Chemically modified nanopores as tools to detect single DNA and protein molecules Stefan Howorka Introduction Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding 1
Introduction Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding Sensing principle current time Stochastic sensing by single channel current recording 2
Single channel current recording The protein pore α-hemolysin (αhl) Bacterial exoprotein Forms heptameric pore 3
Introduction: Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding Generation of a DNA-nanopore cis Cys 17 internal cavity inner constriction transmembrane β-barrel trans αhl modified with a single oligonucleotide attached to an engineered cysteine residue 4
Detection of individual DNA strands by duplex formation inside the pore Howorka, S., Movileanu, L., Braha, O., Bayley, H. (2001). Proc Natl Acad Sci U S A 98, 12996-3001 Introduction: Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding 5
A single mismatch abolishes binding of a DNA strand to the tethered oligonucleotide Howorka, S., Cheley, S., Bayley, H. (2001) Nature Biotechnol 19, 636-39. Sequencing of a complete codon in a single DNA strand tethered to αhl αhl-ss-5 -GCATTCX 5 X 6 X 7-3 serial addition of oligonucleotides of known sequence CGTAAGZ 5 DNA duplexes with matching base pairs have longest lifetimes infer sequence of unknown codon Howorka, S., Cheley, S., Bayley, H. (2001) Nature Biotechnol 19, 636-39. 6
Detection of a mutation in individual DNA strands derived from HIV Detection of common mutation in the reverse transcriptase gene, which confers resistance to the antiviral drug novirapine Kinetics of DNA-duplex formation at the single-molecule level τ on τ off k on = 1 / τ on-mean conc k off = 1 / τ off-mean K d = k off / k on 7
N u m b e r o f e v e n t s Good agreement between singlemolecule and bulk-phase DNA-duplex affinities values derived from nanopore recordings values derived from melting profiles in solution oligo sequence K d [M] K' d [M] oligo-b 5'-GGTGAATG-3' 9.2 x10-8 3.6 x 10-8 oligo-d 5'-TACGTGGA-3' 1.5 x 10-7 1.7 x 10-7 oligo-e 5'-GGTGAAT-3' 1.6 x 10-6 8 x 10-7 DNA-nanopore recording yields details of kinetics difficult to obtain with conventional methods 100 10 τ off-2 Number of events 100 10 τ off-1 τ off-2 1 2000 4000 1 200 Event lifetime [ms] Event lifetime [ms] τ off Lifetime histogram of oligo-b binding events reveals two different types of events Howorka, S., Movileanu, L., Braha, O., Bayley, H. (2001). Proc Natl Acad Sci U S A 98, 12996-3001 8
Thermodynamic data for duplex formation obtained via T-dependence of k on and k off 3.0 k on [M -1 s -1 ] x 10-7 2.5 2.0 1.5 1.0 0.5 O k off k on 10 1 0.1 k off [s -1 ] 10 15 20 25 30 35 40 temperature [ C] weak T-dependence of k on and strong dependence of k off in line with ensemble measurements for duplex formation in solution get activation and equilibrium enthalpies and entropies Introduction: Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding 9
Can DNA-hybridization inside a pore be used as tool to probe the pore? Length DNA strands as molecular rulers? Potential how does transmembrane potential drop off inside the pore lumen? Test approach on protein of known structure Experimental approach Attachment of single oligo-a inside protein pore Channel insertion Addition of complementary oligos with 5 - extension of increasing length (da N -oligo-b and dt N -oligo-b 10
Signature of binding events changes with number, N, in da N -oligo-b 0 1 2 3 4 100 pa 200 ms 5 6 7 8-1 State A State B 20 ms Howorka, S., Bayley, H. (2002). Biophys J 83, 3202-10. Length dependence of binding signature in agreement with molecular modeling DNA-oligos are useful molecular rulers N = 2 : downward spikes indicate contact to inner constriction N 3: increasing current drop due to blockade of bottleneck inner constriction 11
