anopore Technology for the Analysis of Proteins and DNA Liviu Movileanu

Transcription

1 anopore Technology for the Analysis of Proteins and DNA Liviu Movileanu

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5 Courtesy Alek Aksimentiev

6 Single-channel electrical recording Current V = 4 mv 7 ps = 3 pa 1 M KCl cis trans

7 Single-molecule stochastic sensing frequency of events ---> concentration of analyte specific signature: t, g conductance etc. ---> identity of analyte alternative single-molecule detection and manipulation techniques ---> single-molecule fluorescence, single-molecule force spectroscopy

8 Kinetic information is determined from the single-channel electrical trace t on = 1/k on [A] t off = 1/k off K d = k off / k on

9 3.4 kda biotinylated PEG Courtesy A. Aksimentiev S.M. Bezrukov, I. Vodyanoy, V. Adrian Parsegian, 1994, Nature 37, L. Movileanu, S. Howorka, O. Braha and H. Bayley, 2, Nature Biotechnol. 18,

10 Distinct signatures for each type of capture Analyte/cis A Analyte/trans B

11 Quantification of the protein analyte at nm concentration 3 nm nm 2 1/ton(s-1) Current nm W12A streptavidin (nm) 2 Get kon, koff, Kd 1 1 s Events are detected in the microsecond timescale

12 DNA hybridization within a nanopore S. Howorka, L. Movileanu, O. Braha and H. Bayley, 21, Proc. Natl. Acad. Sci. USA 98(23),

13 Inverse Phase Transition (VPGXG) n High Temp Low Temp Transition temperatures can be fine tuned from 2 to 4 o C by controlling the chain length, its sequence and salt concentration

14 A temperature-responsive protein pore Cold Warm Y.H. Jung, H. Bayley and L. Movileanu, 26, J. Am. Chem. Soc. 128,

15 Control of confinement free energy of the peptide by rational protein design Current Current Current Current All-points histogram All-points histogram All-points histogram All-points histogram A 2 No peptide inside the vestibule; large conductance, no flickering events W 7 B Time (ms) Current Short peptide inside the vestibule; low conductance, fast flickering events 1 4 E5 1 W Medium peptide inside the Time vestibule; (ms) lower open-state conductance, Current long-duration blockades C E1 1 W 6 D Time (ms) Current Long peptide inside the vestibule; lowest open-state conductance, stacked polymer 1 4 E2 1 W Time (ms) Current 8 mv, 2 M KCl, 1 phosphate buffer, ph 7.4

16 Current All-points histogram Current All-points histogram Current All-points histogram Temperature-dependent single-channel activity A E1 1 W C B Time (ms) Current C C Time (ms) Current C Time (ms) Current

17 How will we be able to thread single proteins through a nanoscopic pore? L. Movileanu et al., 29, Trends Biotechnol. 27,

18 Catalyzing the translocation of polypeptides Electrostatic traps: rings of negative charges cis 1 Å cap K147D barrel K131D 2 Å trans Courtesy Alek Aksimentiev A.J. Wolfe, M.M. Mohammad, S. Cheley, H. Bayley and L. Movileanu, 27, J. Am. Chem. Soc. 129,

19 cis trans Wild Type K131D 7 K147D 7 Current Current Current Current K131D 7 /k147d ms 1 ms 1 ms Syn B2 1 ms A.J. Wolfe, M.M. Mohammad, S. Cheley, H. Bayley and L. Movileanu, 27, J. Am. Chem. Soc. 129,

20 Time (ms) 4 Insertion of DNA in a Nanopore 3 DNA in Pore Current (na) Position (µm) F k ( Z ) 1 Z U. F. Keyser et al. Nature Physics 2,473 (26)

21 Redesign of a protein nanopore from scratch a L4 Extracellular b L5 L3 L11 65 Å Cork Periplasm 31 Å 44 Ǟ M. Mohammad et al., J. Biol. Chem. 286 (211) 8; M. Mohammad et al., JACS 134 (212) 9521

22 Current Number of insertions The redesigned large-conductance nanopores are uniform c WT FhuA 3.6 nm FhuA ΔC/ Δ4L d kda MW Detergent Boiling LPhC OG DDM Folded 64 Unfolded ns +4 mv 2 1 lower conductance higher conductance Conductance (ns)

23 Is the engineered FhuA better than -hemolysin? a b c HL 1.2 FhuA C/ 4L FhuA C/ 4L 2 Normalized current IN 1 (C) IN 3 IN pa 12 5 pa s 2 1s IN 15 (mv) Low salt 2 mm KCl, ph HL d e FhuA C/ 4L s pa IN 3 IN 3 23 pa 1 3s Low ph 1 M NaCl, ph Normalized current f HL 1 1. FhuA C/ 4L HL

24 Amplitude (pa 2 /Hz) Amplitude (pa 2 /Hz) There is still an imperfection: the low-frequency noise g Low salt 2 mm KCl, ph 7.4 Low ph 1 M NaCl, ph 3.5 h.1.1 1E-3 FhuA C/ 4L.1 HL FhuA C/ 4L HL 1E E k 1k Frequency (Hz) 1E-3 1E k 1k Frequency (Hz)

