Artificial nanopores and uses and methods relating thereto
20220412948 · 2022-12-29
Inventors
Cpc classification
G01N33/48721
PHYSICS
B82Y15/00
PERFORMING OPERATIONS; TRANSPORTING
International classification
B82Y15/00
PERFORMING OPERATIONS; TRANSPORTING
Abstract
The invention relates to the field of nanopores and the use thereof in analyzing biopolymers, including polypeptides and polynucleotides. Provided is an artificial nanopore comprising a multimeric assembly of subunits, each subunit comprising (i) the transmembrane (TM) sequence of a β-barrel or α-helical pore forming protein fused to the amino acid sequence of (ii) a subunit of a ring-forming protein capable of controlling the transport of a polypeptide or polynucleotide across the TM region of the assembly.
Claims
1. An artificial nanopore comprising a multimeric assembly of subunits, each subunit comprising: (i) the transmembrane (TM) sequence of a β-barrel or α-helical pore forming protein fused to the amino acid sequence of (ii) a subunit of a ring-forming protein which controls the transport of a polypeptide or polynucleotide across the TM region of the assembly.
2. Artificial nanopore according to claim 1, comprising the TM sequence of an α-helical pore forming protein, preferably the TM sequence of FraC, ClyA, AhlB or Wza (translocon for E. coli capsular polysaccharides).
3. Artificial nanopore according to claim 1, comprising the TM sequence of a β-barrel pore forming protein, preferably the TM sequence of α-heamolysin, aerolysin or anthrax protective antigen (PA).
4. Artificial nanopore according to claim 3, wherein the TM sequence comprises or consists of the amino acid sequence VHGNAEVHASFFDIGGSVSAGF.
5. Artificial nanopore according to any one of claims 1-4, wherein the TM sequence is N- or C-terminally fused to the subunit of a ring-forming protein.
6. Artificial nanopore according to any one of claims 1-4, wherein the TM sequence is inserted within the sequence of the subunit of a ring-forming protein.
7. Artificial nanopore according to any one of claims 1-5, wherein the TM sequence is flanked on the N- and/or C-terminal side by a flexible linker of at least 3, preferably at least 5, amino acids, more preferably wherein the N-terminal linker comprises or consists of the sequence GSS and/or wherein the C-terminal linker comprises or consists of the sequence SSG.
8. Artificial nanopore according to any one of claims 1-7, wherein the ring-forming protein is a heptameric protein.
9. Artificial nanopore according to claim 8, wherein the ring-forming heptameric protein controls the transport of a polynucleotide across the TM region.
10. Artificial nanopore according to claim 9, wherein the heptameric protein is an ATPase, preferably A. aeolicus ATPase or a homolog or functional equivalent thereof.
11. Artificial nanopore according to claim 8, wherein the ring-forming heptameric protein controls the transport of a polypeptide across the TM region.
12. Artificial nanopore according to claim 11, wherein the heptameric protein is proteasome activator PA28, PA26, or a homolog or functional equivalent thereof.
13. Artificial nanopore according to any one of claims 1-12, wherein the C-terminus of the subunit of the ring-forming protein comprising the TM sequence is genetically fused to the N-terminus of a proteasome α-subunit.
14. Artificial nanopore according to any one of claims 1-12, wherein the N-terminus of the subunit of the ring-forming protein comprising the TM sequence is genetically fused to the C-terminus of a Clp protease (ClpP) subunit.
15. A multi-protein nanopore sensor complex, comprising (i) an artificial nanopore according to any one of claims 1-14, (ii) one or two rings composed of proteasome α-subunits and optionally (iii) one or two rings composed of proteasome β-subunits.
16. A multi-protein nanopore sensor complex according to claim 15, wherein the proteasome α-subunit lacks at least 5 amino acids at its N-terminus.
17. Multi-protein nanopore sensor complex according to claim 15 or 16, wherein the ring composed of proteasome β-subunits is engineered to provide a distinct type of protease activity.
18. Multi-protein nanopore sensor complex according to any one of claims 15-17, further comprising a protein translocase which can bind, unfold, and translocate a polynucleotide or polypeptide through the nanopore sensor complex in a sequential order.
19. Multi-protein nanopore sensor complex according to claim 18, wherein the protein translocase is an NTP-driven unfoldase, preferably an AAA+ unfoldase, more preferably wherein the protein translocase is selected from ClpX, VAT, PAN, AMA, 854, MBA and SAMP.
