Use of a Single Ion Channel to Analyze the Structure & Dynamics of Individual DNA Molecules. Mark Akeson

Transcription

1 Use of a Single Ion Channel to Analyze the Structure & Dynamics of Individual DNA Molecules Mark Akeson Biophysics Laboratory Dept. of Biomolecular Science & Engineering University of California, Santa Cruz OVERVIEW Basics of the nanopore device & some history Discrimination among Watson-Crick base pairs on single DNA molecules Base pair specific end fraying and HIV integration into chromosomal DNA Prospects for high speed sequencing Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz

2 Some History Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz Size Scales in Biological Science & Engineering Ecosystems Higher Organisms Single Cells Molecules (Meters) nanoscale Å Courtesy of Michael Isaacson

3 Single Molecule Nanopore Sequencing Single Channel Biophysics Solid State Pores & Detectors - Cl K + - Cl K + 1 ms

4 a) - Cl K + 1 second b) - ssdna cis trans ssdna dsdna

5 Basic Differences of our Nanopore Device Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz Single Channel Device A/D khz Four Pole Bessel 1-1 khz

6 1 mv 1 M KCl 7 µl µm film

7 Capturing DNA Hairpin Molecules Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz DNA Hairpins loop stem G C C G G C C G 5' 3' Senior, PNAS, v 85,1988

8 ssdna 13 Å dsdna 22 Å 12 polyc 5 mer 12 6bp DNA Hairpin 1 ms

9 a b c A B C 1 ms Tests of the Model: 1) Orientation of the hairpin molecules: stem first? 2) Dwell time predicted by stability in bulk phase? 3)Titration of the pore: Does the duplex stem fit as predicted? Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz

10 Do the Hairpin Stems Enter the Vestibule First as Modeled? 5 bp DNA Hairpin 5 3 Blunt end loop No blunt end 3 5 TA TA Loop Loop s

11 Dwell Time is Predicted by Duplex Stability Average Duration (log ms) 6 8bp 5 7bp 4 6bp 3 5bp 2 4bp 1 3bp D Gº Hairpin Formation (kcal/mol) 6bp 6b 14 loop stem DG : G C C G G C C G kcal/mol G C C G A A G C C G kcal/mol

12 A Base-Pair Mismatch Changes Hairpin Blockades 1% 52% 1% 6bp 6b 14 1 second 1 ms Average Duration (log ms) 6 8bp 5 7bp 4 6bp 3 5bp 2 1 3bp 6b D Gº Hairpin Formation (kcal/mol)

13 Titrating the Pore Vestibule at 3.4 Å Precision Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz

14 B form DNA 3.4 Å 3bp 12bp 22 Å 12 9 One 6bp hairpin molecule Duration (log ms)

15 bp hairpin 2.4 Å Duration (log ms) bp hairpin 6bp hairpin 1.2 Å Å Duration (log ms)

16 bp 4bp 5bp 6bp 7bp 8bp 1.2 Å 13.6 Å 17. Å 2.4 Å 23.6 Å Å Duration (log ms) Vercoutere et al.,nature Biotechnology 21 Stem Resistance in Gohms bp 11bp Stem Length in Base Pairs

17 ~36Å below hairpin loop to reach limiting aperture Discrimination Among Individual Watson-Crick Base-Pairs Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz

18 12 () 7bp 3 ms 7bp 12 () 9bp

19 T A T A C G 5 3 T A T A G C 5 3 T A T A T A 5 3 T A T A 5 3 AT TA Current () hairpin is captured Time (ms) 8 1

20 12 τ LL 9bp LL Number of observations bpTA t LL = 7.1 ms 9bp t LL = 171 ms Duration (log ms)

21 Mechanisms that can account for the Discrete current transitions caused by DNA hairpins 1) Sequence-dependent binding and orientation of the hairpin molecules 2) Dynamic changes in duplex structure Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz 3 prime bound end unbound 3+5 prime bound 12 UL IL LL 9bpC G 2

22 a b c d e 12 9bpC G LL LL S S 2 Observing the internal dynamics of single DNA duplexes: End fraying Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz 22

23 G kcal/mol T A T A C G C G C G T A T A T G C G 1 8 T G 1

24 Test of the fraying model: Do internal mismatches cause impedance spikes? Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz A A T A C G 5 3 A A T A T A C G 5 3

25 TT AT AT TA T A TT AT AA TA T A TT AT AT AA T A 1 spikes s spikes s -1 1 spikes s -1 Does a spike result from a frayed end reaching the limiting aperture? Lysine 147 Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz

26 2Å G:C C:G A:T G:C C:G A:T 31Å 31Å G:C C:G A:T G:C C:G A:T 41Å A:A T:A C:G 6bp A T C A A G 6bp frayed A:T G:C C:G A:A T:A C:G A:T G:C C:G A T C A A G 9bp 9bp frayed G:C C:G A:T A:T G:C C:G A:A T:A C:G G:C C:G A:T A:A T:A C:G 1 1 Both molecules spike at 12mV applied. 9bp 1 3 ms 1 6bp 3 ms 25 spikes s ms 2 spikes s ms

