Molecular hopper

Abstract

Provided herein are methods for moving a molecular hopper along a track, methods for characterizing an analyte using a molecular hopper, kits for characterizing an analyte, and molecular hoppers themselves and systems comprising these hoppers. In particular, the invention relates to the use of such methods and kits in analyte characterization.

Description

Field of the Invention

[0001] The present invention relates to methods for moving molecular hoppers along tracks, methods for characterizing analytes using molecular hoppers, kits for characterizing analytes, and the molecular hoppers themselves and systems containing these hoppers. The present invention particularly relates to the use of such methods and kits in the characterization of analytes such as polynucleotides, polypeptides and polysaccharides. Background of the Invention

[0003] Many desired technological applications require precise control of molecular motion. For example, characterization of analytes at the single molecule level requires precise manipulation of the analyte with nanometer precision. Attempts have been made to achieve this goal and have generally focused on controlling the analyte using complex systems such as molecular or optical tweezers or other physical means. However, in many areas of nanotechnology, particularly in the field of single molecule analyte characterization, the possibility of controlling molecular motion without relying on such equipment has great potential.

[0004] Some attempts to control molecular motion have been inspired by the processivity exhibited by biological machines. For example, replicative DNA polymerases can incorporate thousands of nucleotides before disengaging from their template (1). Molecular motors, such as kinesins and dyneins, can move hundreds of steps along microtubules without leaving their tracks (2-4). However, these systems are very complex and are generally not easily applicable in biotechnology.

[0005] There is a great need for methods and systems for moving molecules that resemble their biomolecular counterparts but use simpler components (5). The ultimate goal is to achieve true processivity, which can be defined as directional motion without derailment, and the ability to perform useful work such as transporting cargo. Ideally, synthetic systems should exhibit the reversibility of walking seen in various biological systems (6, 7) to enable the direction of motion to be switched by external control. SUMMARY OF THE INVENTION

[0007] The inventors have developed a molecular hopper capable of moving along a track comprising a plurality of primary functional groups arranged along a substrate. The hopper itself comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track, such that application of a driving force causes the hopper to be directed between the primary functional groups on the track, thereby moving the hopper along the track. The inventors have recognized that there is a clear need for methods and systems that are simple and capable of demonstrating improved continuous synthesis capabilities, autonomous movement, directional control including the ability to reverse, the ability to move cargo, and / or selective loading / unloading. The methods and systems provided in the present invention address some or all of these needs.

[0008] Therefore, the present invention provides a method of moving a molecular hopper along a track, wherein:

[0009] (a) the track comprises a plurality of primary functional groups arranged along a substrate;

[0010] (b) the hopper comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track; and

[0011] (c) the hopper optionally contains a cargo portion;

[0012] The method comprises the following steps:

[0013] (i) contacting the hopper with the track so that the secondary functional group of the hopper is combined with the first primary functional group on the track;

[0014] (ii) applying a driving force to directionally transfer the hopper from the first primary functional group to the second primary functional group on the track, thereby moving the hopper along the track.

[0015] Typically, the substrate is a surface of the transmembrane pore, such as the inner surface of the transmembrane pore. The hopper may include a cargo portion. Before the hopper is contacted with the track, the hopper can be connected to a positioning portion that positions the hopper relative to the track so that the first primary functional group on the track is combined with the secondary functional group on the hopper. The method may also include, after the hopper moves along the track, (iii) contacting the primary functional group of the track that is combined with the secondary functional group of the hopper with the tertiary functional group on the substrate so that the tertiary functional group is combined with the primary functional group, thereby replacing the secondary functional group and thus releasing the hopper. The method may also include reversing the direction of the driving force relative to the track so that the direction of movement of the hopper is reversed along the track.

[0016] The present invention also provides a method for characterizing an analyte, the method comprising:

[0017] (i) providing (A) a detector; (B) a track comprising a plurality of primary functional groups arranged along a substrate; and (C) a molecular hopper connected to the analyte, wherein the hopper comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track;

[0018] (ii) contacting the hopper with the track so that the secondary functional group of the hopper is combined with the first primary functional group on the track;

[0019] (iii) applying a driving force to directionally transfer the hopper from the first primary functional group to the second primary functional group on the track, thereby causing the hopper to move along the track;

[0020] Wherein the track is positioned such that movement of the hopper along the track causes the analyte to interact with the detector, thereby characterizing the analyte.

[0021] The present invention also provides a kit for characterizing an analyte, the kit comprising: (A) a detector; (B) a track comprising a plurality of primary functional groups arranged along a substrate; and (C) a molecular hopper connected to the analyte, wherein the hopper comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track; wherein preferably the analyte is a polynucleotide, a polypeptide or a polysaccharide.

[0022] The present invention also provides a system comprising: (A) a detector; (B) a track comprising a plurality of primary functional groups arranged along a substrate; and (C) a molecular hopper that binds to a polynucleotide, polypeptide or polysaccharide analyte, wherein the hopper contains a secondary functional group that binds to at least one of the plurality of primary functional groups on the track.

[0023] The present invention also provides a molecular hopper, which comprises:

[0024] - Secondary functional groups for binding to primary functional groups on the track;

[0025] - a polynucleotide, polypeptide or polysaccharide cargo moiety;

[0026] - A linking moiety between the secondary functional group and the cargo moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 shows a molecular hopper on a protein track. (A) The hopper carrying the cargo (red symbol) moves along the track by employing sequential thiol-disulfide interchange reactions. The overall direction is set by the applied voltage. (B) A six-point track containing odd-numbered cysteine ​​residues on the β-strand inside the HL protein nanopore. (C) The applied potential exerts a force on the DNA cargo, which helps to align the three sulfur atoms (yellow) involved in the interchange. The colinear geometry promotes feeding (1) but not the formation of intrachain disulfides, which would release the hopper (2) from the track. Occasionally, back-off (3) is observed. Overrun (4) does not occur. (D) The hopper enters the nanopore as a carrier-hopper disulfide conjugate. More details are given in the Examples.

[0029] FIG2 shows monitoring of individual hopper steps. (A) At +150 mV, a hopper-carrier conjugate capped with traptavidin is pulled from the cis compartment into an αHL nanopore containing cysteines at positions 113, 115, 117, 119, and 121 in one of the seven subunits. The resulting blockade reduces the ionic current from (i) to (ii). Reaction of the disulfide in the hopper-carrier with Cys-115 covalently links the hopper to the track, and the ionic current increases to (iii). (B) Four feeding steps are observed at ±150 mV for the track of five cysteines. Each forward movement moves a portion of the DNA cargo outside the β barrel, thereby increasing the conductance. Alternation of the applied potential drives the hopper repeatedly up and down the track. (C) Hypothetical free energy diagram of controlled feeding motion (not to scale). (D) On an L-shaped track consisting of cysteines at positions 115, 117, 119, and 139, the hopper moves along the track from Cys-115 to Cys-119, where the hopper is released by the side chain of Cys-139. Subsequently, a second hopper is loaded at Cys-115, but its movement is arrested at Cys-117 because Cys-119 is now involved in an interchain disulfide bond. Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, pH 8.5, 20°C ± 1°C. Data are described in the Examples.

[0030] Figure 3 shows the discrimination of different DNA cargoes. (A) Two consecutive abasic nucleotides (dSdS) are substituted at positions 3 and 4 (hopper 2) or at positions 2 and 3 (hopper 3). The number of nucleotides placed inside the β barrel (brown circles, dA; red circles, dS) is modeled based on PyMOL. (B) Four-step feeding is observed with hopper 2 at ±150mV in the trajectory of five cysteines. The current decrease (green arrow) for feeding from 115 to 117 and from 121 to 119 is marked. (C) Four-step feeding using hopper 3 at ±150mV. The current conversion (red arrow) for feeding from 117 to 119 and from 119 to 117 is marked. (D) Top: Overlapping current traces of hoppers 1, 2 and 3 with normalized step times. The current level is given as the residual current (Ires%) relative to the open pore level. Lower part: Step sizes for hoppers 2 and 3 are plotted as ΔIres%. Minima in the curves showing single nucleotide shifts are marked. Conditions: 2M KCl, 20 mM HEPBS, 20 μM EDTA, pH 8.5, 20°C ± 1°C. Data are described in this example.

[0031] Figure 4 shows the LC-MS and HPLC characterization of the carrier. (A) Structure of the carrier. (B) Calculated carrier mass ([M+H] + ) is 1243.7 g mol -1 , measured as 1244.6 g mol-1 (C) The purity of the vector was confirmed by analytical HPLC. The data are described in this example.

[0032] Figure 5 LC-MS characterization of the hopper-carrier conjugate is shown. The two peaks in each chromatogram have the same mass, corresponding to the two positional isomeric conjugates formed by the copper-free click reaction. The mass of the hopper-1-carrier conjugate was calculated to be 14327 g mol -1 and measured to be 14327 g mol -1 The mass of the hopper-2-carrier conjugate and the mass of the hopper-3-carrier conjugate were calculated to be 14059 g mol -1 and measured to be 14061 g mol -1 The data are described in this example.

[0033] Figure 6 shows loading of hopper 1 onto different tracks. At +150 mV, before reacting with Cys-115, a traptavidin-terminated hopper-1-carrier conjugate was passed through the cis side of the nanopore, which covalently linked the hopper to (A) a two-cysteine ​​track (pivot: Cys-115, 117), (B) a three-cysteine ​​track (pivot: Cys-115, 117, 119), (C) a five-cysteine ​​track (pivot: Cys-113, 115, 117, 119, 121). The movement of the hopper was tracked by the position determined by the residual current through the nanopore. For the five-cysteine ​​track, only three step shifts were seen in the first movement at +150 mV. After four steps of feeding at -150 mV, a second shift at +150 mV on the same trajectory has four steps, indicating initial attachment at Cys-115. The trace was filtered at 200 Hz. Conditions: 2M KCl, 20 mM HEPBS, 20 μM EDTA, pH 8.5, 20°C ± 1°C. The data are described in the Examples.

[0034] Figure 7Feeding on the three-cysteine ​​track is shown. For the three-cysteine ​​track (fulcrum: Cys-115, 117, 119), two feeding steps are observed at ±100mV, ±150mV and ±180mV with hopper 1. The feeding cycle is repeated ≥20 times for each voltage, and the rate constant given in Table S1 is obtained using QuB. The application of ±100mV is not enough to quickly flip the oligonucleotide to the opposite direction, and leads to a very noisy level before the oligonucleotide is fully stretched, and the feeding can be restarted. When the noisy level is observed to last for more than 20 minutes, the noisy level is particularly common under negative voltage. The trace is filtered at 200Hz. Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, pH 8.5, 20℃±1℃. The data are described in the examples.

[0035] Figure 8 shows the feeding cycle on the track of five cysteines. When loading hopper 1 onto the track of five cysteines (pivot points: Cys-113, 115, 117, 119, 121) ( Figure 6C ) After that, during a single recording period, hopper 1 moved more than 20 complete cycles under external control (from 113 to 121 at +150mV, then from 121 to 113 at -150mV). (A) Shows a complete cycle at ±150mV, from which kinetic rate constants are obtained using QuB (see Section 11). (B) In the same experiment, various values ​​of externally manipulated voltage (e.g., +100mV) were also applied at non-terminal pivots (e.g., Cys-115). The total number of steps was 282 (counting only the back shift at the non-terminal pivot; not counting the step shift after reaching the final pivot and before the voltage switch). The trace was filtered at 200Hz. Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, pH 8.5, 20°C ± 1°C. The data are described in the examples.

[0036] Fig. 9 Feeding on the six-cysteine ​​track is shown. Five feeding steps were recorded for hopper 1 on the six-cysteine ​​track (pivots: Cys-113, 115, 117, 119, 121, 123) at ±150 mV. Each forward movement moves a portion of the DNA cargo outside the beta barrel, which produces an increase in conductance. Alternation of the applied potential drives the hopper repeatedly up and down the track. The trace was filtered at 200 Hz. Conditions: 2M KCl, 20 mM HEPBS, 20 μM EDTA, pH 8.5, 20°C ± 1°C. Data are described in the Examples.

[0037] Fig.10The kinetic model and duration histogram of two steps on the three-cysteine ​​track (pivot: Cys-115, 117, 119) at 100mV, 150mV, and 180mV are shown. The kinetic model (top) connects the two steps under positive voltage with the two steps under negative voltage to form a closed loop. By assigning an arbitrary duration of 1s to the final pivot (Cys-119 under positive voltage; Cys-115 under negative voltage), two voltage-switching conversions are built into the model. Considering that there is no backward shift on the intermediate pivot during all feeding cycles, QuB is set to treat each forward shift as irreversible. The kinetic rate constants obtained from QuB are given in Table S1. Data were collected from two separate αHL holes. The data are described in this embodiment.

[0038] Figure 11 shows the estimated number of nucleotides within the αHL barrel during forward and backward movement between pivots 115 and 117. (A) Length of the fully stretched linker (left) and the αHLβ barrel (from residues 111 to 129) (right). The average vertical distance between the pivots is assumed to be The linker then spans a distance of about two steps. (B) At a positive potential, hopper 1 moves forward from pivot 115 to 117 and back from 117 to 115 (left). During the forward movement from 115 to 117, it is estimated that four negative charges move in the barrel (see Section 12). At a negative potential, the hopper moves forward from pivot 117 to 115 and back from 115 to 117 (right). In this case, there is almost no charge movement in the barrel because most of the DNA has retreated to the vestibule. The data are described in this embodiment.

[0039] Fig.12 The kinetic model and duration histograms of forward and backward movement on the two-cysteine ​​track (pivot: Cys-115, 117) at 100mV, 150mV, 180mV are shown. According to the two-state kinetic model (top), the forward and backward movement rate constants (Table S2) were derived by using the maximum interval likelihood algorithm of QuB. Data were collected from 4 αHL holes. The data are described in this example.

[0040] Fig.13The kinetic model and duration histogram of four steps on the trajectory of five cysteines (pivot points: Cys-113, 115, 117, 119, 121) are shown. The kinetic model (top) connects four steps at positive voltage with four steps at negative voltage to form a closed loop. Two voltage-switching transitions are built into the model by assigning an arbitrary duration of 1s to the final pivot point of the trajectory (Cys-121 at +150mV; Cys-113 at -150mV). QuB is set to treat forward shifts as irreversible, except for 117 to 115, where backward shifts occurred 29 times. Backward shifts after reaching the final pivot point are not counted. The resulting kinetic rate constants are given in Tables S3 and S5. Data were collected from a single αHL pore. The data are described in this embodiment.

[0041] Fig.14 The kinetic model and duration histogram of forward and backward movement on the last two pivots (Cys-119, 121) at +150mV for the trajectory of five cysteines (pivots: Cys-113, 115, 117, 119, 121) are shown. The forward and backward movement rate constants were derived by using the maximum interval likelihood algorithm of QuB according to the two-state kinetic model (top). The resulting kinetic rate constants are given in Table S4. Data were collected from a single αHL pore. The data are described in this example.

[0042] Fig.15 Shows how the charged oligonucleotides gradually move out of the electric field as the hopper moves along the track. See the Examples for more information.

[0043] Fig.16 Two current patterns are shown for Hopper 3 at pivot 119 at -150 mV. Four-step feeding was observed using Hopper 3 at ±150 mV (n=3 separate experiments). While feeding produced a consistent pattern at +150 mV, feeding at -150 mV produced two ion current signatures. In one ion current signature, the current at pivot 119 is much noisier than the current at the adjacent pivot (top extended trace) using the hopper: RMS noise I RMS (119) = 2.8 ± 0.1 pA; I RMS (121) = 1.2 ± 0.2 pA; I RMS (117) = 1.0 ± 0.1 pA. In the second mode, the current at pivot point 119 using hopper 3 shows two sub-conductance levels (bottom extended trace). No conversion between the two modes was observed. The ratio of dwells at pivot point 119 where noise occurs to dwells with two sub-conductance states is 5:2. The current amplitude is the same for the noise level of the first mode and the higher conductance state (larger negative current) in the second mode (indicated by the blue arrow). This value is used to plot Figure 3D, and are reported in Table S6. These two modes must correspond to two ways in which Hopper 3 interacts with the nanopore, since they are seen not only in separate experiments but also in the same experiment with the same Hopper molecule, which excludes the possibility of impurities in the oligonucleotide or differences between Hoppers. The data are described in this Example.

[0044] Figure 17 shows the thiol-disulfide exchange cycle. a. Cysteine ​​(pink) on one of the seven α-hemolysin (αHL) subunits provides nucleophilic thiolate at a defined site within the transmembrane β barrel. Six single cysteine ​​mutants (113, 115, 117, 119, 121, 143) and two double cysteine ​​mutants (115 / 117 and 115 / 143) were used. b. The substrate consists of biotin at one end and a peptide-oligonucleotide connection formed by click chemistry. A cleavable disulfide bond is set between the click product and the oligonucleotide. The peptide length is manipulated by adding two amino acids (glycine or serine, Δn=2) at the time after the PEG unit: POC1 (n=0); POC2 (n=2); POC3 (n=4); POC4 (n=6). c. Under an applied potential (+150 mV, trans), molecules of the traptavidin-terminated substrate penetrate the nanopore from the cis side (i) and are blocked from translocation by the traptavidin blocker (ii). The favorable arrangement allows for thiol-disulfide interchange between cysteine ​​thiolates and substrate disulfides, generating αHL-peptide adducts (top, iv) or αHL-oligonucleotide adducts (bottom, iii). Reduction of the newly formed disulfides by DTT (5 mM in the trans compartment) returns the nanopore to its open state (i) to initiate the next reaction cycle. The results are described in Example 2.

[0045] Figure 18 shows how the arrangement of substrate disulfides with nanoreactor cysteine ​​sulfurs determines reactivity. a. The calculated transition state for thiol-disulfide interchange has approximately SS bond length and SSS bond angle of about 180°. This produces reaction zone, while in the middle (red) Assuming that the cysteine ​​(Cys) side chain is free to rotate, the thiolate can reach (blue triangles). The overlap between the disulfide-reactive zone and the thiolate-accessible zone leads to thiol-disulfide interchange. b. Reactivity between different substrates and single-cysteine ​​nanoreactors, represented by the ratio of thiol-disulfide interchange events to the total number of crossover events (reactivity = ninterchanges / ncrossovers), expressed as a percentage. Blue indicates that crossovers lead to the formation of adducts. Red indicates direct cleavage of the substrate by DTT (5 mM, trans) without adduct formation. All crossover events lead to adduct formation or cleavage. White indicates untested combinations. c. Relative position of substrate and cysteine ​​within the single-cysteine ​​nanoreactor. The accessible positions of the disulfides are represented by dashed lines, which are inferred from the reactivity results. Due to the tilt of the β-chain relative to the barrel axis, consecutive nanoreactor cysteines are spaced at approximately The disulfides in POC1 are arranged based on their reactivity with Cys-113 and Cys-115. Assuming that the force generated by the electric field is sufficient to fully elongate the base, in the static model, two additional amino acids are created within the barrel at a depth of about 100 Å below the disulfide. The disulfides in POC2, POC3 and POC4 were subsequently aligned on this basis. The results are described in Example 2.

[0046] Figure 19 shows the regioselectivity of single-molecule thiol-disulfide exchange. a. Identification of flipping intermediates from current characteristics. POC1 penetrates the nanoreactor 113 from the cis compartment (i), resulting in partial blockage of ion flow (Ires% = 33%) (ii). The thiol-disulfide exchange between the nanoreactor 113 and POC1 produces two types of electrical patterns (iii, iv), which correspond to the formation of two different covalent adducts within the nanopore (see SI Part 1): αHL-peptide adducts provide three sub-conductance states (iii: pink line: Ires% = 43%, 48%, 51%); αHL-oligonucleotides give a single current state (iv: Ires% = 39%). The subsequent adduct is released by DTT, returning the system to its initial state (i). The lack of arrangement of substrate disulfides and thiolates is unfavorable for the reaction between nanoreactor 117 and POC1, mainly leading to direct cleavage of substrate disulfides by DTT rather than adduct formation (4.5% reactivity). Traces were filtered at 200 Hz. Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, 5mM DTT (trans), pH 8.5, 20°C ± 1°C. b, Sulfur regioselectivity of thiol-disulfide interchange between substrates POC1 (red), POC2 (blue), POC3 (yellow) and POC4 (green) and nanoreactors 113, 115, 143, 117, 119 and 121, expressed as a percentage of the total number of adducts formed (reactions under Sα and Sβ). c, Relative positions of substrates and cysteines within individual cysteine ​​nanoreactors inferred from the regioselectivity data. The regioselectivity ratio (Sβ / Sα) of the corresponding combination of substrate and nanoreactor is measured by the area of ​​each solid circle. The open circles indicate complete Sβ regioselectivity. The disulfide was placed where the Sβ / Sα regioselectivity = 1 extrapolated from the regioselectivity data. The accessible positions of the disulfide are indicated by dashed lines based on the reactivity data ( FIG. 18 c ). The results are described in Example 2.

[0047] Figure 20 shows a tandem thiol-disulfide exchange using a dual cysteine ​​nanoreactor. a. In the case of nanoreactors 115 / 117, POC1 reacts site-selectively and regioselectively with cysteine ​​115 to form an αHL-oligonucleotide adduct (iv). Subsequent regioselective attack on Sβ triggers an intramolecular thiol-disulfide exchange to transfer the adduct to cysteine ​​117 (pink arrow). The adduct moves back and forth between two sites on the same β strand before it is ultimately released by DTT, as evidenced by the intermediate αHL-DTT adduct (blue arrow). b. In the case of nanoreactors 115 / 143, POC1 reacts with 81% site selectivity and 100% regioselectivity at cysteine ​​115 to form an αHL-oligonucleotide adduct (iv). Attack on Cys-143 on the antiparallel strand to Sγ cleaves the adduct from the nanoreactor (pink arrow). Thus, cross-chain disulfide bonds are generated, thereby preventing new substrates from flipping (ii, substrates in the hole) until DTT regenerates two free cysteines through an intermediate αHL-DTT adduct (blue arrows). Occasionally, Cys-143 attacks Sβ, resulting in adduct transfer (see SI Section 2). The trace was filtered at 200 Hz. The small image showing the αHL-DTT adduct was filtered at 1000 Hz. Conditions: 2M KCl, 20 mM HEPBS, 20 μM EDTA, 5 mM DTT (trans), pH 8.5, 20°C ± 1°C. The results are described in Example 2.

[0048] Fig.21 Showing transmembrane communication. In a droplet interface bilayer (DIB) system, components of a mixed substrate in a left droplet are identified by selective reaction with a nanoreactor embedded in the bilayer under an applied potential. Substrate flipping releases oligonucleotide fragments (brown) into the right droplet to trigger various downstream responses. For example, translocated oligonucleotides can be complementary to single-stranded promoter regions to initiate in vitro transcription and translation. The results are described in Example 2.

