMISTRY
383 (2) 331-335 FEB 2002
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FCS is detecting fluctuations only if particle
is much smaller than volume, fluctuation equals
concentration
Adjimatera N, Kral T, Hof M, Blagborough I.
PHARMACEUTICAL RESEARCH 23 (7) 1564 2006
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Fluorescence lifetime changes from 4.2 to 3.5 ns
Picogreen sees in the folded state a different
microenvironment

• Lifetime change is the pre-requisite for FLCS
• FLCS experiments at the titration midpoint
  possible

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PN- normalised ACFs obtained by lifetime filters
(3 ns and 4ns) at the titration midpoint compared
with individual recorded ACFs for condensed and
uncondensed form
The ACFs correspond to the ACFs of the free and
condensed DNA measured independently (except a
small difference on the ms scale)? most of the
molecules are present in the totally collapsed
and non-collapsed state (all or none
transition)
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ACFs and CCFs obtained by lifetime filters (3
ns and 4ns)
ms
-the non-one CCFs suggest that apart from
coexistence of folded and unfolded DNA,
additional processes can be expected -inspection
of CCFs suggests a dynamic interchange in the
range of milliseconds -model assuming ms
dynamics and considering multiple labeling shows
that about 5 separated domains within DNA
collapse independently
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Spermine (4) versus HTAB (1)

• In case of HTAB lifetime pattern of the pure
  condensed form is difficult to determine
  (compaction end point prior to aggregation is not
  existing)
• ? Different Approach
• a) Lifetime patterns of uncondensed form (easy to
  obtain) TTTR file at a spermine/basepairs ratio
  of interest
• b) filtering out of the ACF of uncondensed form
  
• ?ACF of the sum of all condensed forms
• ? Direct characterization of the properties of
  the condensed form at different
  spermine/basepairs ratios

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ACF of compacted/condensed DNA by spermine
circular 10 kbp
spermine/basepairs
orange uncondensed form for comparison
1
before lifetime change
1.25 titration midpoint
2.5 compaction complete
4 aggregation
the size of the condensed particles after the
onset of the compaction does not depend on the
amount of added spermine ? all-or-non transition
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ACF of compacted/condensed DNA by HTAB
circular 10 kbp
spermine/basepairs
orange uncondensed form for comparison
2 before lifetime change
3.5 ongoing lifetime change
4 ongoing lifetime change
6 constant lifetime aggregation
continuous shortening of the diffusion time ?
condensation in smaller domains stabilized by the
hydrophobic interaction of the aliphatic chains
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Summary for FLCS on 10 kbp DNA

• Condensation process dramatic decrease in tD
  and PN
• Condensation leads to change in fluorescence
  lifetime
• Spermine FLCS at titration midpoint shows
  coexistence of both forms Non-or-all transition
• Spermine At this point a small population of the
  DNA molecules undergo gradual conformational
  change on the ms time scale
• Continuous mechanism for HTAB
• Same conclusion for linear 49 kbp DNA

Humpolickova et al, Biophys J 2008 , J Fluor
2008, submitted
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Summary for FLCS and Non Viral Gene Therapy

• Condensation process dramatic decrease in tD
  and PN
• Condensation leads to change in fluorescence
  lifetime
• Mechanism of DNA compaction can be elucidated by
  FLCS
• ? FLCS is a very useful tool when developing
  chemicals (condensers for NVGT

Adjimatera N, Kral T, Hof M, Blagborough I.
PHARMACEUTICAL RESEARCH 23 (7) 1564 2006
45
Background Gene Therapy Liposomes as DNA
Carrier

• DNA has to be encapsulated in liposomes
• Intercellular distance in cancer tissue is larger
• Liposomes accumulate in cancer tissue
• Liposomes give the DNA to cancer cells
  (Endocythosis) and thus initiate the
  programmedcell death

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Background Gene Therapy Liposomes as DNA
Carrier

• Liposomes have diameter of 100 nm
• DNA can be 3000 nm long in its native form
• Positive charged detergents induce DNA collapse
  (3000 nm long wurm turns into 50 nm diameter
  gobular system)