25 IN3 2 IN3 IN3 IN3 I N 3 Current ( p A ) T i m e ( s ) Normalized frequency c _ + Pepsin Time e IgG FhuA C/ 4L Time(s) 225 Time(s) cis trans 2 min 5 s 3 min 2 s Open IgG interaction Pepsin + IgG d Pepsin Time (min) Digestion (%) f Pepsin addition min 3 s - 15 min + IgG + pepsin Time (min) min 48 s 68 min 18 s Time(s) IgG + pepsin 8 3 sec 6

26 12 pa IN 3 IN 3 F/F₀ (%) IN 3 Current a T G G G 1 T T 15 C G G C C G A T 5 T A 2 C G A T G C 5` G C 3` T T G C 1 T A15 C G G C C G A T 5 T A 2 C G A T G C 5` G C 3` C A T A 1C C15 C G G C C G A T 5 T A 2 C G A T G C 5` G C 3` DNA 1 DNA 2 DNA 3 b _ + + charged - charged - charged cis Lipid bilayer trans Open HIV-1 NCp Time FhuA C/ 4L / NCp7 interaction c FhuA C/ 4L 3 4 d DNA DNA 3 DNA NCp7 4 2 DNA NCp7 + DNA 1 (1:2) DNA/NCp sec

27 Silicon Nitride-based Nanopores in a tiny sub 1-nm thick membrane A B 5 nm 1 nm C D 5 nm 1 nm

28 Single-Molecule Captures of Proteins With a 9 nm-diameter nanopore, the long-lived captures of BSA proteins have a noticeable effect on the frequency of the short-lived BSA-induced events Li and Talaga, JACS 131 (29) 9287 Yusko et al., Nature Nanotechnoloy 6 (211) 253

29 Sampling a Protein Biomarker of the HIV-1 K E G A H K E G G I G H C Q C A M N Zn K K Zn W K F N C C C C D K R K T MQKGNFRNQRKTV APRKK ERQAN RNA NCp7 2 nm D.J. Niedzwiecki et al., ACS Nano, 213, In Press.

30 Current IN 3 Test case for small nanopores (< 6 nm) IN 3 Normalized Count Count (N) nm SL3 (GAG) 5 nm SL3 (GAG) & 14 5 nm NCp7 4 s Dwell Time time (ms) Inter-event Time (ms)

31 Test case for large nanopores (> 15 nm) Current Amplitude IN 3 Current IN SL3 (GAG) SL3 (GAG):NCp7 (1:1) s SL3 (GAG):NCp7 6 SL3 (GAG) SL3(GAG):NCp7 (1:1) 4 2 SL3 (GAG) Dwell time (ms)

32 Test case for drug-protein biomarker interactions Current IN 3 IN 3 IN : SL3 (GAG) : NCp7 : NEM 56 1: SL3 (GAG) 2: NCp7 : NEM 56 1: SL3 (GAG) 2: NCp7 12: NEM s D.J. Niedzwiecki et al., ACS Nano, 213, In Press.

33 One-billion dollar question! FhuA FOTS dsdna SiN PDMS SiN SiO 2 Si Hall et al., Nature Nanotechnology, 5 (21) 874.

34 Summary Current Stimuli-responsive nanostructures Polypeptide translocation/sensing K147D Lipid bilayer K131D cis - trans + Low Targeting sequence Globular domain Open Closed Time High Other -barrel protein pores/ Single-molecule science Extracellular P Periplasmic Single-molecule & membrane science of ABC transporters What s next? Nanomedicine, DNA/RNA aptamers, nanofluidics. Other stochastic sensors & synthetic biology

35 David J. Niedzwiecki Syracuse U. Aaron J. Wolfe Mohammad M. Mohammad Carl P. Goodrich Khalil R. Howard Belete R. Cheneke Noriko Tomita Nan Qin Avinash Kumar Thakur Some movies/pictures in this talk Courtesy Alek Aksimentiev, Hagan Bayley and Cees Dekker Andreas Matouscheck U. Texas at Austin Sumit Prakash Dmitrii Makarov U. Texas at Austin Serdal Kirmizialtin Makoto Ohta Tohoku Univ. Noriko Tomita Phil Borer Syracuse U. Mark McPike Raghu lyer Valerica Raicu U. Wisconsin at Milwaukee Julie Oliver Mathias Winterhalter Jakobs U. (Bremen) Catalin Chimerel Bert van den Berg U. Mass Medical School, Worchester National Science Foundation National Science Foundation National Institutes of Health National Institutes of Health - DMR16332 National Biomaterials Initiative; - AGEP-NYU-PR (through Gina Lee-Glausser) - R1 GM85785 (Sub-Contract with U. Mass. Medical School) - R1 GM8843 (National Institute of General Medical Sciences)