[0049] Figure 22 shows the current changes caused by αHL-polymer adducts in response to voltage steps. a. After the thiol-disulfide exchange between POC2 and nanoreactor 119, αHL-oligonucleotide adducts are formed. Steps to +50mV produce different levels of current blockade, depending on whether the step is started from -150mV or +150mV (purple arrows), indicating that the switch in the oligonucleotide orientation occurs at high applied potentials. b. Formation of αHL-peptide adducts after thiol-disulfide exchange between POC2 and nanoreactor 113. After the step to -50mV, the current levels of the three sub-conductance levels are the same, whether the step is from +150mV or -150mV (green arrows), consistent with the presence of covalent adducts locked in the vestibule of the αHL pore by the traptavidin barrier. Traces were filtered at 200Hz. Conditions: Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, 5mM DTT(trans), pH 8.5, 20°C±1°C. The results are described in Example 2.

[0050] Figure 23 shows the thiol-disulfide exchange between nanoreactor 115 / 143 and POC1. a. After passing through, αHL-oligonucleotide adduct is formed at Cys-115. The oligonucleotide is transferred between Cys-115 and Cys-143 by Sβ attack (orange: transferred from 115 to 143; brown: transferred from 143 to 115) until Sγ is attacked to form a cross-chain disulfide (purple). Herein, we show an unusual example of six transfers observed, which is the most we have recorded. When DTT reduces the disulfide to regenerate two free cysteine ​​residues, αHL-DTT adduct (blue) is subsequently observed. b. After passing through, αHL-oligonucleotide adduct is formed at Cys-143. The oligonucleotide is transferred to Cys-115 (brown) and released from it by Sγ attack of Cys-143 (purple). The trace is filtered at 50Hz. The small figure showing the αHL-DTT adduct was filtered at 1000 Hz. Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, 5mM DTT (trans), pH 8.5, 20°C ± 1°C. c. Kinetic model and resulting rate constants for thiol-disulfide exchange observed with nanoreactor 115 / 143 and POC1. Residence time analysis and rate constant estimation were performed by using the maximum interval likelihood algorithm of QuB. The results are described in Example 2.

[0051] Figure 24 shows the regioselectivity of thiol-disulfide interchange between DTT and αHL adducts. (a) Mixed disulfide formation with macromolecular substrates to give αHL-oligonucleotide adducts or αHL-peptide adducts, or (b) mixed disulfide formation with DTNB. When DTT attacks cysteine ​​sulfur (Sγ), transient intermediates can be detected by single-channel electroacoustic recording. Results are described in Example 2.

[0052] Fig.25 The interconversion current levels recorded with POC3 penetrating the nanoreactor 119 are shown. Three interconversion current levels were observed. Specifically, αHL-oligonucleotide adducts and αHL-peptide adducts were not formed by one of the three levels. The trace was filtered at 200 Hz. Conditions: 2M KCl, 20mM HEPBS, 20μM EDTA, 5mM DTT (trans), pH 8.5, 20°C ± 1°C. The results are described in Example 2.

[0053] Figure 26 shows the LC-MS and HPLC characterization of the POC1 peptide. (a) The structure of the peptide. (b) The calculated mass of the peptide ([M+H]+) is 1243.7 g·mol -1 , measured as 1244.6 g·mol -1 (c) The purity of the peptide was confirmed by analytical HPLC. The results are described in Example 2.

[0054] Figure 27 shows the LC-MS and HPLC characterization of the POC2 peptide. (a) The structure of the peptide. (b) The calculated mass of the peptide ([M+H]+) is 1358.8 g·mol -1 , measured as 1358.8 g·mol -1 (c) The purity of the peptide was confirmed by analytical HPLC. The results are described in Example 2.

[0055] Fig.28 LC-MS characterization of the macromolecular substrates POC1-4 is shown. The two peaks in each chromatogram have the same mass, corresponding to the two regioisomer conjugates formed by copper-free click chemistry between peptides (blue) and oligonucleotides (green). The mass of POC1 was calculated to be 14327 g·mol -1 , measured as 14327 g·mol -1 The mass of POC2 is calculated to be 14440 g·mol -1 , measured as 14441 g·mol -1 The mass of POC3 is calculated to be 14615 g·mol -1 and was measured to be 14616 g·mol -1 The mass of POC4 is calculated to be 14729 g·mol -1and measured to be 14730 g·mol -1 The results are described in Example 2.

[0056] Sequence Listing Description

[0057] SEQ ID NO: 1 shows the amino acid sequence of one subunit of wild-type α-HL. Amino acids 2 to 6, 73 to 75, 207 to 209, 214 to 216, and 219 to 222 form an α helix. Amino acids 22 to 30, 35 to 44, 52 to 62, 67 to 71, 76 to 91, 98 to 103, 112 to 123, 137 to 148, 154 to 159, 165 to 172, 229 to 235, 243 to 261, 266 to 271, 285 to 286, and 291 to 293 form a β strand. All other non-terminal amino acids, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, and 287 to 290, form a loop region. Amino acids 1 and 293 are terminal amino acids.

[0058] SEQ ID NO: 2 shows the amino acid sequence of one subunit of wild-type α-HL-D8H6. The same amino acids that form the α-helix, β-strand and loop regions in wild-type α-HL form the corresponding regions in this subunit.

[0059] SEQ ID NO: 3 shows the amino acid sequence of one subunit of 115C117C-α-HL-D8H6. The same amino acids that form the α-helix, β-strand and loop regions in wild-type α-HL form the corresponding regions in this subunit.

[0060] SEQ ID NO: 4 shows the amino acid sequence of one subunit of 115C117C119C-α-HL-D8H6. The same amino acids that form the α-helix, β-strand, and loop regions in wild-type α-HL form the corresponding regions in this subunit.

[0061] SEQ ID NO: 5 shows the amino acid sequence of one subunit of 113C115C117C119C121C-α-HL-D8H6. The same amino acids that form the α-helix, β-strand and loop regions in wild-type α-HL form the corresponding regions in this subunit.

[0062] SEQ ID NO: 6 shows the amino acid sequence of one subunit of 113C115C117C119C121C123C-α-HL-D8H6. The same amino acids that form the α-helix, β-strand and loop regions in wild-type α-HL form the corresponding regions in this subunit.

[0063] SEQ ID NO: 7 shows the amino acid sequence of one subunit of C115C117C119C139C-α-HL-D8H6. The same amino acids that form the α-helix, β-strand and loop regions in wild-type α-HL form the corresponding regions in this subunit. DETAILED DESCRIPTION OF THE INVENTION

[0065] definition

[0066] The following substituent definitions apply to the compounds defined herein:

[0067] Alkyl is an unsubstituted or substituted, straight or branched, saturated hydrocarbon group. Typically, the alkyl group has 1 to 20 carbon atoms, i.e., the alkyl group is C 1-20 Typically, an alkyl group is C 1-10 Alkyl. The alkyl group can be, for example, C 1-6 Alkyl, such as methyl, ethyl, propyl, butyl, pentyl or hexyl, or C 1-4 Alkyl, such as methyl, ethyl, isopropyl, n-propyl, tert-butyl, sec-butyl or n-butyl. When the alkyl group is substituted, it usually carries one or more substituents selected from substituted or unsubstituted C 1-10 alkyl, substituted or unsubstituted aryl (as defined herein), cyano, amino, C 1-10 Alkylamino, di(C 1-10 ) alkylamino, arylamino, diarylamino, arylalkylamino, amide, acylamide, hydroxy, oxo, halogen, carboxyl, ester, acyl, acyloxy, C 1-10 Alkoxy, aryloxy, halogenated alkyl, sulfonic acid, mercapto (i.e., thiol, -SH), C 1-10 Examples of substituted alkyl groups include halogenated alkyl groups, hydroxyalkyl groups, aminoalkyl groups, alkoxyalkyl groups, and alkaryl groups. As used herein, the term "alkaryl group" refers to a C alkyl group in which at least one hydrogen atom is replaced by an aryl group. 1-10 Alkyl. Examples of such groups include, but are not limited to, benzyl (phenylmethyl, PhCH2-), diphenylmethyl (Ph2CH-), triphenylmethyl (triphenylmethyl, Ph3C-), phenethyl (phenylethyl, Ph-CH2CH2-), styryl (Ph-CH=CH-), cinnamyl (Ph-CH=CH-CH2-). Typically, substituted C 1-10 The alkyl group carries 1, 2 or 3 substituents, for example 1 or 2.

[0068] An alkenyl group is an unsubstituted or substituted straight-chain or branched unsaturated hydrocarbon group having one or more, for example one or two, double bonds. Typically, an alkenyl group has 2 to 20 carbon atoms, i.e., an alkenyl group is C 2-20 Typically, alkenyl is C 2-10 Alkenyl. Alkenyl can be, for example, C 2-6 alkenyl, such as ethenyl, propenyl, butenyl, pentenyl or hexenyl, or C 2-4 alkenyl, such as vinyl, isopropenyl, n-propenyl, tert-butenyl, sec-butenyl or n-butenyl. When alkenyl is substituted, it generally carries one or more substituents selected from substituted or unsubstituted C 1-10 alkyl, substituted or unsubstituted aryl (as defined herein), cyano, amino, C 1-10 Alkylamino, di(C 1-10 ) alkylamino, arylamino, diarylamino, arylalkylamino, amide, acylamide, hydroxy, oxo, halogen, carboxyl, ester, acyl, acyloxy, C 1-10 Alkoxy, aryloxy, halogenated alkyl, sulfonic acid, mercapto (i.e., thiol, -SH), C 1-10 Examples of substituted alkenyl groups include halogenated alkenyl, hydroxyalkenyl, aminoalkenyl, alkoxyalkenyl and alkenylaryl. As used herein, the term "alkenylaryl" refers to a C substituted alkenyl group in which at least one hydrogen atom is replaced by an aryl group. 2-10 Examples of such groups include, but are not limited to, styryl (PhCH=CH-), Ph2C=CH-, PhCH=C(Ph)-, and cinnamyl (Ph-CH=CH-CH2-). Typically, the substituted C 2-10 Alkenyl carries 1 , 2 or 3 substituents, for example 1 or 2.

[0069] Alkynyl is an unsubstituted or substituted straight-chain or branched unsaturated hydrocarbon radical having one or more, for example one or two, triple bonds. Typically, an alkynyl radical has 2 to 20 carbon atoms, i.e., an alkynyl radical is C 2-20 Typically, an alkynyl group is a C 2-10 Alkynyl. Alkynyl can be, for example, C 2-6 Alkynyl, such as ethynyl, propynyl, butynyl, pentynyl or hexynyl, or C 2-4 Alkynyl, such as ethynyl, isopropynyl, n-propynyl, tert-butynyl, sec-butynyl or n-butynyl. When alkynyl is substituted, it generally carries one or more substituents selected from substituted or unsubstituted C 1-10 alkyl, substituted or unsubstituted aryl (as defined herein), cyano, amino, C 1-10 Alkylamino, di(C 1-10) alkylamino, arylamino, diarylamino, arylalkylamino, amide, acylamide, hydroxy, oxo, halogen, carboxyl, ester, acyl, acyloxy, C 1-10 Alkoxy, aryloxy, halogenated alkyl, sulfonic acid, mercapto (i.e., thiol, -SH), C 1-10 Examples of substituted alkynyl groups include halogenated alkynyl, hydroxyalkynyl, aminoalkynyl, alkoxyalkynyl, and alkynylaryl. As used herein, the term "alkynylaryl" refers to a C alkynyl group in which at least one hydrogen atom is replaced by an aryl group. 2-10 Examples of such groups include, but are not limited to, Ph-C≡C-, Ph-C≡C-CH2-, HC≡C-CH(Ph)-, and HC≡C-CPh2-. 2-10 The alkynyl group carries 1 , 2 or 3 substituents, for example 1 or 2.

[0070] Cycloalkyl, which may also be referred to as carbocyclyl, is an unsubstituted or substituted hydrocarbon radical which is also a cyclic radical, i.e., a monovalent moiety obtained by removing a hydrogen atom from an alicyclic atom of a carbocyclic ring of a carbocyclic compound. Typically, this moiety has 3 to 10 carbon atoms (unless otherwise specified), including 3 to 10 ring atoms, in which case it is referred to as C 3-10 Cycloalkyl or C 3-10 Thus, the term "cycloalkyl" includes the subclasses cycloalkenyl and cycloalkynyl. 3-10 Examples of the cycloalkyl group include C 3-7 Cyclic hydrocarbon group. 3-10 When the cycloalkyl group is substituted, it generally carries one or more substituents selected from unsubstituted C 1-6 alkyl, aryl (as defined herein), cyano, amino, C 1-10 Alkylamino, di(C 1-10 ) alkylamino, arylamino, diarylamino, arylalkylamino, amide, acylamide, hydroxy, oxo, halogen, carboxyl, ester, acyl, acyloxy, C 1-20 Alkoxy, aryloxy, halogenated alkyl, sulfonic acid, mercapto (i.e., thiol, -SH), C 1-10 alkylthio, arylthio, phosphate, phosphate ester, phosphonic acid and phosphonic acid ester, and sulfonyl. 3-10 The cycloalkyl group carries 1, 2 or 3 substituents, for example 1 or 2.

[0071] C 3-10 Examples of cycloalkyl groups include, but are not limited to, saturated monocyclic hydrocarbon compounds wherein C 3-10Cycloalkyl is unsubstituted or substituted as defined above: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7), methylcyclohexane (C7), dimethylcyclohexane (C8), menthane (C9), cyclohexane (C10), cyclohexane (C11), cyclohexane (C12), cyclohexane (C13), cyclohexane (C14), cyclohexane (C15), cyclohexane (C16), cyclohexane (C17), cyclohexane (C18), cyclohexane (C19), cyclohexane (C20), cyclohexane (C21 10 );

[0072] Unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7), methylcyclohexene (C7), dimethylcyclohexene (C8);

[0073] Saturated polycyclic hydrocarbons: thujone (C 10 ), carane (C 10 ), pinane (C 10 ), camphane (C 10 )、norcarane (C7), norcarane (C7), norbornane (C7), adamantane (C 10 ), decahydronaphthalene (decahydronaphthalene) (C 10 );

[0074] Unsaturated polycyclic hydrocarbon compounds: camphene (C 10 ), limonene (C 10 ), pinene (C 10 );

[0075] Polycyclic hydrocarbon compounds with aromatic rings: indene (C9), indane (e.g. 2,3-dihydro-1H-indene) (C9), tetralin (1,2,3,4-tetralin) (C 10 ).

[0076] A heterocyclyl group is an unsubstituted or substituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound. Typically, the moiety has 3 to 10 ring atoms (unless otherwise specified), of which 1 to 5 are ring heteroatoms, in which case it is referred to as a C 3-10 Preferably, each ring has 3 to 7 ring atoms, of which 1 to 4 are ring heteroatoms. 3-10 When the heterocyclic group is substituted, it generally carries one or more substituents selected from unsubstituted C 1-6 alkyl, aryl (as defined herein), cyano, amino, C 1-10 Alkylamino, di(C 1-10) alkylamino, arylamino, diarylamino, arylalkylamino, amide, acylamide, hydroxy, oxo, halogen, carboxyl, ester, acyl, acyloxy, C 1-20 Alkoxy, aryloxy, halogenated alkyl, sulfonic acid, mercapto (i.e., thiol, -SH), C 1-10 alkylthio, arylthio, phosphate, phosphate ester, phosphonic acid and phosphonic acid ester, and sulfonyl. 3-10 The heterocyclyl group carries 1 , 2 or 3 substituents, for example 1 or 2.

[0077] Examples of heterocyclyl groups include C 5-10 Heterocyclic group, C 3-7 Heterocyclic group, C 5-7 Heterocyclic and C 5-6 Heterocyclic group.

[0078] (Non-aromatic) monocyclic C 3-10 Examples of heterocyclic groups include, but are not limited to, those derived from:

[0079] N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g. 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoxazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);

[0080] O1: ethylene oxide (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxolene (dihydrofuran) (C5), ethylene oxide (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);

[0081] S1: ethylene sulfide (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiohexane (tetrahydrothiopyran) (C6), thioheptane (C7);

[0082] O2: dioxolane (C5), dioxane (C6) and dioxepane (C7);

[0083] O3: trioxane (C6);

[0084] N2: imidazolidine (C5), pyrazolidine (oxadiazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);

[0085] N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);

[0086] N1S1: Thiazoline (C5), Thiazolidine (C5), Thiomorpholine (C6);

[0087] N2O1: Oxadiazine (C6);

[0088] O1S1: oxathiolane (C5) and oxathiolane (thioxane) (C6); and

[0089] N1O1S1: Oxathiazine (C6).

[0090] Examples of substituted (non-aromatic) monocyclic heterocyclyls include those derived from cyclic forms of sugars, for example furanose (C5) such as arabinofuranose, lyxofuranose, ribofuranose and xylofuranose, and pyranose (C6) such as allopyranose, altrose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose and talopyranose.

[0091] C 3-10 Examples of heterocyclic groups which are also aryl groups are the heteroaryl groups described below.

[0092] Aryl is a substituted or unsubstituted monocyclic or fused polycyclic aromatic radical containing typically 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms, in the ring portion. Examples include phenyl (i.e., monocyclic), naphthyl, indenyl and indanyl (i.e., fused bicyclic), anthracenyl (i.e., fused tricyclic) and pyrenyl (i.e., fused tetracyclic). Aryl is unsubstituted or substituted. When an aryl as defined above is substituted, it typically carries one or more substituents selected from unsubstituted C1-C6 alkyl (to form an aralkyl), unsubstituted aryl, cyano, amino, C1-C6 alkyl ... 1-10 Alkylamino, di(C 1-10 ) alkylamino, arylamino, diarylamino, aralkylamino, amide, acylamide, hydroxy, halogen, carboxyl, ester, acyl, acyloxy, C 1-20 Alkoxy, aryloxy, halogenated alkyl, mercapto (i.e., thiol, -SH), C 1-10 Alkylthio, arylthio, sulfonic acid, phosphoric acid, phosphate, phosphonic acid and phosphonic acid esters and sulfonyl. Usually, they carry 0, 1, 2 or 3 substituents. The substituted aryl can be replaced by a single C 1-6 Hydrocarbylene or -XC 1-6 Alkylene or -XC 1-6 The bidentate substitution represented by alkylene-X-, wherein X is selected from O, S and NR, and wherein R is H, aryl or C 1-6 Thus, a substituted aryl group may be an aryl group fused to a cycloalkyl or heterocyclyl group. As used herein, the term "alkaryl" refers to a group in which at least one hydrogen atom (e.g., 1, 2, 3) is replaced by a C 1-6Aryl substituted aryl. Examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), cumyl (or cumyl, from cumene), and duryl (from durene).

[0093] As used herein, heteroaryl is a substituted or unsubstituted monocyclic or fused polycyclic (e.g., bicyclic or tricyclic) aryl group, typically containing 5 to 14 atoms in the ring portion, the ring portion including at least one heteroatom, such as 1, 2 or 3 heteroatoms, selected from O, S, N, P, Se and Si, more typically selected from O, S and N. Examples include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl, pyrazolyl, oxazolyl, isothiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, indolyl, indazolyl, carbazolyl, acridinyl, purinyl, cinnolinyl, quinoxalinyl, naphthyridinyl, benzimidazolyl, benzoxazolyl, quinolinyl, quinazolinyl and isoquinolinyl. Heteroaryl is often a 5- or 6-membered ring. However, as used herein, heteroaryl also includes fused polycyclic ring systems, including, for example, fused bicyclic ring systems in which heteroaryl is fused to aryl. When heteroaryl is such a fused heteroaryl, a preferred example is a fused ring system in which a 5- to 6-membered heteroaryl is fused to a phenyl group. Examples of such fused ring systems are benzofuranyl, isobenzofuranyl, benzothienyl, indolyl, indazolyl, benzimidazolyl, benzoxazolyl, quinolyl, quinazolinyl, and isoquinolyl moieties.

[0094] Heteroaryl may be unsubstituted or substituted, e.g. as specified above for aryl. Typically, it carries 0, 1, 2 or 3 substituents.

[0095] An alkylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from a hydrocarbon compound, both from the same carbon atom or each from two different carbon atoms, the hydrocarbon compound may be aliphatic or alicyclic and may be saturated, partially unsaturated or fully unsaturated. The hydrocarbon compound may have from 1 to 20 carbon atoms, in which case the alkylene group is C 1-20 The hydrocarbon compound may have, for example, 1 to 10 carbon atoms, in which case the alkylene group is C 1-10 Thus, the term "alkylene" includes subclasses such as alkenylene, alkynylene, and cycloalkylene discussed below. However, it is typically a saturated aliphatic (non-cyclic) group. It is typically C 1-6 Alkylene or C 1-4 A hydrocarbylene group such as methylene, ethylene, isopropylene, n-propylene, tert-butylene, sec-butylene, or n-butylene. It may be, for example, C 2-4 Alternatively, it may be, for example, C2-3 Alkylene, such as ethylene, n-propylene, isopropylene. (Although in this article C 2-3 Hydrocarbylene usually refers to ethylene or n-propylene). It can also be pentene, hexene, heptene, octene and various branched isomers thereof. Hydrocarbylene can be unsubstituted or substituted, for example, as specified above for alkyl. Typically, substituted hydrocarbylene carries 1, 2 or 3 substituents, for example 1 or 2.

[0096] Typically, an alkylene group is typically derived from an alkyl group as defined herein, for example, from a C 1-10 An unsubstituted or substituted bidentate moiety derived from an alkyl group by removing two hydrogen atoms, the two hydrogen atoms being derived from the same carbon atom or from two different carbon atoms. Typically, an alkenylene group is obtained by removing two hydrogen atoms from an alkenyl group as defined herein, for example from a C 2-10 An unsubstituted or substituted bidentate moiety derived from an alkenyl group by removing two hydrogen atoms, both from the same carbon atom or each from two different carbon atoms. Typically, an alkynylene group is obtained by removing two hydrogen atoms from an alkynyl group as defined herein, for example from a C 2-10 Alkynyl is an unsubstituted or substituted bidentate moiety derived by the removal of two hydrogen atoms, both from the same carbon atom or each from two different carbon atoms.

[0097] In this context, a prefix (e.g., C 1-4 , C 1-7 , C 1-10 , C 2-7 , C 3-7 , etc.) represents the number of carbon atoms or the range of the number of carbon atoms. For example, as used herein, the term "C 1-4 The term "alkylene" refers to an alkylene group having 1 to 4 carbon atoms. Examples of alkylene groups include C 1-4 Alkylene ("lower alkylene"), C 1-7 Alkylene and C 1-10 Alkylene.