20. An analytical system comprising a hydrophobic membrane separating a fluid chamber into a cis side and a trans side, said membrane comprising an artificial nanopore according to any one of claims 1-14, or a multiprotein nanopore sensor complex according to any one of claims 15-19.
21. A method for single molecule analysis, preferably for identification and/or sequencing of a biopolymer, more preferably for single molecule polypeptide or polynucleotide sequencing, comprising adding a biopolymer to be analyzed to the chamber of an analytical system according to claim 20 and allowing the biopolymer to contact the pore.
22. The use of an analytical system according to claim 20, for single molecule analysis, preferably for identification and/or sequencing of a biopolymer, more preferably for single molecule polypeptide or polynucleotide sequencing.
23. A nucleic acid molecule encoding a subunit of an artificial nanopore according to any one of claims 1-14.
24. An expression vector comprising a nucleic acid molecule according to claim 23.
25. A host cell comprising an expression vector according to claim 24, optionally further comprising a distinct expression vector encoding a proteasome beta-subunit and/or a proteasome alpha-subunit.
Description
LEGEND TO THE FIGURES
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EXPERIMENTAL SECTION
Materials and Methods
[0077] General materials. Oligonucleotides and gBlock gene fragments were obtained from Integrated DNA Technologies (IDT). Phire Hot Start II DNA Polymerase, restriction enzymes, T4 DNA ligase, and Dpn I were purchased from Fisher Scientific. Angiotensin I, dynorphin A, pentane, hexadecane, and Trizma base were obtained from Sigma-Aldrich. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was purchased from Avanti Polar Lipids. Sodium chloride and Triton X-100 was bought from Carl Roth.
[0078] Plasmid Construction for proteins. gBlock gene fragments were ordered for synthesis by IDT, and cloned into pT7-SC1 plasmid.sup.33 using Nco I and Hind III restriction digestion sites. Plasmid and gene were ligated together using T4 ligase (Fermentas). 0.5 μL of the ligation mixture was incorporated into 50 μL E. Cloni® 10G (Lucigen) competent cells by electroporation. Transformants were grown overnight at 37° C. on LB agar plates supplemented with ampicillin (100 μg/mL). Ampicillin-resistant colonies were picked and inoculated into 5 mL LB medium supplemented with of ampicillin (100 μg/mL) for plasmid DNA preparation. The plasmid was extracted with GeneJET Extraction Kit (Fisher Scientific). The identity of the clones was confirmed by sequencing at Macrogen.
[0079] Plasmid Construction for building a sequencing proteasome machine. gBlock gene fragments of Thermoplasma acidophilum α and β were ordered for synthesis by IDT. The gene encoding for the a subunit was cloned upstream of pETDuet-1 vector (Novagen) between the Nco I and Hind III sites with the gene of Strep-tag at the C-terminus. Subsequently, the gene encoding for an untagged β subunit was cloned downstream between the Nde I and Kpn I sites. PA-nanopore was fused to a subunit gene through PCR splicing by overlap extension.sup.34, and cloned into pET-28a vector (Novagen) using Nco I and Hind III restriction digestion sites with His tag at the N terminus.
[0080] Construction of mutants. All mutants were constructed using the QuickChange protocol.sup.35 for site-directed mutagenesis on a circular plasmid template DNA with Phire Hot Start II Polymerase. Partially overlapping primers were used to avoid primer self-extension. PCR amplification was as follows: denaturation at 98° C. for 3 min, followed by 30 cycles of 98° C. for 30 s, 55° C. for 30 s, and 72° C. for 3 min, and a final extension cycle of 72° C. for 5 min. After the PCR reaction, the parental DNA template was digested with Dpn I enzyme for 1 h at 37° C. The PCR amplified plasmid was separated on 1% agarose gel, extracted with GeneJET Gel Extraction Kit (Fisher Scientific), and incorporated into 50 μL E. Cloni® 10G (Lucigen) competent cells by electroporation. Transformants containing the plasmid were grown overnight at 37° C. on LB agar plates supplemented with ampicillin (100 μg/mL). Ampicillin-resistant colonies were picked and inoculated into 5 mL LB medium supplemented with of ampicillin (100 μg/mL) for plasmid DNA preparation. The plasmid was extracted with GeneJET Extraction Kit (Fisher Scientific), and sequenced at Macrogen for confirmation of the mutation.
