lecture 2 application of optical tweezers in single-molecule experiments with dna

Lecture 2Application of optical tweezers in single-molecule experiments

with DNA

DNA and RNA stretching experiments

Entropic elasticity of a single DNA molecule

o

AP 34

A ... TG ... CC ... GT ... A . . .

Nucleotides Adenine, Guanine, Cytosine, Tymine

First experiment by Carlos Bustamante and co-workers Science (1992)

Optical Tweezers

Mazolli, Maia Neto, and Nussenzveig, Proc. R. Soc. London, Ser. A 459, 3021 (2003)Viana, Rocha, Mesquita, Mazolli, Maia Neto, Nussenzveig, Appl. Phys. Lett. (2006) and PRE (2007)Neves, Fontes, Pozzo, Thomaz, Chilce, Rodriguez, Barbosa, Lenz-Cesar, Optics Express (2006)

A. Ashkin, Phys. Rev. Lett. 24, 156 (1970)

Recent theoretical and experimental advances

Visible He-Ne back-scattering profile

4

1

14

12

LzL

z

A

kTF

Marko-Siggia equation

z= end-to-end distanceA= persistence lengthL= contour length

S. Harris et al. BJ 2005 (large scale MD, entropic effects, denaturing bubbles);

G. Weber et al. Nature Phys., 2009 (microscopic elasticity).

Force x extension curve

DNA/Ethidium BromideFit to the neighbor exclusion model

Ethidium bromide Daunomycin

M. S. Rocha et al. JPC 2007

Psoralen

Circles: intercalated

Squares: intercalated +UVA light

Psoralen

Márcio. S. Rocha,doctoral thesis UFMG (2008)

ni

n

bpPnK

nCC

1

1 1

14104.28.8

13.043.1

MK

n

i

77.01

64.0 max

n

0

0

L

LL

neighbor exclusion model

Psoralen intercalated fraction

Persistence length abrupt transition around r ~ 0.4

r = concentration of drug bound/concentration of DNA base-pairs

r=0.4 r=0.5

ab initio DFT calculation DNA/Psoralen

ab initio DFT calculation DNA/Psoralen

“DNA-psoralen: single-molecule experiments and first principles calculations”M. S. Rocha, A. D. Lúcio, S. S. Alexandre, R. W. Nunes, and O. N. Mesquita (APL, 2009)

0

20

40

60

80

100

120

140

160

-2 0 2 4 6 8

cyclodextrin

Cc/Cbp

Water transport in a single MDCK kidney cell

How to measure the volume of a cell?

MDCK Kidney Cell

Isoosmotic medium Hiperosmotic medium

Osmoregulation

Effect of vasopressin (AVP) on water transport

Lucio, Santos, and Mesquita, PRE (2003)

Stretching DNA

Stretching RNA

The 3 Main Classes of Motor Proteins

DNA condensation

Fluctuation theorem

PRL, 050601 (2002)

MORPHOLOGY-DEPENDENT RESONANCES (MDR) MODES

Blood 92, 2975 (1998)

PRL 93, 078102 (2004)

Acustooptical deflectors

•Optical Tweezers is a very convenient tool to manipulate, apply and measure forces on single molecules (pN range). It can be used to measure and change transition states during protein folding, with access to the time evolution of these processes.

•It can be used for manipulating single cells for measurements of membrane elasticity, water transport and for manipulation of organelles inside the cell.

•It has been used as an interesting tool to test new ideas on Statistical Physics of small systems.

•It has been used for manipulation of nanowires, carbon nanotubes and silver nanoparticles.

•Applications of optical tweezers have increased much during the last few years.