[0098] Straight chain saturated C 1-7 Examples of alkylene groups include, but are not limited to, -(CH2) n -, wherein n is an integer from 1 to 7, for example -CH2-(methylene), -CH2CH2-(ethylene), -CH2CH2CH2-(propylene) and -CH2CH2CH2CH2-(butylene).

[0099] Branched chain saturated C 1-7Examples of alkylene groups include, but are not limited to, -CH(CH3)-, -CH(CH3)CH2-, -CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH2CH3)-, -CH(CH2CH3)CH2-, and -CH2CH(CH2CH3)CH2-.

[0100] Straight chain partially unsaturated C 1-7 Examples of alkylene groups include, but are not limited to, -CH=CH-(vinylene), -CH=CH-CH2-, -CH2-CH=CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH2-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-, -CH=CH-CH=CH-CH2-, -CH=CH-CH2-CH=CH-, and -CH=CH-CH2-CH2-CH=CH-.

[0101] Branched partially unsaturated C 1-7 Examples of alkylene groups include, but are not limited to, -C(CH3)=CH-, -C(CH3)=CH-CH2-, and -CH=CH-CH(CH3)-.

[0102] Alicyclic saturated C 1-7 Examples of alkylene groups include, but are not limited to, cyclopentylene (eg, cyclopent-1,3-ylidene) and cyclohexylene (eg, cyclohex-1,4-ylidene).

[0103] Alicyclic partially unsaturated C 1-7 Examples of alkylene groups include, but are not limited to, cyclopentenylene (eg, 4-cyclopentene-1,3-ene), cyclohexenylene (eg, 2-cyclohexene-1,4-ene; 3-cyclohexene-1,2-ene; 2,5-cyclohexadiene-1,4-ene).

[0104] As defined herein, alkylene, alkenylene, alkynylene and alkyl are continuous or interrupted by one or more heteroatoms or heteroradicals, such as S, O or N(R"), wherein R" is H, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, alkylene, Alkyl groups which are uninterrupted or interrupted between adjacent carbon atoms by heteroatoms such as oxygen or sulfur, or by hetero groups such as N(R"), wherein R" is as defined above, or by arylene or heteroarylene (typically arylene, more typically phenylene) groups, or by -C(O)-, -C(O)O- or -C(O)N(R")- groups, wherein R" is as defined above, or for example by one or more carbocyclylene or heterocyclylene groups, each of which may be unsubstituted or substituted.

[0105] For example, a C such as n-butyl 1-10 The alkyl group may be interrupted by heteroaryl N(R") groups such as -CH2N(R")CH2CH2CH3, -CH2CH2N(R")CH2CH3, or -CH2CH2CH2N(R")CH3. Similarly, an alkylene group such as n-butylene may be interrupted by heteroaryl N(R") groups such as -CH2N(R")CH2CH2CH2-, -CH2CH2N(R")CH2CH2-, or -CH2CH2CH2N(R")CH2-. Typically, an interrupted C 1-10 Alkylene or C 1-10 Interrupted alkyl groups are interrupted by 1, 2 or 3 heteroatoms or hetero groups, or by 1, 2 or 3 arylene groups (usually phenylene groups). More typically, such as interrupted C 1-10 Alkylene or C 1-10 Interrupted alkyl groups are interrupted by 1 or 2 heteroatoms or heteroradicals, or by 1 or 2 arylene groups (usually phenylene groups). For example, a C 1-20 The alkyl group may be interrupted by 2 heteroradicals N(R") as follows: -CH2N(R")CH2N(R")CH2CH3.

[0106] Arylene is an unsubstituted or substituted monocyclic or fused polycyclic bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, the moiety having 5 to 14 ring atoms (unless otherwise specified). Typically, each ring has 5 to 7 or 5 to 6 ring atoms. Arylene can be unsubstituted or substituted, for example, as specified above for aryl.

[0107] In this context, a prefix (such as C 5-20, C 6-20 , C 5-14 , C 5-7 , C 5-6 ) represents the number of ring atoms or range of ring atoms, whether carbon atoms or heteroatoms. For example, as used herein, the term "C 5-6 "Arylene" refers to an arylene group having 5 or 6 ring atoms. Examples of arylene groups include C 5-20 Arylene, C 6-20 Arylene, C 5-14 Arylene, C 6-14 Arylene, C 6-10 Arylene, C 5-12 Arylene, C 5-10 Arylene, C 5-7 Arylene, C 5-6 arylene group, C5 arylene group and C6 arylene group.

[0108] The ring atoms may be, for example, a "carboarylene" (e.g., C 6-20 Carboarylene, C 6-14 Carboarylene or C 6-10 All carbon atoms in the carboarylene group.

[0109] C without ring heteroatoms 6-20 Arylene (i.e. C 6-20 Examples of carboarylene groups include, but are not limited to, those derived from the compounds discussed above with respect to aryl groups, such as phenylene, and also include those derived from aryl groups bonded together, such as phenylene-phenylene (diphenylene) and phenylene-phenylene-phenylene (triphenylene).

[0110] Alternatively, the ring atoms may include, for example, a "heteroarylene" (e.g., C 5-14 One or more heteroatoms in a heteroarylene group. 5-14 Examples of heteroarylene groups include, but are not limited to, those derived from the compounds discussed above with respect to heteroaryl groups.

[0111] A carbocyclylene group is an unsubstituted or substituted divalent moiety obtained by removing two hydrogen atoms from the same or different ring carbon atoms of a carbocyclic compound. Typically, this moiety has 3 to 10 carbon atoms, including 3 to 10 ring atoms, in which case it is referred to as a C 3-10 Carbocyclylene. Carbocyclylene may be unsaturated or saturated, and thus includes the subclasses of cycloalkenylene and cycloalkynylene. It may also be unsubstituted or substituted, as specified above for carbocyclyl and cycloalkyl.

[0112] A heterocyclylene group is an unsubstituted or substituted divalent moiety obtained by removing two hydrogen atoms from the same or different ring carbon atoms of a heterocyclic compound. Typically, the moiety has 3 to 10 carbon atoms, including 3 to 10 carbon atoms, of which 1 to 5 are ring heteroatoms, in which case it is referred to as a C 3-10 Heterocyclylene. When a heterocyclylene group is substituted it typically carries one or more, typically 1, 2 or 3, substituents as defined above for a heterocyclyl group.

[0113] As used herein, the term "oxo" represents a group of the general formula =0.

[0114] As used herein, the term "acyl" represents a group of the general formula C(=O)R, wherein R is an acyl substituent, such as a substituted or unsubstituted C 1-20 Alkyl, C 1-20 Perfluoroalkyl, substituted or unsubstituted C 3-10 Cycloalkyl, substituted or unsubstituted C 3-10 Heterocyclic, substituted or unsubstituted aryl, perfluoroaryl or substituted or unsubstituted heteroaryl. Examples of acyl include, but are not limited to -C(=O)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=O)C(CH3)3 (tert-butyryl) and -C(=O)Ph (benzoyl, acylphenyl).

[0115] As used herein, the term "acyloxy" (or reverse ester) represents a group of the general formula -OC(=O)R, wherein R is an acyloxy substituent, such as a substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-10 Heterocyclic group, or substituted or unsubstituted aryl group, usually C 1-6 Examples of acyloxy groups include, but are not limited to, -OC(=O)CH3 (acetoxy), -OC(=O)CH2CH3, -OC(=O)C(CH3)3, -OC(=O)Ph, and -OC(=O)CH2Ph.

[0116] As used herein, the term "ester" (or carboxylate, carboxylate, or oxycarbonyl) represents a group of the general formula -C(=O)OR, wherein R is an ester substituent, such as a substituted or unsubstituted C 1-10 Alkyl, substituted or unsubstituted C 3-20 Heterocyclic group or substituted or unsubstituted aryl group (usually phenyl group).Examples of ester groups include, but are not limited to, -C(=O)OCH3, -C(=O)OCH2CH3, -C(=O)OC(CH3)3 and -C(=O)OPh.

[0117] As used herein, the term "amino" represents a group of the general formula -NH2. 1-10"Alkylamino" means a group of formula -NHR' wherein R' is C as defined above 1-10 Alkyl, preferably C 1-6 The term "di(C 1-10 )alkylamino" represents a group of the general formula -NR'R"-, wherein R' and R" are the same or different and represent a C 1-10 Alkyl, preferably C 1-6 The term "arylamino" represents a radical of the formula -NHR', wherein R' is an aryl radical as defined above, preferably a phenyl radical. The term "diarylamino" represents a radical of the formula -NR'R" wherein R' and R" are the same or different and represent an aryl radical as defined above, preferably a phenyl radical. The term "arylalkylamino" represents a radical of the formula -NR'R" wherein R' is a C 1-10 Alkyl, preferably C 1-6 R" is an alkyl group, and R" is an aryl group, preferably a phenyl group.

[0118] A halogen group is chlorine, fluorine, bromine or iodine (chloro, fluoro, bromo or iodo). Typically, it is chlorine, fluorine or bromine.

[0119] As used herein, the term "amide" represents a group of the general formula -C(=O)NR'R", wherein R' and R" are independently selected from H, C 1-10 Alkyl and aryl. Examples of amide groups include, but are not limited to, -C(=O)NH2, -C(=O)NHCH3, -C(=O)N(CH3)2, -C(=O)NHCH2CH3, and -C(=O)N(CH2CH3)2, as well as amide groups in which R' and R" together with the nitrogen atom to which they are attached form a heterocyclic structure such as piperidinylcarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinecarbonyl.

[0120] As used herein, the term "acylamide" refers to a radical of the formula -NR 1 C(=O)R 2 A group in which R 1 is an amide substituent, such as hydrogen, C 1-10 Alkyl, C 3-20 Heterocyclic or aryl, preferably hydrogen or C 1-10 Alkyl, R 2 is an acyl substituent, such as C 1-10 Alkyl, C 3-20 Preferably, R 1 is hydrogen, R 2 It is C 1-10 Examples of acylamide groups include, but are not limited to, -NHC(=O)CH3, -NHC(=O)CH2CH3, -NHC(=O)Ph, -NHC(=O)C 15 H 31and -NHC(=O)C9H 19 Therefore, the substituted C 1-10 The alkyl group may comprise a group having the general formula -NHC(=O)-C 1-10 Alkyl defines an acylamide substituent, such as -NHC(=O)C5H 11 or -NHC(=O)C9H 19 . R 1 and R 2 A cyclic structure such as in succinimidyl, maleimide and phthalimide groups can be formed together.

[0121]

[0122] C 1-10 Alkylthio is the above C 1-10 Alkyl, preferably C 1-6 Arylthio is an aryl group connected to a thio group, preferably a phenyl group.

[0123] C 1-10 Alkoxy is a substituted or unsubstituted C 1-10 Alkyl. C 1-6 Alkoxy is a substituted or unsubstituted C 1-6 Alkyl. C 1-4 Alkoxy is a substituted or unsubstituted C 1-4 Alkyl. 1-10 , C 1-6 and C 1-4 The alkyl group is optionally interrupted as defined herein. 1-4 Examples of alkoxy groups include -OMe (methoxy), -OEt (ethoxy), -O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy). 1-20 Other examples of alkoxy include -O(adamantyl), -O-CH2-adamantyl and -O-CH2-CH2-adamantyl. Aryloxy is a substituted or unsubstituted aryl group as defined herein attached to an oxygen atom. An example of an aryloxy is -OPh(phenoxy).

[0124] Unless otherwise indicated, the above includes well-known ionic, salt, solvate and protected forms of these substituents. For example, reference to carboxylic acid, carboxyl or carboxyl (-COOH) also includes the anionic (carboxylate) form (-COO-), its salt or solvate, and conventional protected forms. Similarly, reference to amino includes the protonated form (-N + HR 1R 2 ), salts or solvates of amino groups, such as hydrochlorides, and conventional protected forms of amino groups. Similarly, references to hydroxyl groups (-OH) also include anionic forms (-OH). - ), its salts or solvates, and conventional protected forms.

[0125] Certain compounds may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, atriomeric, stereoisomeric, tautomeric, conformational or anomeric forms, including but not limited to cis and trans forms; E and Z forms; c-, t- and r- forms; endo and exo forms; R-, S- and meso forms; D- and L- forms; d- and l- forms; (+) and (-) forms; keto, enol and enolate forms; cis and trans forms; syncline and anticline forms; α- and β- forms; axial and equatorial forms; boat, chair, twist-boat, envelope and half-chair forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomer forms").

[0126] Note that, except as discussed below for tautomeric forms, structural (or constitutional) isomers (i.e., isomers that differ in the connections between atoms rather than in the positions of the atoms in space) are explicitly excluded from the term "isomers" as used herein. For example, a reference to methoxy, -OCH3, would not be construed as a reference to its structural isomer, hydroxymethyl, -CH2OH. Similarly, a reference to o-chlorophenyl would not be construed as a reference to its structural isomer, m-chlorophenyl. However, a reference to a class of structures is likely to include structural isomeric forms belonging to that class (e.g., C 1-7 The alkyl group includes n-propyl and isopropyl; the butyl group includes n-butyl, isobutyl, sec-butyl, and tert-butyl; and the methoxyphenyl group includes o-, m-, and p-methoxyphenyl).

[0127] The above exclusion does not relate to tautomeric forms, such as keto, enol and enolate forms, such as, for example, the following tautomeric pairs: keto / enol (shown below), imine / enamine, amide / imino alcohol, amidine / amidine, nitroso / oxime, thione / enethiol, N-nitroso / hydroxyazo and nitro / aci-nitro.

[0128]

[0129] Unless otherwise indicated, reference to a particular compound includes all such isomeric forms, including (whole or partial) racemic forms and other mixtures. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatography) of such isomeric forms are known in the art or are readily obtained by adapting known methods in a known manner.

[0130] Unless otherwise indicated, reference to a particular compound or complex also includes ionic, salt, solvated and protected forms.

[0131] Molecular Hopper

[0132] As described above, the present invention provides a method for moving a molecular hopper along a track, wherein:

[0133] (a) the track comprises a plurality of primary functional groups arranged along a substrate;

[0134] (b) the hopper comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track; and

[0135] (c) said hopper optionally contains a cargo portion,

[0136] The method comprises the following steps:

[0137] (i) contacting the hopper with the track so that the secondary functional group of the hopper is combined with the first primary functional group on the track;

[0138] (ii) applying a driving force to directionally transfer the hopper from the first primary functional group to the second primary functional group on the track, thereby moving the hopper along the track.

[0139] Hopper movement

[0140] Advantageously, in the present invention, the movement of the hopper along the track can be controlled. In other words, the movement of the hopper along the track is not just a random walk. Instead, the movement of the hopper can be determined by applying a driving force as described herein. In this way, the hopper can perform useful work, such as by directing the movement of cargo molecules toward a desired destination or a desired direction. Non-directional random walks of molecules along tracks and capture by thermodynamic cold sources have been described in the literature, but providing a directional system that determines the direction by applying a driving force is a significantly more complex challenge solved by the present invention.

[0141] Typically, in the present invention, the direction of the driving force relative to the track determines the overall direction of movement of the bucket along the track. Typically, the bucket moves along the track in the same direction as the direction in which the force is applied. For example, if the driving force is applied in the direction from the beginning of the track to the end of the track, the overall movement of the bucket is typically also from the beginning of the track to the end of the track. Similarly, if the direction of the driving force is reversed so that the driving force is applied in the direction from the end of the track to the beginning of the track, the overall movement of the bucket is also reversed, that is, from the end of the track to the beginning of the track. However, the present invention also includes methods in which the movement of the bucket is determined by the direction of the driving force relative to the track, and the overall movement of the bucket is opposite to the driving force.

[0142] In the present invention, the transfer of the hopper from the first primary functional group to the second primary functional group on the track is generally independent of the addition of an exogenous fuel such as a chemical reagent. In other words, a catalyst is generally not required for transferring the hopper from the first primary functional group to the second primary functional group on the track, although the use of such a catalyst is not excluded in the present invention. When there is no catalyst or exogenous "fuel", the movement of the hopper along the track is said to be autonomous. This movement is in contrast to systems that require the use of sacrificial fuel molecules to cause movement.

[0143] Typically, in the present invention, step (ii) of the method includes applying a driving force to the hopper so that the hopper is sequentially transferred between each of the multiple primary functional groups on the track, so that the hopper moves along the track. Those skilled in the art will recognize that the driving force can be applied directly to the hopper, or can be applied to the hopper via the track. For example, if the track is the surface of the hole and the hopper moves along the track, the force can be, for example, a physical or chemical potential applied from one side of the hole to the other side. For example, an electric potential can be applied across the hole. Such a driving force acts on the hopper so that the hopper is sequentially transferred between each functional group on the track to move along the track. From the above discussion, it is obvious that the direction of movement of the hopper is generally the same as the direction of the applied force. By way of non-limiting example, if an electric potential is applied from the "cis" side of the hole to the "trans" side on the entire track in the hole, the movement of the hopper along the track is generally also in the "cis to trans" direction. The applied force causes the hopper to move in a defined direction, and therefore the overall movement of the hopper is not random.

[0144] The movement of the hopper is typically highly progressive. Backing off is highly undesirable and is not typically observed. The hopper does not spontaneously disengage from the track, although release from the track can be controlled by appropriate design of the track to provide a release point as described herein. Typically, the hopper is able to complete more than 10 steps, such as more than 20 steps, such as more than 30 steps, such as more than 50 steps, such as more than 100 steps, such as more than 150 steps, such as more than 200 steps, such as more than 250 steps, such as more than 500 steps, such as more than 1000 steps without disengaging from the track.

[0145] Base

[0146] In the present invention, the track includes a plurality of primary functional groups arranged along the substrate.

[0147] Typically, substrates are organic or inorganic surfaces comprising a plurality of primary functional groups. Any suitable organic or inorganic surface can be used in such methods. The substrate can be the surface of an inorganic substance, for example, a surface of a metal having a semiconductor material such as an oxide coating or such as silicon or a compound thereof. The substrate can be organic, for example, the substrate can be a polymer or a protein or any other organic substrate. The polymer substrate can include naturally occurring or non-naturally occurring polymers. Similarly, the protein substrate can include naturally occurring proteins or non-naturally occurring proteins.

[0148] Non-naturally occurring proteins may contain only naturally occurring amino acids, or may contain one or more non-naturally occurring amino acids (also referred to as atypical amino acids). Naturally occurring proteins can be produced in various ways known in the art, and the methods used for their production are not limitations of the present invention. Known methods include expression in bacteria, yeast or insect cells by suitable plasmids, followed by appropriate processing, such as purification, as needed. Cell-free expression systems (e.g., in vitro transcription / translation systems) can also be used. Synthetic approaches, such as natural chemical ligation methods, can also be used. Similar methods can also be used to produce non-naturally occurring proteins using appropriate plasmids encoding the desired protein sequence or using non-natural amino acids incorporated by synthetic or biological means. Methods for producing proteins are described in references known to those skilled in the art, such as Green and Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press (4 th edition).

[0149] Typically, in the present invention, the substrate is the surface of the transmembrane pore. The surface may be an inner surface or an outer surface. Typically, in the present invention, the substrate is the inner surface of the transmembrane pore.

[0150] As described below, any suitable transmembrane pore can be used in the present invention. The nanopore can be naturally occurring (e.g., pore-forming protein) or can be synthetically produced, such as a hole (pore) produced in a synthetic material such as a silicon compound (e.g., silicon nitride) or graphene. For example, the transmembrane pore can be a protein nanopore, a solid-state nanopore, a DNA nanopore, a polymer nanopore, or a synthetic or semi-synthetic nanopore. The first transmembrane pore may be included in the second transmembrane pore, for example, the protein nanopore may be located in the solid-state nanopore. When the substrate is an organic or inorganic surface, the hole may be a well, a gap, a channel, a groove, or a slit in the surface. Typically, in the present invention, the transmembrane pore is a protein or solid-state nanopore. More often, the transmembrane pore is a transmembrane β-barrel protein nanopore. Common transmembrane pores are described in more detail below.

[0151] When the substrate is a surface of transmembrane pores, the pores may be contained in an array of pores. Thus, an array of tracks may be formed in an array of pores. The pores may be identical or different. The array may be formed, for example, by positioning protein nanopores in an array formed on a solid surface, for example in a solid-state nanopore array.

[0152] hole

[0153] A transmembrane pore is a structure that passes through the membrane to some extent. It allows hydrated ions driven by an applied potential to flow through or within the membrane. A transmembrane pore usually passes through the entire membrane so that hydrated ions can flow from one side of the membrane to the other side of the membrane. However, a transmembrane pore does not necessarily have to pass through the membrane. It can be closed at one end.

[0154] Transmembrane pores suitable for use in the present invention may be solid-state pores. Solid-state nanopores are typically nanometer-sized holes formed in synthetic membranes. Suitable solid-state pores include, but are not limited to, silicon nitride pores, silicon dioxide pores, and graphene pores. Solid-state nanopores may be manufactured, for example, by focused ion beams or electron beams, so that the size of the pores can be freely adjusted. Suitable solid-state pores and methods for preparing them are discussed in U.S. Patent No. 6464842, WO 03 / 003446, WO 2005 / 061373, U.S. Patent No. 7258838, U.S. Patent No. 7466069, U.S. Patent No. 7468271, and U.S. Patent No. 7253434.

[0155] The transmembrane pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936). DNA origami pores are disclosed in WO2013 / 083983.

[0156] The transmembrane pore may be a polymer based pore. Suitable pores can be formed from polymer based plastics, for example polyesters such as polyethylene terephthalate (PET), by track etching.

[0157] Transmembrane pores suitable for use in the present invention may be transmembrane protein pores. Transmembrane protein pores are polypeptides or collections of polypeptides that allow ions driven by an applied potential to flow from one side of the membrane to the other side of the membrane. Transmembrane protein pores are particularly suitable for use in the present invention.

[0158] The transmembrane protein pore can be isolated, substantially isolated, purified or substantially purified. A pore is isolated or purified if it is completely free of any other components, such as lipids or other pores. A pore is substantially isolated if it is mixed with a carrier or diluent that does not interfere with its intended use. For example, a pore is substantially isolated or substantially purified if it is present in a form that contains less than 10%, less than 5%, less than 2% or less than 1% of other components, such as lipids or other pores. The pore is typically present in a membrane, such as a lipid bilayer or a synthetic membrane such as a block copolymer membrane.

[0159] The transmembrane protein pore may be a monomer or an oligomer. The transmembrane protein pore often consists of several repeating subunits, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or at least 16 subunits. The pore is typically a hexameric, heptameric, octameric or nonameric pore.

[0160] The pore may be a homo-oligomer or a hetero-oligomer. The transmembrane protein pore may be a heptameric pore. The transmembrane protein pore may generally include a barrel or channel through which ions may flow. The subunits of the pore generally surround a central axis and contribute chains to a transmembrane beta barrel or channel or a transmembrane alpha helical bundle or channel.