[0081] Expression and purification. The gene of the PA nanopore was transformed into E. coli. BL21 (DE3) pLysS chemically competent cells. Transformants were selected after overnight growth at 37° C. on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The cells were grown at 37° C. (180 rpm shaking). After the optical density reached an absorbance of 0.6 at 600 nm, the expression was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The temperature was lowered to 25° C., and the cell cultures were further grown overnight. The cells were harvested by centrifugation for 20 min (4000×g) at 4° C. and the pellets were stored at −80° C. About 100 mL of cell culture pellet was thawed and solubilized with ˜20 mL lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 units/mL DNase I, 10 μg/mL lysozyme, 1% v/v Triton X-100) and stirred with a vortex shaker for 1 hour at 22° C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000×g at 4° C. for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Strep-Tactin resin (IBA) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (1% v/v Triton X-100, 150 mM NaCl, 15 mM Tris-HCl, pH 7.5). After 1 hour, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). In total, 10 mL of wash buffer (1% v/v Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole) was used to wash the beads. The protein was eluted with approximately 100 μL elution buffer (2.5 mM desthiobiotin, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100).
[0082] The genes encoding for test peptides S1 and S2 were separately transformed into E. coli. BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37° C. on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The cells were grown at 37° C. (180 rpm shaking). After the optical density reached an absorbance of 0.6 at 600 nm, the expression was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37° C. And the cell cultures were further grown 4 hours. The cells were harvested by centrifugation for 20 min (4000×g) at 4° C. and the pellets were stored at −80° C. About 100 mL of cell culture pellet was thawed and solubilized with ˜20 mL lysis buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 units/mL DNase I, 10 μg/mL lysozyme, 0.2% v/v Triton X-100) and stirred with a vortex shaker for 1 hour at 4° C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000×g at 4° C. for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100). After 1 hour at 4° C., the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100). In total, 10 mL of wash buffer (300 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole) was used to wash the beads. The protein was eluted with approximately 200 μL elution buffer (500 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl, pH 7.5).
[0083] The genes encoding for VAT and GFP were separately transformed into E. coli. BL21 (DE3) electrocompetent cells.
[0084] Transformants were selected after overnight growth at 37° C. on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The cells were grown at 37° C. (180 rpm shaking). After the optical density reached an absorbance of 0.6 at 600 nm, the expression was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25° C. And the cell cultures were further grown overnight. The cells were harvested by centrifugation for 20 min (4000×g) at 4° C. and the pellets were stored at −80° C. About 100 mL of cell culture pellet was thawed and solubilized with ˜20 mL lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.1 units/mL DNase I, 10 μg/mL lysozyme) and stirred with a vortex shaker for 1 hour at 4° C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000×g at 4° C. for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). After 1 hour at 4° C., the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). In total, 10 mL of wash buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole) was used to wash the beads. The protein was eluted with approximately 200 μL elution buffer (500 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5).
[0085] Proteasome co-expression and purification. For the assembly of the proteasome-nanopore, the pETDuet-1 containing the gene encoding for the α and β subunits of the proteasome and pET28a containing the gene encoding for the PA28-αΔ20 nanopore plasmids were co-transformed into E. coli BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37° C. on LB agar plates supplemented with ampicillin (100 mg/L) and kanamycin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin and kanamycin. Protein expression was induced by 0.5 mM β-d-thiogalactopyranoside (IPTG) when the A600 reached about 0.6. The temperature was lowered to 25° C. After 12 h induction, the cells were collected, and the pellets were stored at −80° C.
[0086] About 100 mL of cell culture pellet was thawed and solubilized with ˜20 mL lysis buffer (150-1000 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 20 mM imidazole, 0.1 units/mL DNase I, 10 μg/mL lysozyme, 1% v/v Triton X-100) and stirred with a vortex shaker for 1 hour at 22° C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000×g at 4° C. for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (1% v/v Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5). After 1 hour, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 15 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). The protein was eluted with approximately 200 μL elution buffer (500 mM imidazole, 150-1000 mM NaCl, 15 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). Subsequently, the eluted protein was mixed with 50 μL of Strep-Tactin resin (IBA) to a 2 mL tube, which was pre-equilibrated with wash buffer (1% v/v Triton X-100, 150 mM NaCl, 15 mM Tris-HCl, pH 7.5). After 30 minutes, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). In total, 10 mL of wash buffer (150-1000 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole, 0.2% v/v Triton X-100) was used to wash the beads. The protein was eluted with approximately 100 μL elution buffer (2.5 mM desthiobiotin, 150-1000 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100).