27 Lower Level dwell time (t LL ) calculation for 9bp hairpin (12mV) G:C C:G A:T A:T G:C C:G A:A T:A C:G 1 Counts (N) 1 t LL = weighted mean 15ms 3ms For 9bp hairpin: t LL =.35 ms log (LL Dwell time (ms)) G:C 1 C:G A:T A:T G:C C:G A:A T:A C:G Lower Level (LL)-to-spike dwell times 1 9bp LL dwell times 3 ms 15 ms 1 G:C C:G A:T A:A T:A C:G 3 ms 1 6bp LL dwell times 15 ms

28 Lower Level dwell times for 9 and 6 bp hairpins with AA/TA/ ends 1.2 t LL (ms) bp AA3rd y = -.121x bp AA3rd y = -.128x mv ~22Å below hairpin loop to reach strong electric field

29 An Application : Fraying of HIV DNA Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz DNA End Processing by HIV Integrase Diagram F.D. Bushman, Salk Institute

30 2 End Processing Value CA GT CAGT GTCA WT Stabilize CA GTCC Destabilize -1 1 Stability of Duplex End vs Wild Type (Kcal/mol)* 1 P. Brown & Coworkers; * Mfold (M. Zuker) Stabilized G formation (kcal/mol) Processing Value 5 CA GT (WT) 5 CAGT GTCA CA GTCC Destabilized

31 Observing the internal dynamics of single DNA duplexes: Sequence-Dependent Changes in End Dynamics Duplex Biophysics Laboratory, Biomolecular Science & Engineering, U.C. Santa Cruz

32 8 8 C C G A G A 2 8 C C GA T G G C 2

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34 High Speed Structure Analysis 6 bp NMR, X-RAY CRYSTALLOGRAPHY 1 Structure 1, min 4 6 Structures 7+ years NANOPORE 1 Structure 5 min 4 6 Structures 2 weeks Center for Biomolecular Science & Engineering, U.C. Santa Cruz High Speed DNA Sequencing Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz

35 High Speed DNA Sequencing Cost per finished bp in US Dollars E-3 1E Year High Speed DNA Sequencing Cost per finished bp in US Dollars $1 per mammalian genome E-3 1E Year

36 High Speed DNA Sequencing Cost per finished bp in US Dollars $1 per mammalian genome E-3 1E Year Single Molecule Sequencing U.C. Davis, 1991 Advantages: Long read length No cloning and replication

37 x 1 17 Translocations 1 Translocation 1 meter 1 x 1-8 meters 1 Volts / cm 1, Volts / cm Can we distinguish among polymers Of different base composition? Purines vs Pyrimidines Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz

38 Individual RNA Homopolymers Are Distinguishable Poly A Poly C µs/nt 3 µs/nt 12 Blockade Duration (ms) Current () Poly U. Poly A Poly C

39 A 3 C 7 A C 2 ms poly C 1.3 nm poly A 2.1 nm Arnott & coworkers Saenger & coworkers

40 Circular Dichroism of poly rc and poly dc Poly-rC Increasing Order Poly-dC Adler et al Biochemistry 7: Blockade Duration (ms) % of Open Current Poly dc 2 Poly rc Open Channel Current Poly dc Poly rc 11% 8% 4% 1 ms

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42 An Abasic 2mer Can Be Read Between ssdna Segments Abasic 2 µs Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz Limits on Precision of Single Strand Analysis Low S/N at 3 µs interval 12 nt in limiting aperture

43 Slowing Down Translocation Using Processive DNA Modifying Enzymes Biophysics Laboratory, Center for Biomolecular Science & Engineering, U.C. Santa Cruz

44 Exonuclease I Pro Phe Glu Thr 13 nucleotides 2 3 ms Enzyme bound to ssdna

45 Open Dwell Time (ms) Open Dwell Time (ms) λ Exonuclease 3 nm 1.5 nm 5 nm Rate: ~ 1 ms nt -1 Processivity: 1+ kilobases

46 1 8 () Time (ms) I/Io Duration (ms) () 6 4 I/Io Time (ms) Duration (ms) () 6 4 I/Io Time (ms) Duration (ms) () 6 4 I/Io Time (ms) Duration (ms)

47 U.C. SANTA CRUZ DAVID DEAMER** DAVID HAUSSLER** REBECCA BRASLAU W. VERCOUTERE V. DeGUZMAN** BRETON VEACH WESLEY SUGHRUE SAM RIDINO UNIVERSITY OF BRITISH COLUMBIA ANDRE MARZIALI JONATHAN NAGANE HARVARD UNIVERSITY DAN BRANTON LOS ALAMOS NATIONAL LABORATORIES PETER GOODWIN ANDREA SOLBRIG** HELMUT ZEPIK CLARENCE LEE ** FUNDING: HHMI, NHGRI, NSF, AGILENT