[0161] Suitable transmembrane pores for use in the present invention can be β barrel pores, α helical bundle pores or solid state pores. β barrel pores include barrels or channels formed by β strands. Suitable β barrel pores include, but are not limited to, β toxins such as α-hemolysin, anthrax toxin and leukocidin, and other membrane proteins / porins of bacteria, such as Mycobacterium smegmatis porins (Msp), such as MspA, MspB, MspC or MspD, CsgG, outer membrane porins F (OmpF), outer membrane porins G (OmpG), outer membrane phospholipase A and Neisseria autotransporting lipoprotein (NalP) and other pores, such as lysin. α helical bundle pores include barrels or channels formed by α helices. Suitable α helical bundle pores include, but are not limited to, inner membrane proteins and α outer membrane proteins, such as Wza and ClyA toxins. For example, the transmembrane pore may be derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, and the hemolytic protein FraC.

[0162] For example, the pore may be derived from α-hemolysin (α-HL). The wild-type α-HL pore is formed by seven identical monomers or subunits (i.e., it is heptameric). The sequence of one wild-type monomer or subunit of α-hemolysin is shown in SEQ ID NO: 1. Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290, and 293 of SEQ ID NO: 1 form a loop region. Residues 111, 113, and 147 of SEQ ID NO: 1 form part of the constriction of the barrel or channel of α-HL.

[0163] In certain embodiments, the transmembrane protein pore is chemically modified. For example, by adding histidine residues (His tags), aspartic acid residues (Asp tags), streptavidin tags and / or flag tags, or by adding signal sequences to promote their secretion from cells in which the polypeptide does not naturally contain such sequences, the pore (e.g., a monomer derived from α-HL (i.e., SEQ ID NO: 1 or a variant thereof)) can be modified to assist in its identification or purification. For example, a His tag and an Asp tag can be included in the transmembrane pore to assist in purification. Such a strategy is used in the embodiment in which the α-HL monomer is modified with 8 aspartic acid and 6 histidine (D8H6) residues to assist in purification and characterization. The sequence of WT α-HL-D8H6 is given in SEQ ID NO: 2. An alternative to introducing a genetic tag is to chemically react the tag to a natural or engineered position on the pore. An example thereof is reacting a gel shift reagent with a cysteine ​​engineered on the outside of the pore. This has been demonstrated as a method for isolating hetero-oligomers of α-HL (Chem Biol. 1997 Jul; 4(7): 497-505).

[0164] As described above, the track can be present in a transmembrane protein hole (i.e., the substrate can include the surface of a transmembrane protein hole). Therefore, the substrate can be the inner surface of a barrel or channel or lumen of a transmembrane protein nanopore such as a transmembrane protein nanopore described herein. Typically, the substrate is the inner surface of the barrel or channel of the hole. The barrel or channel of the hole is the part of the hole through which ions pass through the membrane through the hole. When the track is in the barrel or channel of the hole, the hopper usually moves along the track through the barrel or channel. Depending on the type of hole, the barrel or channel can be formed by an alpha helix or a beta chain. The track can be located near the constriction of the barrel or channel or can span the constriction of the barrel or channel. In another embodiment, the track can be located at the entrance of the hole. The entrance of the hole is the part of the hole where the analyte enters the barrel or channel. The entrance is usually formed mainly by the ring area in the hole.

[0165] Tracks may be naturally present in naturally occurring proteins or may be introduced. For example, as described in more detail in the Examples, tracks may be generated in transmembrane protein pores such as α-hemolysin, Msp, lysin, CsgG, ClyA, Sp1 and the hemolytic protein hemolytic toxin C (FraC) by introducing appropriate amino acid residues at appropriate sites in the amino acid sequence of the monomers and then oligomerizing the monomers to form hetero-oligomeric or homo-oligomeric pores. For example, with reference to the exemplary protein α-HL, positions 113, 115, 117, 119, 121 and / or 123 of SEQ ID NO: 1 may be modified, for example to contain amino acid residues such as cysteine ​​residues (as shown in SEQ ID Nos: 3 to 7), to generate thiol-containing tracks in SEQ ID NO: 1 monomers; the monomers may then be oligomerized to form transmembrane pores containing the tracks. Typically, such monomers are oligomerized to form hetero-oligomeric pores.

[0166] As is apparent from the above discussion, the present invention includes the use of tracks comprising a plurality of primary functional groups arranged along a substrate. Typically, the track comprises an array of natural and / or non-natural amino acid residues contained in the barrel and / or cavity of a transmembrane β-barrel protein nanopore, wherein each amino acid residue in the track comprises a primary functional group. Natural amino acids and non-natural amino acids are mentioned herein. As used herein, the term "natural amino acid" refers to one of the 20 classical amino acids: i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Selenocysteine ​​may also be considered as a naturally occurring amino acid. When the track comprises natural amino acids, it typically comprises cysteine ​​and / or selenocysteine ​​residues. The amino acids in the track can be D- or L-amino acids, generally L-amino acids. Non-natural (non-classical) amino acids can be synthetically prepared and introduced into proteins as described above. Those skilled in the art will understand that a protein comprising one or more non-natural amino acids is still referred to as a protein despite the incorporation of such non-naturally occurring (non-classical) amino acids.

[0167] When the track is contained on a protein substrate, the track may generally comprise an array of amino acids. The amino acids need not be identical, although identical amino acids are within the scope of the invention. Non-identical amino acids may be different residues (e.g., cysteine ​​and selenocysteine) or may be identical residues (e.g., cysteine) in different chemical environments, for example due to differences in local pH or pKa values ​​of surrounding amino acid residues.

[0168] The amino acid array is typically a one-dimensional array, such as a line of amino acids with equal or approximately equal spacing between amino acids. For more complex movements, the amino acid array can be a 2-dimensional array, such as a grid of amino acids, or a non-linear arrangement, such as a "U", "V", "L" or "Z" shape. Many other track geometries are possible and the present invention is not limited thereto.

[0169] Typically, in the present invention, the track comprises an array of amino acid residues that are approximately uniformly spaced along one or more beta strands in the barrel of a transmembrane beta barrel protein nanopore, wherein each amino acid residue in the track comprises a primary functional group. The amino acids may be natural or non-natural amino acids, and may be the same or different. Those skilled in the art will appreciate that by "uniformly spaced," it is meant that the "step" size between adjacent amino acid residues in the track is approximately constant, for example, the step size may be uniformly between about to about For example, between about to about (As promised ). Without being bound by theory, the inventors believe that when the hopper contains a polymer cargo, the size of the "step" between adjacent pivots, such as amino acid residues in the track, can be advantageously selected to be similar to the distance between adjacent monomer units on the cargo. For example, when the hopper contains a polynucleotide cargo, the inventors believe (without being bound by theory) that the distance between adjacent pivots, such as amino acid residues in the track, can be advantageously selected to be similar to the internucleotide spacing in the polynucleotide.

[0170] An exemplary track with two pivots can be generated by using positions 113 and 115, 115 and 117, 117 and 119, 119 and 121, or 121 and 123 of a monomer of SEQ ID NO: 1 or 2. For example, suitable amino acid residues, such as cysteine ​​or selenocysteine ​​(e.g., cysteine) residues, can be located at such points in the amino acid sequence of a protein monomer, and the monomers can then be oligomerized to form a hetero-oligomeric or homo-oligomeric pore. An example of a track with two pivots is provided in SEQ ID NO: 3, where the track is arranged along the inner surface of an α-HLβ-barrel protein nanopore.

[0171] A track with three pivots can be generated, for example, by using positions 113, 115, and 117; 115, 117, and 119; 117, 119, and 121; or 119, 121, and 123 of SEQ ID NO: 1 or 2 monomers. For example, suitable amino acid residues, such as cysteine ​​or selenocysteine ​​(e.g., cysteine) residues, can be located at such points in the amino acid sequence of protein monomers, and the monomers can then be oligomerized to form a hetero-oligomeric or homo-oligomeric pore. An example of a track with three pivots is provided in SEQ ID NO: 4, where the track is arranged along the inner surface of an α-HLβ-barrel protein nanopore.

[0172] A track with four pivots can be generated, for example, by using positions 113, 115, 117, and 119; 115, 117, 119, and 121; or 117, 119, 121, and 123 of SEQ ID NO: 1 or 2 monomers. For example, suitable amino acid residues, such as cysteine ​​or selenocysteine ​​(e.g., cysteine) residues, can be located at such points in the amino acid sequence of protein monomers, and the monomers can then be oligomerized to form a hetero-oligomeric or homo-oligomeric pore. An example of a track with four pivots is provided in SEQ ID NO: 5, where the track is arranged along the inner surface of an α-HLβ-barrel protein nanopore.

[0173] A track with five pivots can be generated, for example, by using positions 113, 115, 117, 119 and 121; or 115, 117, 119, 121 and 123 of SEQ ID NO: 1 or 2 monomers. For example, suitable amino acid residues, such as cysteine ​​or selenocysteine ​​(e.g., cysteine) residues, can be located at such points in the amino acid sequence of protein monomers, and the monomers can then be oligomerized to form a hetero-oligomeric or homo-oligomeric pore. An example of a track with five pivots is provided in SEQ ID NO: 6, where the track is arranged along the inner surface of an α-HLβ-barrel protein nanopore.

[0174] A track with six pivot points can be generated, for example, by using positions 113, 115, 117, 119, 121 and 123 of the monomers of SEQ ID NO: 1 or 2. For example, suitable amino acid residues, such as cysteine ​​or selenocysteine ​​(e.g., cysteine) residues, can be located at such points in the amino acid sequence of protein monomers, and the monomers can then be oligomerized to form a hetero-oligomeric or homo-oligomeric pore. An example of a track with six pivot points is provided in SEQ ID NO: 7, where the track is arranged along the inner surface of an α-HLβ-barrel protein nanopore.

[0175] Those skilled in the art will appreciate that other positions can be similarly modified to generate alternative tracks. Similarly, analogous positions in other protein sequences can be used (e.g., protein sequences of Msp, lysin, CsgG, ClyA, Sp1, or the hemolytic protein FraC).

[0176] In the present invention, a suitable substrate is a variant of a protein nanopore, such as a variant of any one of SEQ ID NOs: 1 to 7. A variant of a protein pore monomer is a subunit having an amino acid sequence that is different from a subunit of a reference sequence (e.g., a subunit from any one of SEQ ID NOs: 1 to 7) and retains its pore-forming ability. Suitable variants may include additional modifications, such as the introduction of positively or negatively charged (usually positively charged) residues, for example to facilitate interaction with an analyte.

[0177] The ability of the variant to form a pore can be determined using any method known in the art. For example, the variant can be inserted into a membrane together with other suitable subunits, and its ability to oligomerize to form a pore can be determined. Methods for inserting subunits into membranes such as lipid bilayers are known in the art. For example, the subunits can be suspended in a solution containing a lipid bilayer in a purified form so that they diffuse into the lipid bilayer and are inserted by binding to the lipid bilayer and assembling into a functional state. Alternatively, the subunits can be directly inserted into the membrane using the "pick and place" method described in MA Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT / GB2006 / 001057 (published as WO 2006 / 100484).

[0178] Those skilled in the art will appreciate that protein nanopores need not contain only naturally occurring amino acids. For example, non-naturally occurring amino acids can be introduced by any suitable means. The resulting polymer containing naturally occurring and / or non-naturally occurring amino acids comprises a "protein" nanopore. Depending on the specific movement of the hopper desired, the track can contain or consist of non-naturally occurring amino acids, or the substrate can be a protein containing non-naturally occurring amino acids and the track can contain naturally occurring amino acids.

[0179] Methods for adding or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced with arginine (R) by replacing the codon for methionine (ATG) with the codon for arginine (AGA) at the relevant position in the hole encoded by the polynucleotide. Similarly, other amino acids can be similarly introduced by using appropriate codons. The polynucleotide can then be expressed as described above.

[0180] Methods for adding or replacing non-natural amino acids are also well known in the art. For example, non-natural amino acids can be introduced by including synthetic aminoacyl-tRNA in an in vitro transcription / translation (IVTT) system for expressing pores. Alternatively, non-natural amino acids can be introduced by expressing pores in Escherichia coli (E. coli), which are auxotrophic for specific amino acids in the presence of synthetic (i.e., non-natural) analogs of those specific amino acids. If partial peptide synthesis is used to produce pores, non-natural amino acids can also be produced by natural ligation methods. Modified Escherichia coli (E. coli) containing genetically modified tRNA synthetase / tRNA can be used to incorporate non-natural amino acids into proteins, as described in Chin et al, Annu. Rev. Biochem. (2014) 83: 379-408.

[0181] In the present invention, any amino acid may be substituted with an appropriately functionalized amino acid to form tracks on a protein substrate. For example, one or more cysteine ​​amino acids may be substituted with one or more amino acids, such as when the primary functional group is a thiol group. Selenocysteine ​​may also be used, such as when the primary functional group is a selenol. Other amino acids containing other primary functional groups can be used.

[0182] Any number of appropriately functionalized amino acids may be introduced. For example, 1, 2, 3, 4, 5, 10, 15, 20, 25 or more appropriately functionalized amino acids may be introduced. In the case of α-HL (i.e., SEQ ID NO: 1), for example, by replacing amino acids at positions 113, 115, 117, 119, 121 and / or 123 of SEQ ID NO: 1 (as in SEQ ID NOs: 3 to 7), a track comprising, for example, 2, 3, 4 or 5 appropriately functionalized amino acid residues may be generated. When other proteins are used, amino acids at positions corresponding to positions 113, 115, 117, 119, 121 and / or 123 of SEQ ID NO: 1 may be replaced.

[0183] In addition to those described above, amino acid substitutions may be made to the amino acid sequences of SEQ ID NOs: 1 to 7, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties or similar side chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, conservative substitutions may introduce another amino acid of the aromatic or aliphatic family to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected based on the properties of the 20 major amino acids defined in Table 1 below. In the case of amino acids of similar polarity, this can also be determined by reference to the degree of hydrophilicity of the amino acid side chains in Table 2. Non-conservative substitutions can be made while the protein pore retains its structure and function.

[0184] Table 1 - Chemical properties of amino acids

[0185]

[0186] Table 2 - Hydrophilicity

[0187]

[0188] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may also be additionally deleted from the above polypeptides. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

[0189] Variants may also include fragments of SEQ ID NOs: 1 to 7 that retain pore forming activity. Fragments may be at least 50, 100, 200 or 250 amino acids in length. Fragments typically comprise the pore forming domain of SEQ ID NOs: 1 to 7. Fragments typically include residues 113, 115, 117, 119, 121 and / or 123 of SEQ ID NOs: 1 to 7. Alternatively or additionally, one or more amino acids may be added to the above polypeptides. Extensions may be provided at the amino or carboxyl terminus of the amino acid sequence of SEQ ID NOs: 1 to 7, or variants or fragments thereof. Extensions may be provided within the amino acid sequence of SEQ ID NOs: 1 to 7, or variants or fragments thereof, for example, an extension can be incorporated to increase the length of the barrel of the pore. The extension may be relatively short, for example 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids.

[0190] The variant can be a naturally occurring variant, which is naturally expressed by an organism, for example, expressed by Staphylococcus bacteria, or recombinantly expressed by bacteria such as Escherichia coli. Variants also include non-naturally occurring variants produced by recombinant techniques. Over the entire length of any amino acid sequence in SEQ ID NO:1 to 7, based on amino acid identity, variants are generally at least 50% homologous to the reference sequence. More generally, based on amino acid identity over the entire sequence of any amino acid sequence in SEQ ID NO:1 to 7, variant polypeptides can be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and more generally at least 95%, 97% or 99% homologous. There may be at least 80%, such as at least 85%, 90% or 95% amino acid identity over a stretch of 200 or more, such as 230, 250, 270, 280 or more contiguous amino acids ("hard homology").

[0191] Standard methods in the art can be used to determine homology. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology, such as using its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). For example, as described in Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, SF et al (1990) J Mol Biol 215: 403-10, PILEUP and BLAST algorithms can be used to calculate homology or arrange sequences (such as identifying equivalent residues or corresponding sequences (usually based on its default settings)).

[0192] Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive threshold score T when consistent with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. When the cumulative alignment score drops by a number X from its maximum achieved value; due to the accumulation of one or more negative-scoring residue alignments, the cumulative score reaches zero or lower; or when the end of either sequence is reached, the extension of word hits in each direction is paused. The BLAST algorithm parameters W, T, and X determine the sensitivity and rate of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919), alignment (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0193] The BLAST algorithm performs a statistical analysis of the similarity between two sequences, see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two amino acid sequences will occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability of a comparison of a first sequence to a second sequence is less than about 1, usually less than about 0.1, more often less than about 0.01, and most often less than about 0.001.

[0194] membrane

[0195] As described above, in the present invention, the substrate may be the surface of a transmembrane pore. A transmembrane pore is a structure that penetrates the membrane to some extent.

[0196] Any membrane can be used according to the present invention. Suitable membranes are well known in the art. The membrane is generally an amphiphilic layer. An amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids, the amphiphilic molecules having at least one hydrophilic part and at least one lipophilic or hydrophobic part. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles forming a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits polymerized together produce a single polymer chain. Block copolymers generally have properties contributed by each monomer subunit. However, block copolymers may have unique properties that are not possessed by polymers formed by separate subunits. It is possible to engineer block copolymers so that one of the monomer subunits is hydrophobic (i.e., lipophilic), while the other subunits are hydrophilic in an aqueous medium. In this case, the block copolymers may have amphiphilic properties and may form a structure that simulates a biofilm. Block copolymers can be diblock (composed of two monomer subunits), but can also be composed of more than two monomer subunits to form more complex arrangements that behave as amphiphiles. Copolymers can be triblock, tetrablock or pentablock copolymers. Typically, the copolymer is a triblock copolymer comprising two monomer subunits, A and B, in an ABA pattern; typically, the A monomer subunit is hydrophilic and the B subunit is hydrophobic.

[0197] The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer.

[0198] The amphiphilic layer is typically a lipid bilayer. The lipid bilayer is a model of a cell membrane and serves as an excellent platform for a range of experimental studies. For example, the lipid bilayer can be used for in vitro investigations of membrane proteins recorded by single channel. Alternatively, the lipid bilayer can be used as a biosensor to detect the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is typically a planar lipid bilayer. WO2008 / 102121, WO 2009 / 077734 and WO 2006 / 100484 disclose suitable lipid bilayers.

[0199] Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Examples. Lipid bilayers are generally formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), wherein a lipid monolayer is carried on an aqueous solution / air interface on either side of a pore perpendicular to the interface. The method of Montal and Mueller is popular because it is a cost-effective and relatively direct method for forming a high-quality lipid bilayer suitable for protein pore insertion. Other common methods for bilayer formation include tip dipping, spraying bilayers, and patch clamping of liposome bilayers. Lipid bilayers can be formed as described in WO 2009 / 077734. The lipid bilayer can also be a droplet interface bilayer formed between two or more aqueous droplets each containing a lipid shell, so that when the droplets contact, a lipid bilayer is formed at the interface of the droplets.

[0200] In another preferred embodiment, the membrane is a solid-state layer. The solid-state layer is not of biological origin. In other words, the solid-state layer is not derived from or isolated from a biological environment, such as an organism or cell, or a synthetically manufactured version of a biologically available structure. The solid-state layer can be formed of organic and inorganic materials, including but not limited to microelectronic materials, insulating materials such as Si3N4, Al2O3 and SiO, organic and inorganic polymers such as polyamides, such as Plastics or elastomers such as two-component addition-cured silicone rubber and glass. The solid layer can be formed by a single atomic layer or a layer only a few atoms thick, such as graphene. WO2009 / 035647 discloses suitable graphene.

[0201] hopper

[0202] In the present invention, the hopper comprises a secondary functional group capable of bonding with each of a plurality of primary functional groups on the track. The primary and secondary functional groups are described in more detail below.

[0203] The hopper can be any molecule that can be directed from the first primary functional group of the track to the second primary functional group of the track, thereby moving along the track. Typically, the hopper has a single foot (i.e., including a single secondary functional group that can bind to each of the multiple primary functional groups on the track, although there may be additional functional groups that cannot bind to each of the multiple primary functional groups on the track). However, a hopper suitable for use in the present invention may include two or more feet (i.e., two or more secondary functional groups that can bind to each of the multiple primary functional groups on the track, and optionally including additional functional groups that cannot bind to each of the multiple primary functional groups on the track). A hopper including two or more feet may also be referred to as a walker. Although the movement of the hopper from the first primary functional group to the second primary functional group may involve the instantaneous formation of species with two or more fulcrums, the movement of the hopper with a single foot does not involve stable intermediates that simultaneously occupy two or more fulcrums (i.e., simultaneously combining with two primary functional groups of the track to form a stable intermediate). In contrast, the movement of a walker with two or more feet involves the formation of a stable intermediate that occupies at least two fulcrums. Those skilled in the art will appreciate that while the present invention relates primarily to molecular hoppers, it also includes molecular walkers. Those skilled in the art will particularly recognize that the present invention includes applying a driving force to cause the hopper to directionally transfer from a first primary functional group to a second primary functional group on a track, thereby causing the hopper to move along the track, and that the application of the driving force and the movement of the hopper / walker (usually reversible movement) are important aspects of the present invention applicable to walkers and hoppers.

[0204] The hopper typically includes a linking moiety between the secondary functional group and the cargo portion. Any suitable linking moiety may be used. The chemical nature of the linking moiety is not particularly limited, provided that it is capable of linking the secondary functional group to the cargo portion without impeding movement of the hopper along the track.

[0205] Typically, the linking moiety comprises a straight or branched, unsubstituted or substituted alkylene, alkenylene, alkynylene, arylene, heteroarylene, carbocyclylene or heterocyclylene moiety. More often, the linking moiety comprises an unsubstituted or substituted alkylene, alkenylene or alkynylene moiety. More often, the linking moiety comprises an unsubstituted or substituted alkylene or alkenylene moiety. Most often, the linking moiety comprises an unsubstituted or substituted alkylene moiety. Typically, the alkylene group is C 1-10 Typically, an alkenylene group is a C 2-10 Typically, an alkynylene group is a C 2-10 Typically, an arylene group is a C 6-12 Typically, the heteroarylene group is a 5- to 12-membered heteroarylene group. Typically, the carbocyclylene group is a C 5-12Typically, the heterocyclylene group is a 5- to 12-membered heterocyclylene group.