[0087] Proteolytic activity of artificial proteasome-nanopore (complex 3). To determine the proteolytic activity of artificial proteasome-nanopore, β-casein was incubated with purified complex 3 under a variety of incubating time, temperature, and salt concentration (
[0088] Electrical recordings in planar lipid bilayers. The setup consisted of two chambers separated by a 25 μm thick polytetrafluoroethylene film (Goodfellow Cambridge Limited), which contain an aperture of approximately 100 μm in diameter, which was formed by applying a high voltage spark. To form a lipid bilayer, the aperture was pre-treated with a drop of 5% hexadecane/pentane solution. After waiting about 1-5 minutes in order to allow pentane to evaporate, 500 μL of a buffered solution (150 mM NaCl, 15 mM Tris-HCl, pH 7.5) was added to each compartment. Then a drop of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane (˜10 mg/mL) was added to each compartment. After evaporation of the pentane, a lipid monolayer formed spontaneously by pipetting the solution up and down over the aperture. Silver/silver-chloride electrodes were submerged into the solution of each compartment. Nanopores were added to the trans side. All experiments were performed at ˜23° C..sup.36.
[0089] Data recordings and analysis. Electronic signals were recorded by using an Axopatch 200B (Axon Instruments) with digitization performed with a Digidata 1440 (Axon Instruments). Clampex 10.7 software and Clampfit 10.7 software (Molecular Devices) were used for electronic signal recording and subsequent data analysis, respectively. Events were collected using the single-channel search feature in clampfit and events shorter than 0.05 ms were ignored.
[0090] Ion selectivity. The current-voltage (I-V) current traces were recorded with an automated voltage protocol that applied each potential for 0.4 s from −30 to +30 mV with 1 mV steps. Ag/AgCl electrodes were surrounded with 2.5% agarose bridges containing 2.5 M NaCl. Reversal potential was measured from extrapolation from I-V curves collected under asymmetric salt concentration condition. The experiment proceeded as follow: First an individual nanopore was reconstituted using the same buffer in both chambers (1 M NaCl, 15 mM Tris, pH 7.5, 500 μL). This allowed assessing the orientation of the nanopore and allowed balancing the electrodes. Then 500 μL solution containing 4 M NaCl, 15 mM Tris, pH 7.5 was slowly added to cis side and 500 μL of a buffered solution containing no NaCl (15 mM Tris, pH 7.5) was added to trans side (trans:cis, 2.0 M NaCl: 0.5 M NaCl).
Example 1: Design of an Artificial Nanopore
[0091] The 20S proteasome from Thermoplasma acidophilum has a cylindrical structure made of four stacked rings composed of 14 α- and 14 β-subunits (
[0092] The amino acid sequence of a subunit of the artificial PA28-nanopore was as follows:
TABLE-US-00001 10 20 30 40 50 60 MATLRVHPEA QAKVDVFRED LCSKTENLLG SYFPKKISEL DAFLKEPALN EANLSNLKAP 70 80 90 100 110 120 GSSVHGN AEVHASFFDI GGSVSAGFSS G LDI CGPVNCNEK IVVLLQRLKP EIKDVTEQLN PVPDPVKEKEKEERKKQQEKEEKEEKKKGDEDDKGPP 130 140 150 160 170 180 LVTTWLQLQI PRIEDGNNFG VAVQEKVFEL MTNLHTKLEG PHTQISKYFS ERGDAVAKAA 190 200 210 220 230 240 KQPHVGDYRQ LVHELDEAEY QETRLMVMEI RNAYAVLYDI ILKNFEKLKK PRGETKGMIY 250 GSSWSHPQFE K
[0093] The transmembrane region of protective antigen flanked by 2 short linkers (SSG) (indicated in bold) was inserted in the polypeptide sequence of PA28a, which insertion also involved deletion of the stretch of amino acids of PA28 that is indicated in italics.
[0094] In order to optimize the fusion nanopore, the length of the linker was varied by adding or removing residues on each side of the transmembrane region. One deletion mutant (∇2) and five insertion mutants (∇2, ∇4, ∇8, ∇12, and ∇16) were prepared based on the sequence of protective antigen nanopore.sup.15 (
[0095] Remarkably, ∇2 inserted as efficiently and as uniformly as other nanopores found in nature (e.g. alpha hemolysin.sup.16). The individual peptides corresponding to the TM region of anthrax protective antigen could not form nanopores, indicating that a soluble scaffold is required to stabilize the nanopore in lipid bilayers.