Length of extension influences event lifetime da N -oligo-b Event lifetime [ms] 1000 100 long extensions (N>2) feel the trans-membrane potential drop in β- barrel of αhl 0 1 2 3 4 5 6 7 8 N = number of bases 3 3 5 5 + Dependence of voltage on event lifetime Event lifetime [ms] 100 10 da 8 -oligo-b dt 9 -oligo-b Event lifetime [ms] 2000 1600 1200 800 400 oligo-b oligo-d 100 120 140 160 180 200 Voltage [mv] 75 100 125 150 175 200 Voltage [mv] 12
Summary, DNA nanopores I detection of individual DNA strands with single-base resolution based on match/mismatch-dependent lifetimes of DNA duplexes kinetics and thermodynamics of DNA duplex formation at single molecule level good agreement with ensemble measurements kinetic details Summary, DNA-nanopores II DNA-hybridization inside protein pore to probe structure and distance to probe transmembrane potential Good agreement between current signature and known αhl structure Potential drops off at transmembrane region but not in cap 13
Introduction: Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding Sensing of protein analytes Streptavidin tetramer (60 kd) How to sense analytes too big to fit into the pore lumen? 14
Sensing of external analytes using a polymeric linker between ligand and pore Binding of streptavidin to biotin outside of pore Flexible PEG linker generates specific current signature Current flicker caused by reversible threading of free end of PEG chain into transmembrane barrel of pore Howorka, S., Movileanu, L., Lu, X., Magnon, M., Cheley, S., Braha, O., Bayley, H. (2000) J Am Chem Soc 122, 2411-2416. 15
Binding of streptavidin to biotinpolymer modulates current signature Polymer links outside binding to inside current modulation Movileanu, L., Howorka, S., Braha, O., Bayley, H. (2000) Nat Biotechnol 18, 1091-1095. Quantification of protein concentration W120A: Streptavidin mutant with lower binding affinity Movileanu, L., Howorka, S., Braha, O., Bayley, H. (2000) Nat Biotechnol 18, 1091-1095. 16
Recordings of ligand-polymer-modified nanopore provides kinetics at single molecule level Introduction: Sensing principle Overview Single channel current recording Protein pore Nanopores detect individual DNA strands Generation of DNA-nanopores Detection of individual DNA strands by duplex formation Sequence-specific detection Kinetics & thermodynamics of DNA duplex formation DNA-duplex to probe distance and structure of pore Nanopores detect single protein molecules Sensing of streptavidin outside the pore Lectin, multivalent binding 17
Probe multivalent protein ligand interaction with protein pore carrying multiple ligands Protein pore modified with up do seven disaccharide ligands for tetrameric lectin Obtain single-molecule kinetics of mono- and bivalent binding Observation of short, monovalent and long, bivalent events bivalent monovalent Deduce kinetic constants and binding constants for monovalent and bivalent binding Bivalent binding constant in line with ensemble measurements Howorka, S., Nam, J., Bayley, H., Kahne, D. (2004) Angew. Chem. Int. Ed. Engl. 43, 842-846. 18
Summary, protein analytes Detection of protein analytes via flexible linker Binding to ligand-polymer modulates current flowing through the pore Linking of outside binding to inside current change Single-molecule kinetics Multivalent interactions Heptamodified pore Kinetics of monovalent and bivalent binding of tetrameric lectin Department of Medical Biochemistry and Genetics College of Medicine The Texas A&M University System Health Science Center College Station, TX Hagan Bayley Orit Braha Steven Cheley Liviu Movileanu Jonwoo Nam, Daniel Kahne, Department of Chemistry, Princeton University Financial support from U.S. Department of Energy, National Institutes of Health Office of Naval Research Texas Advanced Technology Program Austrian Science Foundation Max-Kade Foundation 19
Generation of DNA-nanopores Generation of 35 S-labeled polypeptides of α HL cysteine mutant 17C with cell-free extract Modification with OPSS-oligo-A Addition of unmodified monomers (H) and assembly to heptamer on erythrocyte membranes Purification by SDS gelelectrophoresis, isolation of heptamer modified with one DNA oligo Autoradiographs of SDS-gels 20