[0206] The alkylene, alkenylene or alkynylene moiety may be uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, phosphate, thiophosphate, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene, unsubstituted or substituted carbocyclylene and unsubstituted or substituted heterocyclylene, wherein R is selected from H, unsubstituted or substituted alkyl and unsubstituted or substituted aryl. Typically, the alkylene, alkenylene or alkynylene moiety may be uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, phosphate and thiophosphate, wherein R is selected from H and unsubstituted or substituted alkyl. More typically, the alkylene, alkenylene or alkynylene moiety may be uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, phosphate and thiophosphorothioate, wherein R is selected from H and methyl.

[0207] For example, the linking moiety is often an unsubstituted or substituted C 1-10 Alkylene, C 2-10 Alkenylene or C 2-10 An alkynylene moiety which is uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, phosphate and thiophosphorothioate, wherein R is selected from H and unsubstituted or substituted alkyl. More generally, the linking moiety is unsubstituted or substituted C 1-10 Alkylene or C 2-10 an alkenylene group which is uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, phosphate and thiophosphorothioate, wherein R is selected from H and methyl. For example, the linking moiety may be unsubstituted or substituted C 1-10 An alkylene moiety interrupted by one or more of O, N(R), S, C(O), C(O)NR, C(O)O, phosphate, and thiophosphorothioate, and terminated in S or thiophosphorothioate. An exemplary linking moiety that links a cargo to a secondary functional group can be represented as:

[0208] [Secondary functional group] -Alkylene-[XPhos]-[Cargo]

[0209] Among them, alkylene is, for example, C 4-10 An alkylene group interrupted by C(O)NR and [XPhos] is selected from phosphate and thiophosphorothioate. Examples of such linking groups are:

[0210] [Secondary functional group] -C2H4-[C(O)NH]-C5H 10 -OP(O)(O – )O-[Goods].

[0211] Typically, in this case, the secondary functional group can be an S atom, enabling the hopper to form a disulfide bond with the thiol-functionalized orbital; for example, the secondary functional group and the linker group together can be

[0212]

[0213] Another exemplary linking moiety for linking a cargo to a secondary functional group can be

[0214] [Secondary functional group] -Alkylene-[Cargo],

[0215] wherein alkylene is, for example, C interrupted by C(O)NR 4-10 Alkylene (e.g., C2H4-[C(O)NH]-C5H 10 ). [Cargo] can be, for example, a polynucleotide, wherein the oxygen atom in the terminal phosphate group of the polynucleotide can be bound to the alkylene group. The secondary functional group and the linking group can be, for example,

[0216]

[0217] goods

[0218] Typically, the hopper includes a cargo portion. The cargo can be any suitable substance that can be carried by the hopper along the track. Suitable cargo includes, but is not limited to, metal ions, inorganic salts, polymers such as polymeric acids or bases, dyes, bleaches, pharmaceuticals, diagnostic agents, recreational drugs, explosives, and environmental pollutants. Such cargo can be advantageously analyzed or characterized using the methods described herein.

[0219] The cargo can be an analyte secreted from the cell. Alternatively, the analyte can be an analyte present in the cell, such that the analyte must be extracted from the cell. The analyte is typically a polymer. The cargo is typically charged. The cargo can be positively or negatively charged, often negatively charged. The cargo is often a polynucleotide, a polypeptide, or a polysaccharide, more often a polynucleotide or a polypeptide, most often a polynucleotide. Without being bound by theory, the inventors believe that the application of the driving force orients the cargo relative to the track and orients the hopper to move along the track. Without being bound by theory, the inventors believe that the movement of the cargo typically precedes the hopper, i.e., the hopper "pushes" the cargo along the track, rather than "pulling" the cargo along the track.

[0220] Goods can be polynucleotides, i.e. nucleic acid sequences. Nucleic acids are negatively charged. Nucleic acids are macromolecules comprising two or more nucleotides. Nucleotides can be naturally occurring or artificial. Nucleotides generally contain a nucleobase, a sugar and at least one phosphate group. Nucleobases are generally heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically, adenine, guanine, thymine, uracil and cytosine. Sugars are generally pentoses. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. Nucleotides are generally ribonucleotides or deoxyribonucleotides. Nucleotides generally contain monophosphate, diphosphate or triphosphate. Phosphoric acid can be attached to the 5' or 3' side of the nucleotide.

[0221] Suitable nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), Cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The nucleotide is usually selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.

[0222] When the cargo is a polynucleotide, the polynucleotide can be single-stranded or double-stranded. Typically, when the cargo is a polynucleotide, the polynucleotide is single-stranded, such as cDNA or RNA.

[0223] When the cargo is a single-stranded polynucleotide, the polynucleotide may comprise about 2 to about 1000 nucleotides, such as about 5 to about 500 nucleotides, such as about 10 to about 100 nucleotides, such as about 20 to about 60 nucleotides, such as about 30 to about 50 nucleotides. When the cargo is a double-stranded polynucleotide, each strand of the double-stranded polynucleotide may independently comprise about 2 to about 1000 nucleotides, such as about 5 to about 500 nucleotides, such as about 10 to about 100 nucleotides, such as about 20 to about 60 nucleotides, such as about 30 to about 50 nucleotides.

[0224] The cargo can be a polypeptide. The polypeptide can be a protein or a fragment thereof. The polypeptide can be naturally occurring or non-naturally occurring. The polypeptide can include its synthetic or modified amino acids. Many different types of modifications to amino acids are known in the art. Suitable amino acids and modifications thereof are discussed herein. For the purposes of the present invention, it will be understood that polypeptides can be modified by any method available in the art. For example, polypeptides can be modified by post-translational modifications or sequence variations, which are produced by alternative splicing of RNA during biological peptide synthesis.

[0225] When the cargo is a polypeptide, the polypeptide can be a polypeptide secreted from the cell. Alternatively, the polypeptide can be a polypeptide present within the cell, such that it must be extracted from the cell before the present invention can be practiced. The polypeptide can be extracted by using antibodies or by binding of an affinity tag introduced on the protein.

[0226] As used herein, a polypeptide can be a shorter peptide, which is typically a polymer of about 2 to about 50 amino acids, or can be a longer amino acid polymer. A protein is typically a polypeptide that folds into a functional conformation or forms part of a functional complex. For example, a polypeptide can be about 2 to about 1000 amino acids, such as about 5 to about 500 amino acids, such as about 10 to about 100 amino acids, such as about 20 to about 60 amino acids, such as about 30 to about 50 amino acids.

[0227] When the cargo is a polypeptide, any polypeptide may be used. Suitable polypeptides include, but are not limited to, proteins such as enzymes, antibodies, hormones, growth factors, or growth regulatory proteins such as cytokines, or fragments of such proteins. The polypeptide may be bacterial, archaebacterial, fungal, viral, or parasitic. The polypeptide may be plant derived. The polypeptide is typically mammalian, more typically human.

[0228] The cargo may be a polysaccharide. A polysaccharide is a polymeric carbohydrate molecule composed of chains of monosaccharide units bound together by glycosidic bonds. Polysaccharides may be linear or branched. Polysaccharides may be homogeneous (containing only one repeating unit) or heterogeneous (containing modifications of the repeating units). Polysaccharides include callose or laminarin, chrysophyllan, xylans, arabinoxylans, mannans, fucoidans, and galactomannans.

[0229] The cargo may be a polysaccharide produced by a bacterium such as a pathogen. The polysaccharide may be a capsular polysaccharide having a molecular weight of 100 to 2000 kDa. The polysaccharide may be synthesized from nucleotide-activated precursors (called nucleotide sugars). The polysaccharide may be a lipopolysaccharide. The polysaccharide may be a therapeutic polysaccharide. The polysaccharide may be a toxic polysaccharide. The polysaccharide may be suitable for use as a vaccine. The polysaccharide may be, for example, bacterial or derived from a plant. The polysaccharide may be used as an antibiotic, such as streptomycin, neomycin, paromomycin, kanamycin, chalmycin, erythromycin, tamycin, spiramycin, oleandomycin, ember erythromycin and eumycin, or a derivative of any of the foregoing compounds.

[0230] When the cargo is a polysaccharide, the polysaccharide may comprise from about 2 to about 1000 monosaccharide units, such as from about 5 to about 500 monosaccharide units, such as from about 10 to about 100 monosaccharide units, such as from about 20 to about 60 monosaccharide units, such as from about 30 to about 50 monosaccharide units.

[0231] The cargo may include more than one polymer, for example two or more polymers, for example two or more polymers disclosed herein may be linked together. Thus, the two or more polymers may be selected from polynucleotides, polypeptides and polysaccharides.

[0232] Primary and secondary functional groups

[0233] In the present invention, the hopper is in contact with the track so that the secondary functional groups of the hopper are combined with the first primary functional groups on the track.

[0234] Typically, in the present invention, the secondary functional groups of the hopper are capable of forming chemical bonds with each of the primary functional groups on the track. The present invention is not limited to covalent chemistry, and includes strong non-covalent interactions. As will be appreciated by those skilled in the art, the primary criterion is that the hopper does not detach from the track except at an optional defined release point.

[0235] Typically, the chemical bond is (i) a covalent bond, preferably a dynamic covalent bond; (ii) a coordinate bond; (iii) a hydrogen bond; or (iv) a hydrophobic interaction. More often, the chemical bond is a covalent bond, preferably a dynamic covalent bond, a coordinate bond or a hydrogen bond; even more often, the chemical bond is a dynamic covalent bond.

[0236] The formation or destruction of chemical bonds can be spontaneous, catalyzed by a catalyst, or driven by subjecting the primary and / or secondary functional groups to suitable reagents such as light. More often, the formation or destruction of chemical bonds is spontaneous. Within the meaning of the present invention, the example of the spontaneously formed covalent bond is a disulfide bond, a diselenide bond or a sulfide-selenide bond. Other similar chemical bonds are known to those skilled in the art.

[0237] Many examples of hopper / track chemistries suitable for use with the present invention are known in the art, and the present invention is not particularly limited in this regard.

[0238] For example, the chemical bond between the hopper and the track is usually based on disulfide (SS) chemical composition. Selenium can be used to replace sulfur, on the track or the hopper or both. In other words, the bond between the hopper and the track can be a SS bond (disulfide), S-Se bond (sulfide-selenide), Se-Se bond (diselenide bond), sulfur can be on the track and selenium can be on the hopper, or selenium can be on the track and sulfur can be on the hopper. Primary and / or secondary functional groups are usually included in suitable groups, such as thiol (sulfide) groups can be part of thiophosphoric acid or dithiophosphoric acid groups, and selenol (selenide) groups can be part of selenophosphoric acid or diselenphosphoric acid groups. Phosphoric acid, dithiophosphoric acid, selenophosphoric acid or diselenphosphoric acid groups are usually terminal thiophosphoric acid, dithiophosphoric acid, selenophosphoric acid or diselenphosphoric acid groups. Phosphoric acid, dithiophosphoric acid, selenophosphoric acid or diselenphosphoric acid groups can be included in DNA or RNA phosphodiester bonds.

[0239] The chemical bond between the hopper and the track can be based on As(III)-thiol chemistry. For example, the track can include a primary thiol functional group and the hopper can include an As(III) functional group, such that the hopper forms an As-S bond with the track. The formation of such a bond can be catalyzed, for example, by a free thiol group.

[0240] The chemical bond between the hopper and the track may be based on a thioacetal chemistry, such as a thioacetal bond formed between a (di)ketone or (di)aldehyde functional group and a (di)thiol functional group. For example, the track may include a primary thiol group and the hopper may include a secondary diketone or dialdehyde functional group, or the track may include a primary dithiol group and the hopper may include a secondary ketone or aldehyde functional group, such that a thioacetal bond is formed between the hopper and the track.

[0241] The chemical bond between the hopper and the track can be based on the formation of a boronate ester group by reaction of a boronic acid-containing secondary functional group on the hopper with a primary hydroxyl functional group on the track. For example, the track can include threonine, serine, or tyrosine amino acids, which contain a hydroxyl primary functional group for reaction with the boronic acid secondary functional group.

[0242] The chemical bond between the hopper and the track can be based on the transimidation reaction chemical composition. For example, the hopper can include an aldehyde or ketone secondary functional group, and the track can include an amine, hydroxylamine, hydrazine or semicarbazide primary functional group, thereby forming an imine, oxime, hydrazine or semicarbazide bond (respectively) between the hopper and the track. Similarly, the track can include an aldehyde or ketone secondary functional group, and the hopper can include an amine, hydroxylamine, hydrazine or semicarbazide primary functional group, thereby forming an imine, oxime, hydrazine or semicarbazide bond (respectively) between the hopper and the track.

[0243] The chemical bond between the hopper and the track can similarly be based on the binding of a hopper containing an α-methylene-4-nitrostyrene secondary functional group to a nucleophilic track such as an track containing a secondary amine functional group.

[0244] The chemical bond between the hopper and the track can similarly be based on aldol / anti-aldol chemistry; Diels Alder / anti-Diels Alder chemistry; ene- or alkyne-catalyzed metathesis reactions; thioester or selenoester exchange reactions in which the track includes a thiol or selenol primary functional group, respectively; reversible urea formation by combining an isocyanate or isothiocyanate secondary functional group of the hopper with a primary amine functional group on the track; cyanobenzothiazole chemistry in which the track includes a primary thiol and / or amine functional group; thiaMichael chemistry and dibromomaleimide and related chemistries involving 5-[bis(methylthio)methylene]-2,2-dimethyl-1,3-dioxane-4,6-dione.

[0245] The chemical bond between the hopper and the track can similarly be based on the formation of a metal complex. For example, the track can include a primary histidine or histidine functional group, and the simulated hopper can include a metal (e.g., Ni 2+ 、Co 2+ The track may include a pyridine primary functional group and the hopper may include Pd(II) to form a complex between the track and the hopper. The track may include a chelating agent or a π ligand and the hopper may include a derivatized transition metal ion to form a complex between the track and the hopper.

[0246] Typically, in the present invention, the primary functional group and the secondary functional group are independently selected from thiol groups and selenol groups, wherein optionally, the thiol group is contained in thiophosphoric acid or dithiophosphoric acid groups, and / or the selenol group is contained in selenophosphoric acid or diselenylphosphoric acid groups. Therefore, the chemical bond between the primary and secondary functional groups is generally a covalent bond, which is a disulfide bond, a diselenide bond or a selenosulfur bond. Most often, the primary functional group and the secondary functional group are each a thiol group, so that the chemical bond between the primary functional group and the secondary functional group is a disulfide covalent bond. Disulfide bonds can be formed, for example, between sulfhydryl-containing amino acids. For example, amino acids such as cysteine ​​may be present in proteins, for example in protein nanopores, and thiol-containing groups may be present on hoppers, thereby forming disulfide bonds between the cysteine ​​of the hole and the thiol of the hopper. The cysteine ​​amino acid of the hole may be present in the track defined herein.

[0247] Thus, typically in the present invention, the substrate is the surface of a protein nanopore, and the track comprises an array of amino acid residues contained in the protein nanopore, each amino acid residue of the track comprises a reactive side chain having a primary functional group, and wherein the secondary functional group of the hopper is capable of forming a chemical bond, preferably a covalent bond, with each of the primary functional groups of the reactive side chains of the amino acid residues of the track.

[0248] More typically, the substrate is a surface of a protein nanopore, and the track comprises an array of amino acid residues contained in the protein nanopore, wherein each amino acid residue in the track comprises a primary thiol functional group, and wherein the hopper comprises a secondary thiol functional group, such that the hopper forms a disulfide bond with the amino acid residues of the track.

[0249] position

[0250] Particular aspects of the invention include the ability for a hopper to be preferentially loaded at a precise point on a track. This ability is particularly useful in embodiments when the track has multiple potential loading points, and it is desirable to control movement of the hopper between specific loading points (pivots) on the track, such as from the beginning of the track to the end of the track. Knowledge of the loading position of the hopper on the track can be particularly useful in methods of the invention involving characterizing cargo analytes carried by the hopper.

[0251] As described herein, the hopper may be loaded at a precise point on the track. Alternatively, the hopper may be loaded randomly onto the track. Advantageously, if the hopper is loaded randomly onto the track, the hopper can be moved along the track by applying the driving force of the present invention to a defined point on the track, such as the start of the track. Once at the start of the track, the hopper is in a known position and further movement can therefore be monitored accordingly. Generally, in the present invention, the hopper may be loaded at a precise point on the track.

[0252] Thus, the present invention provides a method wherein the hopper is connected to a positioning portion prior to contacting the hopper with the track, wherein the positioning portion positions the hopper relative to the track such that a first primary functional group on the track bonds with a secondary functional group on the hopper.

[0253] For example, in the present invention, the substrate can be the surface of the transmembrane hole. The track can be included on the transmembrane hole, such as on the inner surface of the hole. Before the hopper is contacted with the track, the secondary functional group of the hopper can be connected to the positioning part so that once the hopper is in contact with the track, the position of the hopper relative to the track can be controlled. Typically, the positioning part includes a barrier for preventing the hopper from passing through the hole. Any suitable barrier can be used.

[0254] For example, the barrier can be a physical barrier that cannot pass through the hole, for example due to having a size larger than the internal size of the hole. The size of the barrier can be determined in many suitable ways known in the art, including dynamic light scattering. For example, the barrier can comprise or consist of a protein, nanoparticle or polymer, such as a polynucleotide, a polypeptide or an organic polymer such as polyethylene glycol.

[0255] The barrier can be a chemical barrier that cannot pass through the hole due to a chemical interaction. For example, the barrier can be a chemical group that binds to the hole, for example, binds to the opening of the hole and thus prevents the hopper from passing through the hole.

[0256] When a positioning portion is used, the present invention generally includes contacting the hopper with the hole so that the barrier prevents the hopper from passing through the hole, thereby maintaining the positioning portion in such a position that the first primary functional group on the track combines with the secondary functional group on the hopper, thereby releasing the positioning portion from the secondary functional group.

[0257] Most often, if present, the barrier comprises or consists of a protein such as streptavidin, neutravidin or traptavidin, typically traptavidin.The barrier moiety may be comprised in the positioning moiety by binding to a suitable group such as a biotin group.

[0258] The positioning portion may include a connecting portion between the barrier and the functional group that is coupled to the hopper, such as by coupling to a secondary functional group of the hopper. The connecting portion may be configured to position the secondary functional group of the hopper at a desired location relative to the track, such as to position the secondary functional group of the hopper so that a first primary functional group on the track is coupled to a secondary functional group on the hopper. If the connecting portion of the positioning portion is coupled to a secondary functional group on the hopper, the coupling of the first primary functional group on the track to the secondary functional group on the hopper typically releases the positioning portion from the secondary functional group of the hopper.

[0259] Therefore, in the present invention, often

[0260] - The substrate is the surface of the transmembrane pore;

[0261] - before the hopper is brought into contact with the track, the secondary functional groups of the hopper are connected to the positioning part;

[0262] - The positioning part includes a blocking body for preventing the hopper from passing through the transmembrane hole;

[0263] Step (i) of the method comprises contacting the hopper with the hole so that the barrier prevents the hopper from passing through the hole, thereby holding the positioning portion in such a position that the first primary functional group on the track combines with the secondary functional group on the hopper, thereby releasing the positioning portion from the secondary functional group.

[0264] Any suitable connecting part can be used to connect the barrier to the hopper. The chemical nature of the connecting part is not particularly limited, provided that it is capable of connecting the barrier to the hopper. The connecting part connecting the barrier to the hopper is also called a carrier.

[0265] Typically, the linking moiety comprises a linear or branched, unsubstituted or substituted alkylene, alkenylene, alkynylene, arylene, heteroarylene, carbocyclylene or heterocyclylene moiety. Such groups are as defined herein. More often, the linking moiety comprises an unsubstituted or substituted alkylene, alkenylene or alkynylene moiety. More often, the linking moiety comprises an unsubstituted or substituted alkylene or alkenylene moiety. Most often, the linking moiety comprises an unsubstituted or substituted alkylene moiety. The linking moiety can be interrupted by any suitable group to position the hopper at the desired location on the track. For example, the alkylene, alkenylene or alkynylene moiety may be uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, unsubstituted or substituted arylene, arylene-alkylene, heteroarylene, heteroarylene-alkylene, carbocyclylene, carbocyclylene-alkylene, heterocyclylene and heterocyclylene-alkylene, wherein R is selected from H, unsubstituted or substituted alkyl and unsubstituted or substituted aryl. Heterocyclyl groups, such as 8,9-dihydro-1H-dibenzo[b,f][1,2,3]triazolo[4,5-d]azocine, can be used to control the rigidity, geometry, length or spatial arrangement of the linking moiety. Such groups can also be used to aid in the synthesis of the linking moiety. Branched substituents on the linking moiety, such as [X] or -alkylene-[X], where [X] is, for example, -C(O)NR2 or aryl, heteroaryl, carbocyclyl or heterocyclyl, and where each R is the same or different and is as defined above, can be used as desired.

[0266] The length of the linking portion can be determined to position the hopper at a desired location. The length of the linking portion can be conveniently controlled by including a polyethylene glycol (PEG) group in the linking portion. For example, including 1 to 30, such as 5 to 20, such as 10 to 15, such as 12 ethylene glycol units in the linker can allow the hopper to be preferably loaded onto the track at a desired location.

[0267] When the blocking body is a protein, such as neutravidin, streptavidin or traptavidin, and the blocking moiety is comprised in the positioning moiety by binding to a group such as a biotin group, the biotin group is typically the terminal group of the linking moiety.

[0268] An exemplary connection portion connecting the barrier to the hopper can be represented as:

[0269] [Blocker]-[Linker 1]-[PEG]-[Linker 2]-[Functional Group]-[Hopper]

[0270] wherein [Blocker] is a blocker such as neutravidin, streptavidin or traptavidin protein bound to a biotin group, [Linker 1] and [Linker 2] are each independently an alkylene-containing group, which is substituted or unsubstituted as defined herein and is uninterrupted or interrupted as defined herein, but is typically interrupted by groups selected from NHC(O) and heterocyclylene, and is typically substituted by -[X] or alkylene-[X] as defined above, [PEG] is a polyethylene glycol group comprising 1 to 30 ethylene glycol units, and [Functional Group] is a functional group bound to the hopper, for example by binding to a secondary functional group of the hopper.

[0271] Examples of such groups are shown below (a blocker (not shown) is bound to the biotin group on the left hand end of the linking moiety shown, and the terminal S atom is suitable for binding to a secondary functional group on the hopper, such as a thiol group or a selenol group, typically a thiol group):

[0272]

[0273] release

[0274] Particular aspects of the present invention include the ability of a hopper to be controllably released from a track at a predetermined location. This capability is particularly useful in embodiments where the hopper is intended to carry cargo from a loading point on the track to a location on the track and be released from the track. For example, according to the present invention, a hopper can be controllably loaded at the beginning of a track and controllably released at the end of the track.