[0096] Molecular dynamics (MD) simulations were performed on the ∇2 PA-nanopore (hereafter PA-nanopore) to better understand the electrostatic and hydrophobic Interactions between the nanopore and the lipid bilayer. As shown in
Example 2: Electrical and Functional Properties of the Optimized Artificial Pore
[0097] Similar to other β-barrel nanopores such as αHL.sup.18 and aerolysin.sup.19 nanopore, the artificial PA-nanopore showed an asymmetric current-voltage (I-V) relationship (
Example 3: Design of an Artificial Transmembrane Proteasome
[0098] In cells, PA28 docks onto the 20S proteasome and controls the translocation of substrates into the catalytic cavity.sup.21. We found, however, that when the proteasome was added to the cis side of individual PA28-nanopores in 1 M NaCl solutions, no clear interaction was observed. Most likely, the high ionic strength used do not allow such interaction.sup.22. The crystal structure of the Thermoplasma acidophilum proteasome in complex with PA26 from Trypanosoma brucei.sup.23, a homolog of PA28, shows that the carboxy-terminal tails of PA26 slide into a pocket on the 20S proteasome, near the amino-terminus of the a subunit (
Example 4: Real-Time Protein Processing
[0099] The activity of the transmembrane proteasome was tested using substrates containing a C-terminal ssrA tag, which mediates the interaction with VAT (Valosin-containing protein-like ATPase of Thermoplasma acidophilum).sup.25, an unfoldase that threads substrate proteins through the proteasome chamber. The first substrate, named S1, was 123 amino acid long and was designed to be unstructured and to contain four stretches of 15 serine residues flanked by a group of 10 arginines and three hydrophobic residues. The second substrate was S2, a longer polypeptide of 210 amino acids. The third substrate was green fluorescent protein (GFP).sup.25 carrying 10 arginines and an ssrA tag (AANDENYALAA) at the C-terminus.
TABLE-US-00002 S1: 1 11 21 31 41 51 61 MGHHHHHHSS RRRPRRRPRR SSSSSSSSSS SSSSSFGYGW SSSSSSSSSS SSSSSRRRRR RRRRPSSSSS 71 81 91 101 111 121 SSSSSSSSSS FGYGWSSSSS SSSSSSSSSS RRRRRRRRRR SSAANDENYA LAA S2: 1 11 21 31 41 51 61 MGHHHHHHSS RRRRRRVPLP IPVPLPIPVP LPIPRRRRRS SSSSSSSSSS SSSSSSSSSS SSSSSSSSSS 71 81 91 101 111 121 131 SSSSSSSSSS SSSSSSSSSR RRRRPVPLPI PVPLPIPVPL PIPRRRRRSS SSSSSSSSSS SSSSSSSSSS 141 151 161 171 181 191 201 SSSSSSSSSS SSSSSSSSSS SSSSSSSSEE EEEPVPLPIP VPLPIPVPLP IPEEEEESSA ANDENYALAA S3: 1 11 21 31 41 51 61 MGHHHHHHSS SKGEELFTGV VPILVELDGD VNGHKFSVSG EGEGDATYGK LTLKFICTTG KLPVPWPTLV 71 81 91 101 111 121 131 TTLTYGVQCF SRYPDHMKRH DFFKSAMPEG YVQERTISFK DDGNYKTRAE VKPEGDTLVN RIELKGIDFK 141 151 161 171 181 191 201 EDGNILGHKL EYNYNSHNVY ITADKQKNGI KANFKIPHNI EDGSVQLADH YQQNTPIGDG PVLLFDNHYL 211 221 231 241 251 STQSALSKDP NEKRDHMVLL EFVTAAGITH GMDELYKSSA ANDENYALAA
[0100] Initial tests were performed using a transmembrane proteasome, in which the proteolytic activity was removed by substituting the amino-terminal threonine 1 in the active site with alanine.sup.26. Reactions were performed in 1 M NaCl, 15 mM Tris-HCl, pH 7.5, 20 mM MgCl.sub.2 solutions. The addition of 20.0 μM of S1 to the cis compartment of an inactive proteasome-nanopore induced both short (average dwell time is 0.62±0.11 ms) and second-long current blockades (
[0101] When a GFP was used instead of S1, the current blockades became longer (average dwell time is 22.1±20.2 ms) and the current signature was strikingly different compared with S1 (
[0102] When the active proteasome was used in the presence of S1 but in the absence of VAT and ATP, uniform and short blockades were observed (
Example 5: PA26-Artificial Nanopore
[0103] This example describes the design and characterization of an artificial nanopore comprising the ring-forming multimeric proteasome activator protein PA26, which is a homolog of PA28.
