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Apr 8, 2009 ... http://www.cartage.org.lb/en/themes/sciences/Physics/ ... References: J. Lippincott-Schwartz, Nature Methods (2009); www.bioplek.org.
Dit zijn de slides van de oratie van Prof. dr. N.H. Dekker. Dit materiaal is uitsluitend voor persoonlijk gebruik bestemd. Voor overig gebruik van dit materiaal dient toestemming te worden verkregen van N.H. Dekker. Stuurt u in dit geval een e-mail naar [email protected] These are the slides accompanying Prof. dr. N.H. Dekker’s inaugural lecture. This material is intended for personal use only. For all other usage, permission from N.H. Dekker is required. Please contact [email protected] for further information.

http://nynkedekkerlab.tudelft.nl

De revolutie van een enkel molecuul The revolution of a single molecule Nynke Dekker 8 april 2009

Overview of this lecture • Why physicists and biologists, despite appearances, can work together • The molecular level in biology • How to measure the dynamics of a single molecule • Breakthroughs in single-molecule science

• Where are we going? • How to equip ourselves for the journey?

The approach of the physicist •

Reduce system to a simple one that can be manipulated and modeled & Try to derive general principles and laws



Related, there’s been a quest for looking for smaller and smaller constituents of matter

References: http://www.bu.edu/core/cc105/lectures/L07-E&M/HydrogenAtom.gif http://www.cartage.org.lb/en/themes/sciences/Physics/QuantumPhysics/ParticlePhysics/ParticlePhysics.htm CMS detector at CERN

Choosing a simple, isolated system in biology

Charles Darwin

Reference: Wikipedia

Biology starts to zoom in Object goes on pin here Focus knob

Antoni van Leeuwenhoek

Lens

References: J. Lippincott-Schwartz, Nature Methods (2009); www.bioplek.org

Quick refresher: Cells

Quick refresher: Size scales 0.3 nm

~10 µm

2 nm Human: 3,000,000,000 basepairs Onion: 18,000,000,000 basepairs

Ability to handle biological material has yielded spectacular advances The crystal structure of DNA

References: Watson and Crick, Nature (1953); Franklin et al., Nature (1953)

Ability to handle biological material has yielded spectacular advances The purification and crystallization of proteins

hemoglobin

Reference: Max Perutz and John Kendrew, Nobel Prize 1962.

Ability to handle biological material has yielded spectacular advances The enzymatic amplification of DNA

Reference: Kary Mullis, Nobel Prize 1993.

Single molecules

Single-molecule experiments on ion channels

Passage of ions through a channel that is alternatively open, closed

Reference: Neher and Sakmann, Nobel Prize 1991

Detection of single molecules within a solid Strategy: 1.Make molecules very cold, to reduce influence of the environment 2. Shine light on them 3. Detect via absorption or fluorescence (latter easier in practice)

Regime of single molecules

Reference: Moerner and Kador, PRL (1989); Orrit and Bernard, PRL (1990)

Reduce sample size

Pulling on Single Molecules Atomic force microscopy

Optical tweezers

Magnetic tweezers

Force ranges: Atomic force microscopy Optical tweezers Magnetic tweezers

References: K.C. Neuman and A. Nagy A, Nature Methods (2008).

10 – 10000 pN 0.1 – 100 pN 0.001 – 100 pN

Force (pN)

First experiment to pull on a single molecule

Extension (μm)

References: Smith et al., Science 1992; C. Bustamante et al., Science 1994.

First experiment to pull on a single molecule 100

1

Force (pN)

10

0.1

0

10

20

Extension (μm)

References: Smith et al., Science 1992; C. Bustamante et al., Science 1994.

30

0.01

Application: DNA-protein dynamics

Motor stops

Single-molecule techniques allow us to see variability between different molecules, influenced by their environment

References: Yin et al., Science 1995; schematic from Wang et al., Science 1998.

Number

Force at which the motor stops:

Twisting Single Molecules of DNA

References: Strick et al., Science (1996); Vilfan et al., Springer Single-Molecule Handbook of Biophysics (2009).

DNA-protein dynamics

References: Koster et al., Nature (2005); Koster et al., Nature (2007).

High-resolution experiments Following a motor walk along the bases of the DNA:

Basepair resolution achieved! References: M. Rief et al., Nature (1997); E.A. Abbondanzieri et al., Nature (2005).

Different Types of Single-Molecule Tools • Force Spectroscopy

• Fluorescence Imaging

• Patch clamping and pores

Detection Molecules Passing through Nanopores

ΔI

References: Chang et al., Nanoletters (2004); Storm et al, PRE (2005).

Distinguish Different Types of Molecules

Reference: Skinner, van den Hout, et al. (2009)

Why a Revolution?

Reference: Time Magazine, March 23, 2009, & Bureau of Labor Statistics

Numbers of papers in the PubMed database with “single molecule” in the title: Exponential growth with doubling time of 2.2 years Reference: W.E. Moerner, PNAS (2007)

Example 1: DNA Sequencing Staggering pace of innovation in human genome sequencing Year 2001a 2001b 2007 2008 2008 2008 2008 2009

Cost $300,000,000 $100,000,000 $10,000,000 $2,000,000 $1,000,000 $500,000 $250,000 $30,000

Technology Machine runs Authors Sanger (ABI) 251 Sanger (ABI) 100,000 274 Sanger (ABI) 100,000 31 Roche(454) 234 27 Illumina 98 48 Illumina 35 77 Illumina 40 196 Helicos 2 4

Coverage 4 5 7 7 33 36 30 10

References: (a) Venter et al., Science (2001); (b) Human Genome Project, Nature (2001). Table courtesy of Stephen Quake.

Development of Single -Molecule Sequencing Single-Molecule

2003: 4bp read length, ~ 30 templates,