[0275] The controlled release of the hopper from the track can be achieved in any suitable manner. For example, the tertiary functional groups on the substrate can be positioned relative to the track so that when the hopper reaches a release point on the track and therefore connects with the primary functional groups at this release point on the track, the tertiary functional groups on the substrate combine with the primary functional groups, thereby replacing the secondary functional groups and thereby releasing the hopper. For example, the primary functional group at the release point on the track can be the final primary functional group in the track, i.e., the end point of the track. Therefore, the present invention may include, after the hopper moves along the track, step (iii): contacting the primary functional groups of the track that are bound to the secondary functional groups of the hopper with the tertiary functional groups on the substrate, so that the tertiary functional groups combine with the primary functional groups, thereby replacing the secondary functional groups and thereby releasing the hopper.

[0276] It is apparent from the above discussion that the common substrate in the present invention is the surface of the transmembrane pore. The track may be contained on the transmembrane pore, for example on the inner surface of the pore. The tertiary functional group may be included in the pore at a position close to the last of the primary functional groups on the track. For example, if the track extends from the cis side of the barrel of the pore to the trans side of the barrel of the pore, the tertiary functional group may be located near the trans outlet of the barrel of the pore. For example, if the track extends from the trans side of the barrel of the pore to the cis side of the barrel of the pore, the tertiary functional group may be located near the cis outlet of the barrel of the pore.

[0277] Thus, in the present invention, the substrate is often the surface of a protein nanopore, and the track comprises an array of amino acid residues contained in the protein nanopore, wherein each amino acid residue of the track comprises a reactive side chain having a primary functional group, and wherein the secondary functional group of the hopper is capable of forming a covalent bond with each of the primary functional groups of the reactive side chains of the amino acid residues of the track; and the tertiary functional group is an additional amino acid residue of the protein nanopore, which comprises a reactive side chain capable of forming a covalent bond with the reactive side chain of the final amino acid residue of the track, thereby displacing the secondary functional group from the primary thiol group of the track and thereby releasing the hopper.

[0278] The tertiary functional group may be the same as or different from the primary functional group. The tertiary functional group may be contained in an amino acid, which is a natural amino acid or a non-natural amino acid. For example, the track may contain a cysteine ​​or selenocysteine ​​residue, and the tertiary functional group may be a cysteine ​​or selenocysteine ​​residue. Other functional groups are within the scope of the present invention. For example, in the present invention, typically, the substrate is the surface of a protein nanopore, and the track includes an array of amino acid residues contained in the protein nanopore, wherein each amino acid residue in the track includes a primary thiol functional group, and the hopper includes a secondary thiol functional group, so that the hopper forms a disulfide bond that binds to the amino acid residue of the track; and wherein the protein nanopore includes additional amino acid residues, which include a tertiary thiol functional group and are positioned so that the tertiary thiol group can form a disulfide bond that binds to the primary thiol group of the track, thereby replacing the secondary functional group from the primary thiol group of the track and thus releasing the hopper.

[0279] Typically, in the present invention, the distance between the tertiary functional group and the primary functional group of the track at the release position (e.g., the final primary functional group in the track) is less than the step length between adjacent pivot points on the track. For example, in the present invention, when the substrate is the surface of a transmembrane protein pore and the track is an amino acid array on the inner surface of the pore, the amino acid residue containing the tertiary functional group is typically separated from the primary functional group of the final amino acid residue in the track by less than about 100 Å. Thus, for example, a track may comprise an array of amino acid residues each comprising a primary functional group and being approximately evenly spaced along one or more beta strands in the barrel of a transmembrane beta barrel protein nanopore, and having a step length between adjacent amino acids that includes between about to about The primary functional groups (Cα-Cα) between to about Between, for example And the amino acid containing the tertiary functional group may be located less than about (Cα-Cα), for example, between about to about Between, for example And the tertiary functional group may be the same as or different from the primary functional group, and usually the tertiary functional group is the same as the primary functional group.

[0280] For example, an exemplary substrate including a track with three pivot points at positions 115, 117, and 119 of a SEQ ID NO: 1 or 2 monomer may include appropriate amino acid residues at such positions, such as cysteine ​​or selenocysteine ​​(e.g., cysteine) residues. An amino acid residue comprising a tertiary functional group may be located at position 139 of a SEQ ID NO: 1 or 2 monomer. The distance between positions 119 to 139 of SEQ ID NO: 1-7 is about (Cα-Cα). An example of such a track is provided in SEQ ID NO:7, wherein the track is aligned along the inner surface of the α-HLβ barrel protein nanopore.

[0281] Driving force

[0282] The present invention relates to applying a driving force to transfer the hopper from the first primary functional group to the second primary functional group on the track in a directionally manner, thereby causing the hopper to move along the track. Generally, in the present invention, the driving force is a physical or chemical potential. For example, if the substrate is a transmembrane pore, an electric potential can be applied across the pore to move the hopper along the track. The driving force applied can be a physical potential such as an electric (voltage) potential. The electric potential can be an electrophoretic or electroosmotic potential. Another example of a physical potential is a temperature gradient. Alternatively, the applied potential can be a chemical potential. An example is the use of a salt gradient across an amphiphilic layer. A salt gradient is disclosed in Holden et al., J Am Chem Soc. 2007 Jul 11; 129 (27): 8650-5. Other examples of chemical potentials are pH gradients and concentration gradients.

[0283] Reversal of direction

[0284] Because the present invention involves applying a driving force to cause the hopper to transfer directionally from the first primary functional group to the second primary functional group on the track, thereby causing the hopper to move along the track, the direction of the hopper on the track can be changed by changing the direction of the driving force relative to the track. For example, when the hopper moves along the track or follows the movement of the hopper along the track, the direction of the driving force can be reversed, thereby reversing the direction of movement of the hopper. Therefore, the present invention can involve reversing the direction of the driving force relative to the track to reverse the direction in which the hopper moves along the track. For example, if the track is contained in a transmembrane pore, and the initial direction of the driving force is from the cis side of the pore to the trans side, so that the hopper moves in the direction from the cis side of the pore to the trans side, then the reversal of the direction of the driving force causes the force to be applied from the trans side of the pore to the cis side to reverse the movement of the hopper, that is, the movement of the hopper can be in the direction from the trans side of the pore to the cis side. Those skilled in the art will understand that the movement of the hopper along the track can therefore be cyclic, that is, the hopper can be moved back and forth along the track multiple times.

[0285] Methods for characterizing analytes

[0286] The present invention also provides a method for characterizing an analyte, the method comprising:

[0287] (i) Provide

[0288] (A) Detector;

[0289] (B) a track comprising a plurality of primary functional groups arranged along a substrate; and

[0290] (C) a molecular hopper connected to the analyte, wherein the hopper comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track;

[0291] (ii) contacting the hopper with the track so that the secondary functional group of the hopper is combined with the first primary functional group on the track;

[0292] (iii) applying a driving force to directionally transfer the hopper from the first primary functional group to the second primary functional group on the track, thereby causing the hopper to move along the track;

[0293] Wherein the track is positioned such that movement of the hopper along the track causes the analyte to interact with the detector, thereby characterizing the analyte.

[0294] The detector may be any suitable detector for characterizing an analyte. Typically in the present invention, the detector is a transmembrane pore, such as one of the pores described herein. Most often, the detector is a transmembrane protein pore as described herein. When the detector is a transmembrane pore as described herein, the substrate is typically the surface of the transmembrane pore, as described in more detail herein, and the track is arranged along the surface, for example, the track may be an array of amino acids comprising primary functional groups as defined herein.

[0295] Typically, the analyte is a polynucleotide, a polypeptide, or a polysaccharide. More often, the analyte is a polynucleotide or a polypeptide. Typically, the analyte is a polynucleotide. The analyte can, for example, be any of the goods described herein.

[0296] The driving force may be a chemical or physical potential as described herein. Typically, the driving force is an electrical (voltage) potential.

[0297] Therefore, in this method, usually:

[0298] -The substrate is the surface of the transmembrane protein pore;

[0299] - a track comprising an array of amino acid residues contained in a protein nanopore, wherein each amino acid residue of the track comprises a reactive side chain, and wherein the hopper comprises an functional group capable of forming a covalent bond with the reactive side chains of the amino acid residues of the track;

[0300] Energy group, optionally, wherein the track and hopper are defined herein;

[0301] - the analyte is a polynucleotide, a polypeptide or a polysaccharide; and

[0302] -The driving force is a physical or chemical potential.

[0303] The method may involve moving the hopper back and forth along the track for multiple cycles, thereby allowing the analyte to interact with the detector multiple times. This can improve the characterization of the analyte.

[0304] Thus, in certain aspects, the present invention provides a method of moving a molecular hopper along a track, wherein:

[0305] (a) the track comprises a plurality of primary functional groups arranged along a substrate; wherein the substrate is a surface of a transmembrane β-barrel protein nanopore, and the track comprises an array of amino acid residues contained in the protein nanopore, wherein each amino acid residue in the track comprises a primary thiol functional group; wherein optionally, the amino acid residues in the track are located at positions corresponding to positions 113, 115, 117, 119, 121 and / or 123 in the sequence of SEQ ID NO: 1 or 2; and

[0306] (b) the hopper comprises a thiol secondary functional group connected via a linker to a cargo moiety, such that the hopper is capable of forming a disulfide bond to the track, wherein the cargo moiety is a polynucleotide and the linker is a group of the form: -alkylene- or -alkylene-[XPhos]-, wherein alkylene is C 4-10 an alkylene group interrupted by C(O)NR, and [XPhos] is selected from phosphate and thiophosphorothioate.

[0307] The method comprises the following steps:

[0308] (i) contacting the hopper with the track so that the thiol group of the hopper combines with the first thiol group on the track;

[0309] (ii) applying an electrical potential to cause the hopper to directionally and reversibly transfer from the first thiol group of the track to a second thiol group on the track, thereby causing the hopper to move along the track; and

[0310] (iii) contacting the primary thiol group of the track bound to the secondary thiol group of the hopper with a tertiary thiol group on a substrate, so that the tertiary thiol group binds to the primary thiol group, thereby replacing the secondary thiol group and thus releasing the hopper, wherein optionally, the tertiary thiol group is included in the amino acid at the position corresponding to position 139 in the sequence of SEQ ID NO: 1 or 2;

[0311] wherein prior to contacting the hopper with the track, the secondary thiol group of the hopper is linked to a positioning moiety of the form: [Blocker]-[Linker 1]-[PEG]-[Linker 2]-[Functional Group]; wherein [Blocker] is a blocker such as neutravidin, streptavidin or traptavidin protein conjugated to a biotin group, [Linker 1] and [Linker 2] are each independently an alkylene-containing group which is substituted or unsubstituted as defined herein and is uninterrupted or interrupted as defined herein, but is typically selected from NH The groups are interrupted by C(O) and heterocyclylene and are typically substituted by -[X] or alkylene-[X] as defined herein, [PEG] is a polyethylene glycol group comprising 1 to 30 ethylene glycol units, [functional group] is a thiol group that combines with the secondary thiol functional group of the hopper to form a disulfide bond; and step (i) of the method comprises contacting the hopper with the hole so that the barrier prevents the hopper from passing through the hole to hold the positioning part in a position so that the first primary thiol group on the track combines with the secondary thiol functional group on the hopper, thereby releasing the positioning part from the secondary functional group.

[0312] Equipment and conditions

[0313] Any suitable equipment can be used to carry out the methods of the present invention.

[0314] Electrical measurements can be made using standard single-channel recording devices such as Stoddart, DS, et al., (2009), Proceedings of the National Academy of Sciences of the United States of America 106, p7702-7707, Lieberman KR et al, J Am Chem Soc. 2010; 132(50): 17961-72 and International Application WO-2000 / 28312. Alternatively, electrical measurements can be made using a multi-channel system, such as described in International Application WO-2009 / 077734 and International Application WO-2011 / 067559.

[0315] In embodiments of the present invention including the use of nanopores (e.g., as substrates or detectors), any device suitable for investigating membrane / pore systems can be used to implement the present invention, wherein the pores are inserted into the membrane. Any device suitable for transmembrane pore sensing can be used to implement the present invention. For example, the device can include a chamber comprising an aqueous solution and a barrier that divides the chamber into two parts. The barrier has pores, wherein a film containing pores is formed. The present invention can also be implemented using a droplet interface bilayer (DIB). Two water droplets are placed on an electrode and immersed in an oil / phospholipid mixture. The two droplets are in close contact and form a phospholipid membrane at the interface, wherein the pores are inserted.

[0316] The invention may be carried out using the apparatus described in International Application No. PCT / GB08 / 000562 (WO 2008 / 102120).

[0317] The method of the present invention generally involves measuring the current flowing through the pore. Therefore, the device may also include a circuit that can apply an electric potential and measure the electrical signal across the membrane and the pore. The method can be implemented using a patch clamp or a voltage clamp. The method generally involves using a voltage clamp.

[0318] The method can be implemented on silicon-based well arrays, where each array includes 128, 256, 512, 1024 or more wells.

[0319] The method can be implemented using a hole array as described herein. Use of a hole array can allow monitoring of the method by monitoring signals such as electrical or optical signals. Optical detection of analytes using nanopore arrays can be performed using techniques known in the art, such as the techniques described in Huang et al, Nature Nanotechnology (Natural Nanotechnology) (2015) 10:986-992.

[0320] The method of the present invention may be related to measuring the current flowing through the hole. Suitable conditions for measuring the ionic current through the transmembrane pore are known in the art and disclosed in the embodiments. The method is usually implemented using a voltage applied across the membrane and the hole. The voltage used is usually from +2V to -2V, usually -400mV to +400mV. The voltage used is usually in the range of having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV and independently selected from the upper limit of +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV. The voltage used is more often in the range of 100mV to 240mV, and most usually in the range of 120mV to 220mV.

[0321] The method of the present invention is usually implemented in the presence of a carrier, such as a metal salt, such as an alkali metal salt, a halide salt, such as a chloride salt, such as an alkali metal chloride salt. The carrier may include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride. In the above exemplary device, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferrocyanide are usually used. A mixture of KCl, NaCl and potassium ferrocyanide and potassium ferrocyanide is preferred. The salt concentration may be saturated. The salt concentration may be 3M or less, and is typically 0.1M to 2.5M, 0.3M to 1.9M, 0.5M to 1.8M, 0.7M to 1.7M, 0.9M to 1.6M or 1M to 1.4M. The salt concentration is typically 150mM to 1M. The method is typically carried out using a salt concentration of at least 0.3 M, e.g., at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. The salt concentration used on each side of the membrane may be different, e.g., 0.1 M on one side and 3 M on the other side. The salts and compositions used on each side of the membrane may also be different.

[0322] The method is usually carried out in the presence of a buffer. In the exemplary apparatus described above, the buffer is present in an aqueous solution in the chamber. Any buffer can be used in the method of the present invention. Usually, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. Usually, the method is carried out at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8 or 7.5 to 8.5. The pH used is usually about 7.5.

[0323] The method can be carried out at 0° C. to 100° C., 15° C. to 95° C., 16° C. to 90° C., 17° C. to 85° C., 18° C. to 80° C., 19° C. to 70° C., 20° C. to 60° C. The method is usually carried out at room temperature.

[0324] Reagent test kit

[0325] The present invention also provides a kit for characterizing an analyte, the kit comprising:

[0326] (A) Detector;

[0327] (B) a track comprising a plurality of primary functional groups arranged along a substrate; and

[0328] (C) a molecular hopper that binds to the analyte, wherein the hopper comprises a secondary functional group that can bind to each of the plurality of primary functional groups on the track.

[0329] Typically, the detector, track, primary functional group, substrate, molecular hopper, analyte, and secondary functional group are each preferably as defined herein. Typically, the analyte is a polynucleotide, polypeptide or polysaccharide, typically a polynucleotide or polypeptide, more typically a polynucleotide.

[0330] Typically, in the cartridge, the track is positioned relative to the detector such that movement of the hopper along the track allows the analyte to interact with the detector, enabling the detector to characterize the analyte.

[0331] system

[0332] The present invention also provides a system, comprising:

[0333] (A) Detector;

[0334] (B) a track comprising a plurality of primary functional groups arranged along a substrate; and

[0335] (C) a molecular hopper that binds to a polynucleotide or polypeptide analyte, wherein the hopper comprises a secondary functional group that binds to at least one of the plurality of primary functional groups on the track.

[0336] Detectors, tracks, primary functional groups, substrates, molecular hoppers, analytes, and secondary functional groups are generally as defined herein.

[0337] hopper

[0338] The present invention also provides a hopper molecule as described herein. The hopper comprises:

[0339] -Secondary functional groups that are bound to primary functional groups in orbit;

[0340] - a polynucleotide, polypeptide or polysaccharide cargo moiety;

[0341] - A linking moiety between the secondary functional group and the cargo moiety.

[0342] The secondary functional groups, primary functional groups, track, cargo and connecting moieties are generally as defined herein. The hopper may also include a positioning portion for positioning the hopper relative to the track so that the first primary functional groups on the track can bind to the secondary functional groups on the hopper. The positioning portion may be as defined herein. The positioning portion may include a barrier as defined herein for preventing the hopper from passing through the transmembrane pore. Typically, the barrier comprises or consists of a protein.

[0343] chemical communications

[0344] The present invention also provides a method of chemical communication across a barrier spanned by a track, the method comprising contacting the track with a molecular hopper under conditions where the hopper moves along the track across the barrier, and wherein movement of the hopper transmits information across the barrier.

[0345] Tracks and hoppers are generally as defined herein. The barrier may be any suitable barrier, such as an energy barrier across a nanopore. The barrier may be between two or more compartments comprising an aqueous medium, such as between two droplets. The compartments or droplets may, for example, be components of a synthetic tissue or a synthetic organism such as a synthetic cell. The droplets may be coated with amphiphilic molecules such as lipids or block copolymers as defined herein. The droplets may thus be or comprise a droplet interface bilayer. Droplet interface bilayers are known in the art.

[0346] Typically, the movement of the hopper is selectively controlled by a track. Movement of the hopper along the track is described in more detail herein. The track can be on a substrate, such as a surface; suitable substrates are described herein. The hopper typically carries cargo. Suitable cargo is described herein. For example, the cargo can be a polynucleotide, such as a polynucleotide complementary to a promoter region in a vector. The polynucleotide can be complementary to a promoter region in a vector suitable for in vitro transcription / translation, and the transport of the cargo across the barrier includes initiating an in vitro transcription / translation reaction. IVTT is described in more detail herein.

[0347] The communication can be of any suitable information, such as, but not limited to, information that can be encoded by a polynucleotide or a polypeptide.

[0348] Acknowledgements

[0349] The work leading to the present invention received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7 / 2007-2013) / ERC Grant Agreement No. 294443.

[0350] Sequence Listing

[0351] SEQ ID NO:1:WT

[0352] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0353] SEQ ID NO:2:WT-D8H6

[0354] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNDDDDDDDDHHHHHH

[0355] SEQ ID NO:3:115C117C-D8H6

[0356] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSCLCYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNDDDDDDDDHHHHHH

[0357] SEQ ID NO:4:115C117C119C-D8H6

[0358] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSCLCYCFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNDDDDDDDDHHHHHH

[0359] SEQ ID NO:5:113C115C117C119C121C-D8H6

[0360] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYCSCLCYCFCGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNDDDDDDDDHHHHHH

[0361] SEQ ID NO:6:113C115C117C119C121C123C-D8H6

[0362] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYCSCLCYCFCGCVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNDDDDDDDDHHHHHH

[0363] SEQ ID NO:7:115C117C119C139C-D8H6

[0364] ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSCLCYCFNGNVTGDDTGKIGGLIGACVSIGHTLKYVQPDF KTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNDDDDDDDDHHHHHH

[0365] The following examples illustrate the invention. However, the examples do not limit the invention in any way. In particular, there are many ways to control and / or monitor the movement of the hopper along the track, and therefore a negative result in any particular assay is not limiting.

[0366] Example 1

[0367] This example demonstrates that a molecular hopper controlled by a chemical ratchet can advance along a track and change direction on command.Key details are set out below, and further information is provided under the heading "Supplementary Materials" (SM) immediately following the main text of the example.

[0368] Scientists have long sought to control molecular motion, attracted by the potential of the technology. This example describes how a single 0.7 nm step of a single molecular hopper can be monitored as it moves in an electric field along a track in a nanopore controlled by a chemical ratchet. The hopper exhibits the desired features in a moving molecule: defined start and end points, progressiveness, fuel autonomy, directional motion, and external control. The hopper is easily functionalized to carry cargo. For example, DNA can ratchet along the track in either direction, a prerequisite for nanopore sequencing.

[0369] Processivity is at the heart of biological machinery. Replicative DNA polymerases are able to incorporate thousands of nucleotides before detaching from their template (1). Molecular motors such as kinesins and dyneins move directionally along microtubules over hundreds of steps without leaving the track (2-4). For many years, scientists have attempted to build mobile molecules that resemble their biomolecular counterparts but use simpler components (5). The ultimate goal is to achieve true processivity, which can be defined as directed motion without leaving the track, as well as the ability to perform useful work such as transporting cargo. Ideally, synthetic systems should exhibit the reversibility of travel seen in various biological systems, so that the direction of motion can be switched by external control (6,7).

[0370] The one-legged molecular hopper reported in this example moves by ratcheting along a protein track via dynamic covalent chemistry and displays a level of processivity unprecedented in previous synthetic systems (Table S7), exceeding that of some biomolecular motors ( Figure 1A ). In addition, the direction of the hopper movement is externally controlled by an electric potential. The track is constructed inside the protein nanopore, α-hemolysin (αHL), and the track consists of a series of cysteine ​​branches facing the lumen of the transmembrane β barrel ( Figure 1B ). The average distance between cysteine ​​branches is (Cα-Cα) and the average vertical spacing is The β-strands of the DNA cargo are evenly spaced. By employing sequential thiol-disulfide interchange reactions, the hopper moves in the direction directed by the applied potential.

[0371] To execute S N 2 reaction, the three participating sulfur atoms are arranged in a nearly linear configuration (8-10). Under an applied potential, the DNA inside the bucket is pulled in the electric field with a force of about 10 pN (see SM Section 12). This force sets the overall direction of motion by flipping the DNA (see below) and helps orient the disulfide for cleavage by the adjacent downstream cysteine ​​thiolate, which moves the bucket forward one step, although other forces can contribute to forward motion ( Figure 1C ). Backtracking is disadvantageous, and overrunning is impossible. No release of the hopper from the linear track has been observed, presumably because the track is too rigid to accommodate the resulting disulfide bonds between adjacent pivots on the same beta strand. In short, each step is chemically directed, as the hopper's "feet" are positioned to favor forward reactions. Furthermore, movement is autonomous, requiring no chemical fuel.