[0104] The transmembrane sequence (bold) of anthrax protective antigen (PDB ID: 3J9C) was fused in the middle of a subunit of PA26 (PDB ID: 1YA7), from which the 12-amino acid sequence shown in italics was deleted, via 2 linkers (GSSSE----SNSSG).
[0105] The complete sequence of an N-terminally Strep-tagged subunit of the artificial PA26-nanopore is as follows:
TABLE-US-00003 10 20 30 40 50 60 70 MGWSHPQFEK SSGPFKRALL IQNLRDSYTE TSSFAVIEEW AAGTLQEIEG IAKAAAEAHG VIRNSTYGRA 70 80 90 100 110 120 QAEKSPEQLL GVLQRYQDLC HNVYCQAETI RTVIAIRIPE HKEEDNLGVA VQHAVLKIID ELEIKTLGSG 130 140 150 160 170 180 GSSSEVH GNAEVHASFF DIGGSVSAGF SNSSG EKGGSGGAPT PIGMYALREY LSARSTVEDK LLG SQSPS SVDAESGKTKGG 220 230 240 250 260 LLLELRQIDA DFMLKVELAT THLSTMVRAV INAYLLNWKK LIQPRTGSDH MVS
[0106]
Example 6: ATPase-Artificial Nanopore
[0107] This example describes the design and characterization of an artificial nanopore comprising the ring-forming multimeric Aquifex aeolicus ATPase (PDB ID: 3M0E), as an example of a protein capable of transporting a polynucleotide.
[0108] The transmembrane sequence (bold) of anthrax protective antigen (PDB ID: 3J9C) was inserted in the middle of a subunit of the ATPase, from which the amino acid sequence indicated in italics was deleted (insertional replacement). The inserted TM sequence was flanked on both sides with a linker (SSSSS) as indicated in bold. The complete sequence of an N-terminally Strep-tagged a subunit of the artificial ATPase-nanopore is as follows:
TABLE-US-00004 10 20 30 40 50 60 MGWSHPQFEK SSGRKENELL RREKDLKEEE YVFESPKMKE ILEKIKKISC AECPVLITGE 70 80 90 100 110 120 SSSSSV HGNAEVHASF SGVGKEVVAR LIHKLSDRSK EPFVALNVAS IPRDIFEAEL FGYE KGAFTGAVS 130 140 150 160 170 180 FDIGGSVSAG FSSSSS SKEG FFELADGGTL FLDAIGELSL EAQAKLLRVI ESGKFYRLGG 190 200 210 220 230 240 RKEIEVNVRI LAATNRNIKE LVKEGKFRED LYYRLGVIEI EIPPLRERKE DIIPLANHFL 250 260 270 280 290 300 KKFSRKYAKE VEGFTKSAQE LLLSYPWYGN VRELKNVIER AVLFSEGKFI DRGELSCLVN
[0109]
[0110] Herewith, another example of an artificial nanopore is provided that is based on the fusion of a beta barrel to a toroidal protein.
Example 7: ClpP-Artificial Nanopore
[0111] This example describes the design of an artificial nanopore for single-molecule protein analysis. It is based on an artificial PA28-nanopore as described in Example 1, fused at its N-terminus to a subunit of ClpP. ClpP (PDB ID: 1TYF) is the caseinolytic Clp protease (ClpP) from E. coli. Wang et al. (1997) Cell 91: 447-456) determined the structure of ClpP at 2.3 Å resolution. The active protease resembles a hollow, solid-walled cylinder composed of two 7-fold symmetric rings stacked back-to-back. Its 14 proteolytic active sites are located within a central, roughly spherical chamber approximately 51 Å in diameter. Access to the proteolytic chamber is controlled by two axial pores, each having a minimum diameter of approximately 10 Å.