[0372] At +150 mV, the hopper was delivered from the cis compartment to the orbital as a hopper-carrier conjugate capped with a single traptavidin ( Figure 1D ), the conjugate is blocked at the pore entrance ( Figure 2AThe disulfide in the construct reacts strictly regioselectively with Cys-115 of the release vector and places the hopper-DNA cargo on the starting fulcrum ( Figure 2A , 6). The position of the hopper is determined by the residual current passing through the nanopore, which reflects the length of the DNA located inside the β barrel when the hopper is at a specific pivot point (Figure 6). By monitoring the current changes, we monitor the step-by-step feeding motion in real time at the single-molecule level.

[0373] The voltage-controlled feeding motion is directional and processive. On a track containing five cysteine ​​pivots (113, 115, 117, 119, 121), a hopper carrying an oligoadenylate 40-mer (A40, hopper 1) moves from cis to trans at +150 mV and from trans to cis at -150 mV. Figure 2B ). When the hopper reaches the terminal pivot point, the applied potential signal is reversed in order to reorient (flip) the DNA cargo and, therefore, reorient the hopper. Alternating between positive and negative potentials repeatedly drives the hopper toward either the trans or cis end of the track.

[0374] In order for the DNA to experience a force, at least one negatively charged phosphodiester bond must be located within the electric field that decreases along the length of the barrel of the pore (11). Thus, in the present nanopore configuration, voltage-controlled feeding is demonstrated with up to six pivot points ( Fig. 9 ). The ability to change feed direction by reversing the applied force at any pivot point demonstrates full control over directionality and the ability to move the hopper back to the initial pivot point after departure.

[0375] In this system, the applied potential provides an external energy source to produce directional motion ( Figure 2C , see SM Section 12). Limited only by bilayer stability, the longest record of processive feeding was recorded with hopper 1, which completed 249 forward steps in 93 minutes on a track of five cysteines (113-121), with an average dwell time of about 22 s at each pivot point (Figure 8). The hopper was never observed to disengage from the track (≥30 departures on different tracks), which means that significantly higher step numbers will be observed with more stable bilayers. Other bilayer systems are described herein and are able to achieve significant stability improvements. In comparison, previous synthetic small molecule walkers have directed movements of less than 10 steps (5, 12). Wild-type kinesins typically have an average number of steps of 75 to 175 before disengaging (2, 3).

[0376] At pH 8.5, the feeding rates for each of the four steps on the trajectory of the five cysteines were derived for both the cis-to-trans and trans-to-cis directions and showed less than 40-fold difference in the feeding rates (0.0081 to 0.30 s -1, Table S3). Because thiolates are reactive nucleophiles in disulfide interchanges, the rate difference may be due to the pK of the branch-point thiols. a The voltage dependence of the charge transfer was investigated by the arsenic(III) walker, which was caused by changes in the value of the charge transfer, which was affected by neighboring residues. Previously, arsenic(III) walkers showed up to 50-fold differences in the connection rate with pivots on the same five cysteine ​​trajectories (113-121) at pH 8.0 (12). The thiol trajectories can be engineered to have optimized inter-pivot distances and enhanced chemical reactivity to accelerate the feeding process. Alternatively, the properties of the reactive sulfur atoms in the hopper can be manipulated by flanking functional groups. For trajectories with two and three cysteines, the effect of voltage on feeding was examined at ±100 mV, ±150 mV, and ±180 mV (Tables S1, S2). The rates showed a weak non-exponential voltage dependence, suggesting that the applied potential may not be the only source of propulsion (see SM Section 12). However, the potential is necessary to 1) flip the DNA over a large barrier to set the direction of movement; 2) help orient the three participating sulfur atoms so that the "forward" reaction is better than the backward reaction. With regard to the latter, the expected “effective concentration” of participating downstream mercaptans is not particularly high (see SM Section 15) (13), and is not actually necessary to produce overall forward motion (see below).

[0377] Even though unfavorable, retreat was occasionally detected and was attributed to the conformational variability of the hopper within the nanopore even under the applied potential. During recording with hopper 1 on the trajectory of the five cysteines (113-121), there were 33 retreats (12%) out of a total of 282 steps at non-terminated pivots. Of the 33 retreats, 29 occurred from 115 to 117 at -150 mV (Table S5). Although the forward equilibrium constant from 117 to 115 is large (K = k 117-115 / k 115-117 =22), but a retreat from 115 to 117 is observed, which is due to the relatively slow forward movement to the next branch 113 (at -150 mV, k 115-117 =0.0094s -1 ;k 115-113 =0.0081s -1 , Tables S3, S5). When the hopper was left on the terminal pivot, backward movement was observed because no forward pivot was left ( Figure 2B These backward movements are rapidly reversed by a hopper, which is preferably located at the terminal (K = k 119-121 / k 121-119 = 5.2, Table S4). The overall motion of the hopper is determined by the product of the k values ​​at each step. Moderate values ​​(K>1) at each step produce a considerable overall tendency to move forward.

[0378] Each subunit of the αHL pore contributes two antiparallel β-strands to the transmembrane β-barrel, which has a diameter of approximately The interchain distance of (Cα-Cα) is 2.3 Å. Considering that the formation of cross-chain disulfides has been reported (14), we reasoned that the addition of cysteines on adjacent chains would force the hopper to release from the track at a given pivot point. Indeed, for an L-shaped track consisting of cysteines at positions 115, 117, 119, and 139, when the hopper reaches pivot point 119, it is connected to the track and detached from the track by regioselective disulfide formation at pivot point 115. The release is initiated by Cys-139 through thiol-disulfide interchange to form a cross-chain disulfide bridge, which prevents subsequent hoppers from entering pivot point 119 ( Figure 2D ). The preference for hopper release over hopper transfer to an adjacent chain is attributed to the failure of the three participating sulfur atoms to form the colinear arrangement necessary for transfer. In the future, the engineering of fulcrums on the surface will allow the construction of more complex feeding paths, where the hopper is transferred to a new track at a designed junction and the cargo is released at a pre-specified depot.

[0379] The ability to translocate a stretched DNA cargo while maintaining a covalent bond to the nanopore suggests an approach for chemically phasing nucleic acids during nanopore sequencing (15), which was explored in a proof-of-concept experiment. To provide a marker, two adjacent abasic residues (1',2'-dideoxyribose, dS) (16) were incorporated into the cargo oligonucleotide carried by hoppers 2 and 3, and the current pattern was recorded during a four-step feed between Cys-113 and Cys-121 ( Figure 3A By comparing with Hopper 1, Hoppers 2 and 3 show different current modulation patterns ( Figure 3B , C, 16, Table S6). The conductance pattern generated by the four-step feeding motion can be repeated with different molecules in hoppers 2 and 3 (n = 3 for each hopper), establishing the pattern as a clear signature for each cargo sequence. The residual current (Ires%, residual current as a percentage of the opening current) of the three hoppers at each pivot point is plotted for comparison ( Figure 3D At -150 mV, hoppers 1 and 2 gave almost the same current blockade at each of pivots 115 and 113, which means that the dSdS sequence has been well transported out of the sensing area by hopper 2. In addition, hoppers 2 and 3 have a single nucleotide shift at the dSdS position, and we observed a one-step shift between hoppers 2 and 3 of ΔIres%, i.e., the Ires% difference between two consecutive steps ( Figure 3D ; Vertical step length The distance between nucleotides in stretched single-stranded DNA Similar (17)).

[0380] These observations suggest that the hopper system reported here has the potential to be used for base discrimination for sequencing purposes (16). An advantage of the processive hopper that could improve sequencing accuracy is the ability to reverse the chemical ratcheting process and thereby obtain many-fold coverage of a single DNA strand. This could be further improved by using pores with longer β barrels (18,19). Perhaps by using fulcrums on extended crystal surfaces or internal phosphorothioate feet to transport long replicates on relatively short tracks, feasible sequencing processes could involve delayed phasing of large numbers of parallel DNA strands.

[0381] Additional information

[0382] 1. Synthesis and Characterization of Hopper-Carrier Conjugates

[0383] 1.1 Chemicals

[0384] Acetic anhydride (Ac2O), acetonitrile (HPLC grade), 5-azidopentanoic acid, N,N-diisopropylethylamine (DIPEA), diethyl ether, dimethylformamide (DMF), hexafluoro-2-propanol (HFIP), N,N-hydroxybenzotriazole (HOBt), 1-methyl-2-pyrrolidone (NMP), Nα-Fmoc-Nε-biotinyl-L-lysine, piperidine, triethylamine (TEA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), Fmoc-Phe-OH, and Rink amide MBHA resin LL (100 to 200 mesh) were from Novabiochem. Fmoc-NH-PEG(12)-COOH was purchased from IrisBiotech GmbH.

[0385] 1.2 Preparation of carriers using solid phase peptide synthesis

[0386] The vector was prepared by manual solid-phase peptide synthesis using a protocol adapted from a previously reported protocol ( Figure 4A )(20).

[0387] The Fmoc method was used using HBTU and DIPEA as coupling reagents on 0.25 mmol Rink amide MBHA resin LL (0.37 mmol g -1) was used for peptide chain extension. The resin was washed and swollen in DMF overnight and Fmoc was removed at room temperature by treatment with 20% (v / v) piperidine in NMP. The carrier was assembled by standard chain extension using reactants Nα-Fmoc-Nε-biotinyl-L-lysine, Fmoc-Phe-OH, Fmoc-NH-PEG(12)-COOH and 5-azidopentanoic acid. For each of the four coupling reactions, a solution of 1.0 mmol reactant, 0.95 mmol HBTU and 2.0 mmol DIPEA in 1.9 mL DMF was added to the resin, which was then shaken for 12 minutes. After each coupling, the resin was washed with NMP and capped with 3 mL DMF solution of 1.5 mmol Ac2O, 0.044 mmol HOBt and 0.39 mmol DIPEA for 2 minutes. The capped resin was then washed with NMP again before the next coupling. After chain assembly is complete, the resin is washed with DMF and ethanol in sequence and dried in vacuo. The peptide is cleaved from the resin by treatment with TFA for 2 hours. TFA is evaporated in a stream of nitrogen and the peptide is precipitated with cold ether, followed by centrifugation and several grindings with ether.

[0388] 1.3 Vector purification and characterization

[0389] The crude peptide was dissolved in DMF and purified by preparative RP-HPLC (Dionex UltiMate 3000, Vydac C18 column: 250×22 mm, 10 to 15 μm (particle size), linear gradient: 5% to 95% of eluent B in eluent A over 45 min, flow rate: 15 mL min -1 , eluent A: 0.1% TFA in water, eluent B: 0.1% TFA in acetonitrile). Purified by analytical LC-MS (Waters LCT accurate mass time-of-flight instrument (ESI-positive), Chromolith RP-18e column: 50 mm × 2 mm, linear gradient: 5% to 100% eluent B in eluent A over 8 min, flow rate: 1 mL min -1 The identity and purity of the fractions were determined by HPLC (Agilent 1260 InfinityHPLC; Polaris C18 column: 150×4.6 mm, 5 μm (particle size), linear gradient: 5% to 95% of eluent B in eluent A over 10 min, flow rate: 1 mL min -1, eluent A: 0.1% TFA in water, eluent B: 0.1% TFA in acetonitrile) characterization ( Figure 4B ).

[0390] 1.4 Generation and characterization of hopper-carrier conjugates

[0391] Oligonucleotides modified with dibenzocyclooctyne-disulfide (DBCO-SS) at the 5' end were purchased from biomers.net.

[0392]

[0393] 5'DBCO-SS-A40 oligonucleotide (10 μL, 1 mM MilliQ water) was added to the carrier (1 μL, 10 mM MilliQ water). The mixture was kept at room temperature for 30 minutes to form the hopper-1-carrier conjugate by a copper-free click reaction. Similarly, the hopper-2-carrier and hopper-3-carrier conjugates were assembled with 5'DBCO-SS-AAdSdSA36 and 5'DBCO-SS-AdSdSA37, respectively.

[0394] The conjugate was purified by semi-preparative HPLC (Agilent 1260 Infinity; Supelco Discovery BIO wide-pore C18 column: 250×10 mm, 10 μm (particle size); linear gradient: 10% to 90% of eluent A in eluent B over 30 min, flow rate: 4.5 mL min -1 ; eluent A: 0.1% TFA in water; eluent B: 0.1% TFA in acetonitrile) and purified by LC-MS (UPLC-MS Waters XEVO G2-QTOF (ESI-negative); ACQUITY UPLC Oligonucleotide BEH C18 column: 2.1×50 mm, 1.7 μm (particle size); linear gradient: 0% to 70% of eluent B in eluent A over 8 min, flow rate: 0.2 mL min -1 ; Eluent A: 8.6 mM TEA, 200 mM HFIP in 5% methanol / water (v / v) solution; Eluent B: 20% methanol solution of eluent A) identification.

[0395]

[0396] See also Figure 5 .

[0397] 2. Preparation of αHL Monomers with Multi-cysteine ​​Tracks

[0398] Our group has previously reported the construction of pT7–αHL–115C117C-D8H6 and pT7–αHL–113C115C117C119C121C-D8H6 (pT7–αHL-5C-D8H6), encoding αHL mutants containing cysteines at positions 115 and 117 or at positions 113, 115, 117, 119, and 121 and an octaaspartic acid tail and a histidine tag at the C-terminus ( 12 ).

[0399] pT7–αHL–6C-D8H6 encodes an αHL mutant containing cysteines at positions 113, 115, 117, 119, 121, and 123 and an octa-aspartate tail and a histidine tag at the C-terminus. It was generated from pT7–αHL-5C-D8H6 by in vivo homologous recombination. Two sets of PCR reactions were performed. For the first reaction, the template was first linearized with NdeI (New England Biolabs) prior to PCR using the forward mutagenic primer: 5'-CTGCTTCTGCGGTTGTGTTACTGGTGATGATACAGG-3' and the reverse non-mutagenic primer (SC47): 5'-CTGCTTCTGCGGTTGTGTTACTGGTGATGATACAGG-3'. For the second reaction, the template was linearized by HindIII (New England Biolabs) with reverse mutant primer: 5'-CCTGTATCATCACCAGTAACACAACCGCAGAAGCAG-3' and forward non-mutant primer (SC46): 5'-ATAAAGTTGCAGGACCACTTCTG-3' before PCR. The linearized template and primer were mixed with 1XPhusion Flash HF Mastermix (New England Biolabs) and placed in the following cycle program: 94°C for 5 minutes, then 94°C (30 seconds), 50°C (30 seconds), 72°C (30 seconds) for 30 cycles, and finally 50°C for 5 minutes. After PCR, 5 μL of each reaction was mixed and used to transform XL10-Gold cells (Agilent). The transformed cells were cultured overnight at 37°C on LB (Luria Broth)-carbenicillin plates. Plasmid DNA was isolated from colonies by using QIAprep Spin Miniprep kit (QIAGEN). Successful mutagenesis was confirmed by DNA sequencing.

[0400] Similarly, pT7–αHL–115C117C119C-D8H6 was made from pT7–αHL-WT-D8H6 using the mutant primer pair: 5′-CAAAAGAGTATATGAGTTGCTTATGCTATTGCTTCAACG-3′ (forward); 5′-CGTTGAAGCAATAGCATAAGCAACTCATATACTCTTTTG-3′ (reverse). pT7–αHL–115C117C119C139C-D8H6 was made from pT7–αHL–115C117C119C-D8H6 using the mutant primer pair: 5′-CCTTATTGGTGCATGTGTTTCGATTGGTCATACACTG-3′ (forward); 5′-CAGTGTATGACCAATCGAAACACATGCACCAATAAGG-3′ (reverse).

[0401] 3. Preparation of αHL heptamers containing a polycysteine ​​track on one of the seven subunits

[0402] The engineered αHL polypeptides were expressed using a commercially available in vitro transcription and translation (IVTT) kit: E. coli T7 S30 Extract System for Circular DNA (Promega). To inhibit transcription by E. coli RNA polymerase, the T7 S30 extract provided in the kit was treated with rifampicin (1 μg mL -1 , final concentration). The standard reaction includes: DNA template (3.2 μg), amino acid mixture without methionine (provided by the kit, 5 μL), S30 premix without amino acids (provided by the kit, 20 μL), [ 35 S]Methionine (2 μL, 1,200 Ci mmol -1 ,15mCi mL -1 ,MP Biomedicals), 15 μL T7 S30 extract (provided by the kit, 15 μL), and nuclease-free water to a final volume of 50 μL. To prepare heteroheptamers, plasmids encoding WT αHL and mutant αHL were mixed in a ratio of 6:1 (WT: mutant). The IVTT mixture was incubated at 37°C for 1 hour.

[0403] Rabbit erythrocyte membranes (3 μL, about 1 mg protein mL -1 ) was added to the IVTT reaction mixture (50 μL), followed by an additional incubation at 37°C for 1 h for heptamerization. The mixture was then centrifuged at 25,000 × g for 10 min. The supernatant was removed and the pellet was resuspended in MBSA buffer (200 μL, 10 mM 3-morpholinopropane-1-sulfonic acid (MOPS), 150 mM NaCl, 1 mg mL-1 Bovine serum albumin, pH 7.4). The MBSA wash was repeated without heating before the pellet was resuspended in 2X Laemmli sample buffer (50 μL) and electrophoresed in a 5% SDS polyacrylamide gel at 70 V for 15 h.

[0404] αHL heteroheptamers containing different numbers of mutant subunits were separated in the gel according to their different electrophoretic mobilities, which were determined by the number of octaaspartate (D8) tails (21). The top and bottom bands correspond to WT7 and (mutant-D8H6)7, respectively. The second band from the top is the desired heteroheptamer containing a single mutant subunit.

[0405] To extract the heptamer pores, the gel was first vacuum dried at room temperature for 5 hours without being fixed on Whatman 3M filter paper. After visualization by autoradiography using Kodak Biomax MR film, the desired bands were cut from the gel with a scalpel. Each excised band was rehydrated in TE buffer (300 μL, 10 mM Tris·HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) at room temperature for 1 hour. The filter paper was then removed and the rehydrated gel was impregnated with a pestle. The resulting slurry was filtered through a 0.2 μm hydrophilic membrane filter (Proteus mini clarification spin column, Generon). The filtrate was stored in 10 μL aliquots at -80°C.

[0406] 4. Single channel recording

[0407] 4.1. Overview

[0408] 1,2-Diphytanoyl-sn-glycero-3-phosphatidylcholine (dPhPC) was purchased from Avanti Polar Lipids. All other chemicals were purchased from Sigma-Aldrich unless otherwise stated.

[0409] Planar bilayer recordings were performed according to the method established by Montal and Muller (22). The two Delrin compartments were separated by a 25 μm thick Teflon membrane containing a pore (60 μm in diameter). The pores were pretreated with a 1% (v / v) hexadecane solution in pentane. Each compartment was then filled with a buffer solution (500 μL, 2M KCl, 20 mM N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), 20 μM EDTA, pH 8.5). A pentane solution of DPhPC (5 mg mL -1) was added dropwise to each compartment. Repeated up and down pipetting of the buffer solution resulted in the formation of a lipid bilayer across the pore. The trans-bilayer potential was applied using two Ag / AgCl electrodes, each contained within a salt bridge formed by 3% (w / v) low melting point agarose in 3M KCl.

[0410] Ionic currents were recorded using a patch clamp amplifier (Axopatch 200B, Axon Instruments) at room temperature (20°C ± 1°C) with a 4-pole low-pass Bessel filter (80 dB / decade). Signals were digitized using a Digidata 1320A digitizer (Molecular Devices) connected to a computer running the pCLAMP 9.2 software suite (Molecular Devices). Unless otherwise stated, signals were filtered with a corner frequency of 5 kHz and sampled at 25 kHz.

[0411] 4.2. Monitoring the feeding process

[0412] The hopper-carrier conjugate (400 μM in 1 μL MilliQ water) was added to a solution of traptavidin (Kerafast) (40 μM, 10 μL phosphate buffered saline, pH 7.4) and incubated at room temperature (20°C ± 1°C) for 15 minutes to form the traptavidin-tagged hopper-carrier. The cis compartment of the device containing the engineered αHL construct of 2M KCl, 20mM HEPBS, 20 μM EDTA, pH 8.5 (500 μL) was stirred until the hole was inserted into the bilayer. The traptavidin-tagged hopper-carrier conjugate (3 μL 36 μM solution) was then added to the cis compartment and driven into the hole with a transmembrane potential of +150 mV (cis at ground). In the case of hopper 1, the passage of the conjugate reduced the current to Ires% = 33% ± 1% (n = 20). Subsequent loading of hopper 1 onto Cys-115 by disulfide interchange gave a current increase of Ires% = 45% ± 1% (n = 20). Under a positive potential (e.g. +150 mV), the hopper moves from Cys-115 toward the trans end (downward) of the track. When the hopper reaches the final pivot point, a negative potential (e.g. -150 mV) drives the hopper in the opposite direction toward the cis end (upward) of the track. Alternation between positive and negative potentials repeatedly drives the hopper back and forth on the track.

[0413] We define cis-to-trans movement as downward and trans-to-cis as upward. At a positive potential (e.g., +150 mV), forward movement is downward; backward movement is upward. At a negative potential (e.g., -150 mV), forward movement is upward; backward movement is downward.

[0414] 4.3. Single channel data analysis

[0415] The current traces were idealized by using Clampfit 10.3 (Molecular Devices). www.qub.buffalo.edu ) to analyze the idealized data (23). Dwell time analysis and rate constant estimation were performed by using the maximum interval likelihood (MIL) algorithm of QuB (24). In the case of feeding on the three-cysteine ​​trajectory and the five-cysteine ​​trajectory, an arbitrary duration of 1 s was assigned to the final pivot in either direction (cis to trans or trans to cis) to complete the kinetic model including step shifts at positive and negative voltages ( Fig.10 , 13 ). The two-state dynamic model is used for feeding between two pivot points ( Fig.12 , 14 ).

[0416] 5. Load the hopper onto the di-, tri- and penta-cysteine ​​tracks

[0417] See Figure 6.

[0418] 6. Feeding mode on the tricysteine ​​track at ±100mV, ±150mV, ±180mV see Figure 7 .

[0419] 7. Feeding mode on the pentacysteine ​​track

[0420] See Figure 8.

[0421] 8. Feeding mode on the hexacysteine ​​track

[0422] See also Fig. 9 .

[0423] 9. Feeding rates at ±100 mV, ±150 mV, and ±180 mV for two steps of the tricysteine ​​trajectory

[0424] Table S1. ±100mV, ±150mV, and ±180mV [b] The three-cysteine ​​track (support points: Cys-115, 117, 119) is composed of QuB [a] The feed rate (k) and average residence time (<τ>) of hopper 1 are obtained.