[0112] The complete sequence of a C-terminally Strep-tagged subunit of the artificial ClpP-nanopore is as follows:
TABLE-US-00005 10 20 30 40 50 60 MGSYSGERDN FAPHMALVPM VIEQTSRGER SFDIYSRLLK ERVIFLTGQV EDHMANLIVA 70 80 90 100 110 120 QMLFLEAENP EKDIYLYINS PGGVITAGMS IYDTMQFIKP DVSTICMGQA ASMGAFLLTA 130 140 150 160 170 180 GAKGKRFCLP NSRVMIRQPL GGYQGQATDI EIHAREILKV KGRMNELMAL HTGQSLEQIE 190 200 210 220 230 240 RDTERDRFLS APEAVEYGLV DSILTHRNAT LRVHPEAQAK VDVFREDLCS KTENLLGSYF 250 260 270 280 290 380 PKKISELDAF LEKPALNEAN LSNLKAPLDI GSSSEVHGNA EVHASFFDIG GSVSAGFSNS 310 320 330 340 350 360 SGCGPVNCNE KIVVLLQRLK PEIKDVTEQL NLVTTWLQLQ IPRIEDGNNF GVAVQEKVPE 370 380 390 400 410 420 LMTNLHTKLE GFHTQISKYF SERGDAVAKA AKQPHVGDYR QLVRELDEAE YQEIRLMVME 430 440 450 480 IPNAYAVLYD IILKNFEKLK KPRGETKGMI YGSSWSHPQF EK
[0113] Residues 1-208 (italics) represent the primary sequence of ClpP from E. coli; residues 209-462 is the PA-nanopore including the C-terminal Strep-tag peptide WSHPQFEK; underlined residues 271-273 and 300-302 are linkers; and residues 274-299 (bold) represent the TM region.
[0114]
[0115] SDS-PAGE analyses of the purified ClpP-nanopore the presence of two unique bands corresponding well the molecular weights of active ClpP-PApore, active ClpP, inactive ClpP-PApore, and inactive ClpPPAαΔ20 (data not shown).
[0116]
[0117]
REFERENCES
[0118] 1. Manrao, E. A., Derrington, I. M., Laszlo, A. H., Langford, K. W., Hopper, M. K., Gillgren, N., Pavlenok, M., Niederweis, M. and Gundlach, J. H. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat. Biotechnol. 30, 349-353 (2012). [0119] 2. Noakes, M. T., Brinkerhoff, H., Laszlo, A. H., Derrington, I. M., Langford, K. W., Mount, J. W., Bowman, J. L., Baker, K. S., Doering, K. M., Tickman, B. I. and Gundlach, J. H. Increasing the accuracy of nanopore DNA sequencing using a time-varying cross membrane voltage. Nat. Biotechnol. 37, 651-656 (2019). [0120] 3. Cressiot, B., Oukhaled, A., Patriarche, G., Pastoriza-Gallego, M., Betton, J. M., Auvray, L., Muthukumar, M., Bacri, L. and Pelta, J. Protein transport through a narrow solid-state nanopore at high voltage: Experiments and theory. ACS Nano 6, 6236-6243 (2012). [0121] 4. Burns, J. R., Göpfrich, K., Wood, J. W., Thacker, V. V., Stulz, E., Keyser, U. F. and Howorka, S. Lipid-bilayer-spanning DNA nanopores with a bifunctional porphyrin anchor. Angew. Chemie—Int. Ed. 52, 12069-12072 (2013). [0122] 5. Spruijt, E., Tusk, S. E. & Bayley, H. DNA scaffolds support stable and uniform peptide nanopores. Nat. Nanotechnol. 13, 739-745 (2018). [0123] 6. Wei, B., Dai, M. & Yin, P. Complex shapes self-assembled from single-stranded DNA tiles. Nature 485, 623-626 (2012). [0124] 7. Mitchell, J. S., Glowacki, J., Grandchamp, A. E., Manning, R. S. & Maddocks, J. H. Sequence-dependent persistence lengths of DNA. J. Chem. Theory Comput. 13, 1539-1555 (2017). [0125] 8. Manning, G. S. The persistence length of DNA is reached from the persistence length of its null isomer through an internal electrostatic stretching force. Biophys. J. 91, 3607-3616 (2006). [0126] 9. Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Earnest, T. N., Cate, J. H. D., & Noller, H. F. Crystal structure of the ribosome at 5.5 Å resolution. Science 292, 883-896 (2001). [0127] 10. Mishra, R., Upadhyay, A., Prajapati, V. K. & Mishra, A. Proteasome-mediated proteostasis: Novel medicinal and pharmacological strategies for diseases. Med. Res. Rev. 38, 1916-1973 (2018). [0128] 11. Becker, S. H., & Darwin, K. H. Bacterial proteasomes: mechanistic and functional insights. Microbiol. Mol. Biol. Rev. 81, 1-20 (2017). [0129] 12. Lowe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Å resolution. Science. 268, 533-539 (1995). [0130] 13. Forster, A. & Hill, C. P. Proteasome Activators. Protein Degrad. 2, 89-110 (2007). [0131] 14. Huber, E. M. & Groll, M. The Mammalian Proteasome Activator PA28 Forms an Asymmetric α4β3 Complex. Structure 25, 1473-1480 (2017). [0132] 15. Jiang, J., Pentelute, B. L., Collier, R. J. & Hong Zhou, Z. Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature 521, 545-549 (2015). [0133] 16. Maglia, G., Restrepo, M. R., Mikhailova, E. & Bayley, H. Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge. Proc. Natl. Acad. Sci. U.S.A 105, 19720-19725 (2008). [0134] 17. Chen, B., Sysoeva, T. A., Chowdhury, S., Guo, L., De Carlo, S., Hanson, J. A., Yang, H. and Nixon, B. T., 2010. Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase. Structure 18, 1420-1430 (2010). [0135] 18. Stoddart, D., Ayub, M., Hofler, L., Raychaudhuri, P., Klingelhoefer, J. W., Maglia, G., Heron, A. and Bayley, H. Functional truncated membrane pores. Proc. Natl. Acad. Sci. U.S.A 111, 2425-2430 (2014). [0136] 19. Piguet, F., Ouldali, H., Pastoriza-Gallego, M., Manivet, P., Pelta, J., & Oukhaled, A. Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore. Nat. Commun. 9, 966 (2018). [0137] 20. Gu, L. Q., Braha, O., Conlan, S., Cheley, S. & Bayley, H. Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter. Nature 398, 686-690 (1999). [0138] 21. Sugiyama, M., Sahashi, H., Kurimoto, E., Takata, S. I., Yagi, H., Kanai, K., Sakata, E., Minami, Y., Tanaka, K. and Kato, K. Spatial arrangement and functional role of a subunits of proteasome activator PA28 in hetero-oligomeric form. Biochem. Biophys. Res. Commun. 432, 141-145 (2013). [0139] 22. Kuehn, L. & Dahlmann, B. Proteasome activator PA28 and its interaction with 20 S proteasomes. Arch. Biochem. Biophys. 329, 87-96 (1996). [0140] 23. Förster, A., Masters, E. I., Whitby, F. G., Robinson, H. & Hill, C. P. The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Mol. Cell 18, 589-599 (2005). [0141] 24. Benaroudj, N., Zwickl, P., Seemüller, E., Baumeister, W. & Goldberg, A. L. ATP hydrolysis by the proteasome regulatory complex PAN serves multiple functions in protein degradation. Mol. Cell 11, 69-78 (2003). [0142] 25. Huang, R., Ripstein, Z. A., Augustyniak, R., Lazniewski, M., Ginalski, K., Kay, L. E. and Rubinstein, J. L. Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study. Proc. Natl. Acad. Sci. U.S.A 113, E4090-W4199 (2016). [0143] 26. Seemuller, E., Lupas, A., Stock, D., Lowe, J., Huber, R. and Baumeister, W. Proteasome from Thermoplasma acidophilum: A Threonine Protease. Science 268, 579-582 (2016). [0144] 27. Kim, Y. I., Burton, R. E., Burton, B. M., Sauer, R. T. & Baker, T. A. Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. Mol. Cell 5, 639-648 (2000). [0145] 28. Akopian, T. N., Kisselev, A. F. & Goldberg, A. L. Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum. J. Biol. Chem. 272, 1791-1798 (1997). [0146] 29. Huang, G., Voet, A. & Maglia, G. FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution. Nat. Commun. 10, 1-10 (2019). [0147] 30. Kisselev, A. F., Songyang, Z. & Goldberg, A. L. Why does threonine, and not serine, function as the active site nucleophile in proteasomes? J. Biol. Chem. 275, 14831-14837 (2000). [0148] 31. Huber, E. M., Heinemeyer, W., Li, X., Arendt, C. S., Hochstrasser, M. and Groll, M. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome. Nat. Commun. 7, 1-10 (2016). [0149] 32. Ripstein, Z. A., Huang, R., Augustyniak, R., Kay, L. E. & Rubinstein, J. L. Structure of a AAA+ unfoldase in the process of unfolding substrate. Elife 6, 1-14 (2017). [0150] 33. Miles, G., Cheley, S., Braha, O. & Bayley, H. The staphylococcal leukocidin bicomponent toxin forms large ionic channels. Biochemistry 40, 8514-8522 (2001). [0151] 34. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68 (1989). [0152] 35. Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 8, 91 (2008). [0153] 36. Maglia, G., Heron, A. J., Stoddart, D., Japrung, D. & Bayley, H. Analysis of single nucleic acid molecules with protein nanopores. In Methods in enzymology 475, 591-623 (2010).