[0425]

[0426] [a] Dwell time analysis and rate constant estimation were performed by using the maximum interval likelihood algorithm of QuB. An arbitrary duration of 1 s was assigned to the final pivot point in either direction (Cys-119 for cis to trans feed at positive voltage or Cys-115 for trans to cis feed at negative voltage) to complete the kinetic model ( Fig.10 ).

[0427] [b] Data collected from two αHL pores.

[0428] See also Fig.10 .

[0429] 10. Feeding rates at ±100 mV, ±150 mV, and ±180 mV for one step of the dicysteine ​​track

[0430] A dicysteine ​​track consisting of Cys-115 and Cys-117 was used to study the rates of forward and backward feeding at various applied potentials (100 mV, 150 mV, 180 mV). Hopper 1 moved forward from pivot 115 to 117 at positive applied potentials and from pivot 117 to 115 at negative applied potentials. Occasional backward movement was also observed. The rate constants (k) for the feeding steps were obtained at positive and negative voltages (Table S2). The equilibrium constant K was then calculated for each potential, where K = k 向前 / k 向后 .

[0431] Under positive applied potentials, the weak voltage dependence of K is consistent with DNA cargo being driven toward higher potentials. The field is primarily down across the barrel (11), and the four negatively charged phosphodiester groups are located within the barrel based on PyMOL modeling ( Fig. 11B ). The trend is less clear at negative potentials, but in this case, only one or zero phosphodiester is located inside the barrel ( Fig. 11B Despite the presence of a weak electric field in the αHL vestibule ( 11 ), the directionality of feeding under negative applied potentials is more likely to result from the DNA helices in the cis vestibule, which disfavor backward movement of the hopper (see Section 12 ).

[0432] See Figure 11.

[0433] Table S2. ±100mV, ±150mV, and ±180mV [b] The next step is to form a double cysteine ​​track (support point: Cys-115, 117) by QuB [a] The feed rate (k) and average residence time (<τ>) of hopper 1 are obtained.

[0434]

[0435]

[0436] [a] Based on the two-state kinetic model, residence time analysis and rate constant estimation were performed by using the maximum interval likelihood algorithm of QuB ( Fig.12 ).

[0437] [b] Data collected from 4 individual αHL pores.

[0438] [c] Equilibrium constant K = k 向前 / k 向后 .

[0439] See also Fig.12 .

[0440] 11. Feeding rates at ±150 mV for four steps of the five-cysteine ​​trajectory

[0441] Table S3. At ±150mV [b] The next step is to form a 5-cysteine ​​orbital (support points: Cys-113, 115, 117, 119, 121) by QuB [a] The feed rate (k) and average residence time (<τ>) of hopper 1 are obtained.

[0442]

[0443] [a] Dwell time analysis and rate constant estimation were performed by using the maximum interval likelihood algorithm of QuB. An arbitrary duration of 1 s was given to the final pivot point (Cys-121 for cis-to-trans transport at +150 mV or Cys-113 for trans-to-cis transport at -150 mV) in either direction to complete the kinetic model ( Fig.13 ).

[0444] [b] Only the steps from the complete cycle (from 113 to 121 at +150 mV and then from 121 to 113 at -150 mV) (Figure 8) were used to fit the kinetic model ( Fig.13 ). Data were collected from a single αHL pore.

[0445] Table S4. At +150mV [b] The last two pivots (Cys-119, 121) of the lower five cysteine ​​tracks (Cys-113, 115, 117, 119, 121) are formed by QuB [a] The feed rate (k) and average residence time (<τ>) of hopper 1 are obtained.

[0446]

[0447] [a] Residence time analysis and rate constant estimation were performed using the maximum interval likelihood algorithm of QuB. Since there was no 119 to 117 back-off during any feeding cycle, a two-state kinetic model was used to determine the step-movement dynamics on the last two pivots ( Fig.14 ).

[0448] [b] Data collected from a single αHL pore.

[0449] Table S5. At -150mV [b] The lower five cysteine ​​tracks (support points: Cys-113, 115, 117, 119, 121) are composed of QuB [a] The obtained feeding rate (k) and average residence time (<τ>) of the backward movement from 115 to 117 of hopper 1 are obtained.

[0450]

[0451] [a] Residence time analysis and rate constant estimation were performed using the maximum interval likelihood algorithm of QuB. With the exception of the 115 to 117 backshift, which occurred 29 times, the other backshifts were rare and no meaningful rate constants could be derived. Therefore, QuB was set up so that forward shifts were considered irreversible ( Fig.13 ).

[0452] [b] Data collected from a single αHL pore.

[0453] See also Fig.13 and 14 .

[0454] 12. Energetics of directional mobility

[0455] In the case of a forward shift from pivot 115 to 117 at +150 mV on the di-cysteine ​​track (pivots: Cys-115, 117) (as discussed in Section 10), the force exerted on the hopper-oligonucleotide is

[0456] F=n(qe)E

[0457] n = number of phosphodiester esters in the field

[0458] q = fractional charge on the phosphodiester

[0459] e = elementary charge (1.60 × 10 -19 C)

[0460] E = Electric field

[0461] Previously, it was suggested that the applied transmembrane potential drops primarily across the transmembrane β barrel (5 nm) of the αHL pore (11). Thus, at ±150 mV, E is 0.03 V nm -1 .

[0462] Let n = 4 ( Fig. 11B ) and q=1

[0463] F = n(qe)E = 4 × 1.60 × 10 -19 C×0.03V nm -1 =1.92×10 -11 N=19.2pN

[0464] Let n = 4 and q = 0.4 (due to the shielding effect of high salt (25, 26))

[0465] F = n(qe)E = 4 × 0.4 × 1.60 × 10 -19 C×0.03V nm -1 =7.68×10 -12 N=7.68pN

[0466] The work done by the electric field W = Fd per molecule = FdN per mole A (d=0.56nm)

[0467] q=1,W=1.08×10 -20 J = 6.47 kJ mol -1

[0468] q=0.4,W=4.32×10 -21 J = 2.59 kJ mol -1

[0469] Similarly, the force and work to move the hopper-oligonucleotide from 117 to 115 at -150 mV can be calculated using n = 1 ( Fig. 11B ).

[0470] q=1, F=4.80pN, W=1.62kJ mol -1

[0471] q=0.4, F=1.92pN, W=0.648kJ mol -1

[0472] According to the experimentally determined equilibrium constant (K = k 向前 / k 向后 )(Table S2), can be calculated according to ΔG = -RTlnK (R = 8.314JK -1 mol -1 ,T=293.15K) calculate the corresponding ΔG.

[0473]

[0474]

[0475] The discrepancy between the obtained ΔG and the calculated W can be explained by other variable thermodynamic terms (positive or negative), which include the aforementioned DNA helix in the vestibule of the pore. After all, the system is highly complex, with 1) non-uniform side chains; 2) non-uniform cross-section of the β barrel; 3) non-uniformity across the polymer.

[0476] The non-electrostatic terms will be more important for the balance of each step at low applied voltages. However, progressive multi-step feeding requires only the overall equilibrium constant (K 总 ) is greater than 1, and can be calculated by multiplying the equilibrium constants of each step (e.g. K 总 =K1K2K3K4). For example, for all four steps on the five-cysteine ​​trajectory, the experimentally derived K at +150 m V was used. 115-117 ,

[0477] K 总 =K 4 =(4.2) 4 =311

[0478] The Big K 总 The observed progressive and directional feeding is explained.

[0479] It is worth pointing out that the calculations shown here apply only to the step between pivot points 115 and 117. All thermodynamic contributors, including electrostatic work, are likely to vary between pivot points. Fig.15 As shown, as the hopper moves along the track, the charged oligonucleotides gradually move away from the electric field. Therefore, as the hopper approaches the terminal pivot point, the electrostatic contribution to ΔG becomes smaller.

[0480] 13. Residual current patterns of three hoppers on the pentacystis track.

[0481] Table S6. Residual current (Ires%) pattern for each hopper on the five-cysteine ​​trajectory (pivot points: Cys-113, 115, 117, 119, 121).

[0482]

[0483]

[0484] [a] Standard deviation of Ires% was less than 0.5% in all cases (n=3 individual experiments from each hopper).

[0485] 14. -150mV current pattern of hopper 3 at fulcrum 119.

[0486] See also Fig.16 .

[0487] 15. Effective concentration of thiol pivots on cysteine ​​tracks

[0488] For thiol-disulfide interchange:

[0489] RSSR+R'SH(RSSR'+RSH

[0490] The apparent rate constant k is pH-dependent (for the reaction with the thiolate anion) and is estimated to be approximately 10 M at pH 8.5. -1 s -1 The estimates are based on reported rate constants for thiol-disulfide interchanges between L-cysteine, DL-dithiothreitol, L-glutathione and their disulfide forms, e.g. (27-29).

[0491] In the case of the αHL pore, assuming that the disulfide (RSSR) is attached to the protein, and R'SH is in solution, we know that:

[0492] <τ>=1 / (k[R'SH])

[0493] When R'SH is in solution.

[0494] If R'SH is attached to a protein, [R'SH] is the effective concentration in the intramolecular reaction.

[0495] For various values ​​of <τ> (see previous section), assuming k = 10M -1 s -1 , we found that:

[0496]

[0497] The effective concentration of thiols on the track relative to upstream disulfides is in the mM range. Effective concentrations in this range were found to be useful for the second arm of the chelator in the nanoreactor (30) and for intramolecular binding of enzyme inhibitors (31).

[0498] 16. Comparison with other 'mobile molecules'

[0499] In order to highlight the progress presented by our molecular hopper, its properties were selected for comparison with the best results of various reported examples from three different categories.

[0500] Table S7. Comparison of Hopper and Three Classes of “Mobile Molecules” [a] .

[0501]

[0502]

[0503] [a] Here, we only compare the molecular 'walker'. Other 'moving molecules' include: sliders (48), rotors (49, 50), pumps (51).

[0504] [b] Progressive is defined as directional walking without leaving the track. The forward rate of directional walking is faster than the backward rate, that is, K = k 向前 / k 向后 >1.

[0505] In previous small-molecule systems, delocalization from the track has been prevented by various strategies, such as the maintenance of covalent bonds between the walker and its track (32,35,36) and the development of mechanically interlocked architectures (33,34). For DNA-based walker systems, programmable hydrogen bonding allows the walker to remain hybridized to track components (44).

[0506] Directionality has been demonstrated in small-molecule systems for transient travel on trajectories terminated with thermodynamically cold sources (12,35,36) or on trajectories with switchable conformations (37), either by alternating chemical reactions of orthogonal walker trajectories (32,38), or by sequential installation and removal of stops on the trajectories (33). Unidirectional motion of DNA-based walkers has been achieved via a ‘burnt bridge’ mechanism through cycles of DNA hybridization and hydrolysis at the expense of reversibility (40,41,44).

[0507] [c] Due to limited track length, up to 5 steps in each direction were demonstrated. More than 100 progressive steps were achieved by 'flipping the potential' after the hopper reached the final pivot point.

[0508] [d]Autonomous means movement without chemical fuels or accelerator molecules (12).

[0509] Autonomous eight-step forward movement has been demonstrated using a nine-branch trajectory that was terminated using a thermodynamic cold source via dynamic covalent chemistry—reversible Michael addition of secondary amines to α-methylene-4-nitrostyrene units (36). The fastest forward movement rate reported was 5 × 10 -5 s -1 (about 5.6 hours per step). The equilibrium constant (K) for the first six steps is reported as 1, consistent with the initial random steps on the track. The last two steps have K values ​​of 1.3 and 2.5, respectively, to which the overall biased directionality is attributed. In contrast, the hopper of the present invention has a feed rate of about 22 seconds per step and a forward equilibrium constant (K) of about 5 at +150 mV and about 20 at -150 mV.

[0510] [e] All steps demonstrated with the hopper system have equilibrium constants greater than 1 and are therefore directional. On the pentacysteine ​​trajectory at -150 mV, the charge from 117 to 115 has K = k 117-115 / k 115-117 =22, which indicates that the observed retreat is caused by the slower step rate of the next step to the downstream pivot 113.

[0511] [f] On the five-cysteine ​​track, the average step time at -150 mV was approximately 22 s.

[0512] [g] Due to the constraints of current nanopore-based tracks, the macromolecular cargo that can be moved through hopper systems is limited to linear molecules such as oligonucleotides and peptides. On the other hand, DNA walkers moving on surfaces are able to transport cargo such as nanoparticles (42,45).

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[0565] Example 2

[0566] This example demonstrates site-selective thiol-disulfide interchange via controlled spatial arrangement.

[0567] Thiol-disulfide exchange occurs extensively in cellular processes including redox sensing and homeostasis, protein folding, cell signaling and apoptosis regulation. Nature utilizes this chemistry in a highly selective manner, which is challenging to reproduce in vitro. In the present embodiment, the inventors report site-selective and regioselective thiol-disulfide exchange on macromolecular disulfide substrates extended in protein nanoreactors, wherein they react with cysteine ​​thiols presented at different positions along the length of β-chains in tubular structures. Many individual reaction events are detected by promoting substrate turnover. For each substrate, most reactive cysteines on β-chains and which sulfur atom in disulfide is attacked are defined, indicating that chemical reactions can be controlled with atomic precision.

[0568] Controlling selectivity has been a long-standing challenge for synthetic chemists. Two important aspects of this endeavor are site selectivity - the ability to discriminate between two (or more) similarly reactive sites in a molecule, and regioselectivity - the ability to discriminate between two (or more) sites within a given functional group. 1a,2a Achieving chemical selectivity often requires exploiting or disrupting the inherent spatial arrangement and electronic properties of molecules or functional groups. 2a A common strategy involves installing a reagent-positioning group on a substrate, which can then be removed 3a , or recruiting catalysts (organic or transition metal based) that bind to substrates to favor specific reaction pathways 1a,2a Alternatively, in micelles 4a or supramolecular cage 5a , using pre-organization of reactants in order to redirect regioselectivity. Recently, it was demonstrated that site selectivity is caused by the spacing of reactive sites within metal-organic frameworks. 6a In the context of enzymology, selectivity has been well demonstrated 7a For example, a switch in the regioselectivity of L-tryptophan nitration by cytochrome P450 has been attributed to a change in substrate orientation that depends on a single amino acid substitution. 8a Despite these impressive achievements, the complex molecular interactions underlying many site-selective or regioselective chemistries were elucidated only after their serendipitous discovery and in some cases are still not fully understood. 2a .

[0569] Thiol-disulfide chemistry is universal in nature, where it enhances key biological activities involving enzymes such as thioredoxin and protein disulfide isomerase. In this work, the inventors used a single-molecule approach to examine thiol-disulfide interchange. Previously, thiol-disulfide chemistry was studied using single-molecule force clamp spectroscopy by monitoring the unfolding of a titin domain mutant containing two disulfides. 9a Unfolding is initiated by L-cysteine, which reduces the number of exposed disulfides. Remarkably, under applied force, the cysteine ​​side chain released from the first disulfide regioselectively reacts with the remaining disulfides, making one sulfur atom about four times more likely to be exposed than the other. Subsequent computational simulations attributed the regioselectivity primarily to protein conformation. 10a , which in the bulk solution 11a-14a Until now, no system has been reported for programming a nucleophile to react with one of the two chemically equivalent sulfur atoms in a disulfide.

[0570] The system reported in this paper consists of a nanoreactor - a cysteine ​​mutant of α-hemolysin (αHL) Fig.17A )—and linear macromolecular substrates with disulfide bonds in the backbone ( Fig. 17B). The substrate consists of a biotin-traptavidin binding motif at the end of a peptide that is coupled to a DNA oligonucleotide via a strain-promoted azide-alkyne cycloaddition. A disulfide is installed between the peptide and the oligonucleotide, with the oligonucleotide having a sulfur atom Sα close to the peptide and a sulfur atom Sβ close to the oligonucleotide. The disulfides are flanked by ethylene groups, and therefore Sα and Sβ are expected to be very similar in terms of intrinsic reactivity and spatial arrangement.

[0571] Four substrate constructs, POC1 to POC4 (Table 1A), were tested in which the length of the peptide segments was adjusted two amino acid residues (Gly and Ser) at a time. When the substrate was fully stretched, each additional dipeptide further reduced the disulfide content by about into the barrel of the αHL pore. Seven single-cysteine ​​αHL mutants were generated at positions 113, 115, 117, 119, 121, 123, and 143 (on one of the seven αHL subunits) with the side chain pointing into the lumen of the transmembrane β barrel. Adjacent cysteines along the β strand are separated by an average distance of approximately (Cα-Cα), the vertical distance is Cys-143 on the antiparallel strand is about 2.5 times lower than Cys-115 in the perpendicular direction. 15a,16a Experimental and computational evidence for a colinear trisulfide-like transition state in thiol-disulfide interchange 17a-19a , we reasoned that the proximity and longitudinal alignment of the substrate disulfides to the cysteine ​​side chains of the nanoreactor would be critical in achieving site- and regioselective control over the reaction.

[0572] Table 1A

[0573]

[0574]

[0575]

[0576] a. All substrates contain oligoadenosine 40mer (A40).

[0577] b.bK, biotinyl-L-lysine; F, phenylalanine; PEG, polyethylene glycol; S, serine; G, glycine.

[0578] c. Regioselectivity is expressed as the number of sites that result in αHL-oligonucleotide adducts (S β ) or αHL-peptide adduct (S α ) The DTT cleavage was not counted.

[0579] d. Residence time analysis and rate constant estimation were performed by using the maximum interval likelihood algorithm of QuB.

[0580] e. / , cannot be measured.

[0581] f. DTT cleavage occurred <5 times and was therefore ignored in the kinetic model.

[0582] Results and discussion

[0583] Flipping of thiol-disulfide interchange. To monitor the site- and regio-selectivity of thiol-disulfide interchange, a substrate flipping method was first established. When capped with traptavidin, the substrate enters the αHL pore from the cis compartment under an applied positive potential and elongates within the electric field. Subsequent alignment-mediated thiol-disulfide interchange with the nanoreactor cysteine ​​side chains leads to the formation of covalent adducts ( Fig. 17C ), when the top or bottom of the substrate detaches, this is revealed by a step shift in the ionic current through the pore. The adduct is then released from the nanoreactor by the reducing agent dithiothreitol (DTT) present in the trans compartment, allowing a new cycle to begin. No sub-step shifts were observed during substrate detachment.

[0584] Controlled reaction between thiolates and substrate disulfides in the nanoreactor. For the time being (see Conclusions), we assume that the system is static, i.e., there is minimal conformational motion of the protein pore and the substrate remains extended. In previous computational studies, it has been proposed that thiol-disulfide interchange proceeds via a nearly linear trisulfide-like transition state with adjacent sulfur atoms separated by approximately 18a,19a Assuming that the cysteine ​​side chain of the nanoreactor rotates freely, the perpendicular distance drawn by the nucleophilic thiolate ( Fig.18A ) is about (CS bond length is approx. and CCS bond angle is about 109.5°). In order to make the interchange, The region must be connected to the disulfide at one or the other end. Reaction zone overlap ( Fig.18A ). When the transition state arrangement is difficult to achieve, in which the nucleophilic thiolate resides at one end of the disulfide Outside the radius, the interchange is unfavorable ( Fig.18A ).

[0585] The system reported here is able to deliver disulfide bonds to nucleophilic thiolates with angstrom precision. When the alignment is favorable, the interchange between the substrate disulfide and the nanoreactor thiolate generates a covalent adduct—either an αHL-peptide adduct or an αHL-oligonucleotide adduct (as discussed later)—which is subsequently released by DTT ( Fig. 17C ,19A ). When the alignment is unfavorable, DTT (5 mM) diffused into the nanopore from the trans compartment cleaved the substrate disulfide bond before exchange could occur, and the two resulting fragments escaped from the pore, the DNA propelled by the applied potential and the peptide by diffusion ( Fig.19A Based on these observations, regardless of regioselectivity, reactivity was defined as the number of thiol-disulfide interchange reactions observed under specified conditions divided by the total number of substrate crossing events, all of which resulted in either interchange or cleavage (reactivity = n 反应 / n 穿过 )( Fig.18B ).

[0586] Using the above four substrates (POC1 to POC4) and seven nanoreactors (nanoreactors 113, 115, 117, 119, 121, 123, and 143), we observed a wide range of reactivity from 0% to 100% (Table 1A, Figure 18). In particular, the extension of the peptide segment (approximately ) can significantly affect the reactivity. For example, once penetrated into nanoreactor 117, the disulfide in POC1 was almost exclusively cleaved by DTT (4.5% reactivity), indicating an unfavorable arrangement. In contrast, the disulfide in POC2, two longer amino acids, always formed a covalent adduct with nanoreactor 117 (100% reactivity) (Table 1A, Figure 18). Similar phenomena were observed with nanoreactor 119 (POC1, 0% reactivity; POC2, 78%) and nanoreactor 121 (POC3, 0% reactivity; POC4, 29%). Similarly, moving the cysteine ​​position along the β-strand by two residues (approximately 1% in the vertical direction) increased the reactivity of the cysteine. ) is sufficient to substantially alter or prevent reaction with the substrate. For example, when comparing nanoreactor 119 to nanoreactor 121, reactivity dropped from 78% to 0% with POC2, from 96% to 0% with POC3, and from 100% to 29% with POC4. Thus, manipulation of the position of the disulfide within the substrate or within the position of the cysteine ​​within the nanoreactor determines whether the thiol-disulfide interchange proceeds.

[0587] The sulfur atom in the substrate disulfide is selected. The substrate disulfide connects the neutral fragment (peptide) to the negatively charged polymer (oligonucleotide) ( Fig. 17B ). Once the thiol-disulfide exchange occurs within the cysteine ​​nanoreactor, an αHL-peptide adduct or an αHL-oligonucleotide adduct is formed, depending on whether the Sα or Sβ atom of the disulfide bond is attacked. The two adducts show unique current characteristics under applied potential ( Fig.19A), the adducts were assigned based on their behavior in response to a voltage step (see SI Section 1). A stable current level control was associated with the αHL-oligonucleotide adducts (attacked Sβ), while rapid switching between three discrete levels was observed for the αHL-peptide adducts (attacked Sα). We speculate that in the electric field within the αHL barrel 20, the charged oligonucleotide experiences a constant pulling force that locks its conformation in an extended state, while the neutral peptide is less constrained and switches between several conformations. By counting the adducts formed after multiple substrate flips, we determined the sulfur regioselectivity of each nanoreactor-substrate combination and expressed it as the percentage of flips that resulted in attack on Sα or Sβ ( Fig.19B , Table 1A).

[0588] In general, when the cysteine ​​in the pore is located within the reactive region of the substrate disulfide, lowering its position (e.g., from 113 to 115) reduces the chance of forming an αHL-peptide adduct and increases the chance of forming an αHL-oligonucleotide adduct. Complete regioselectivity for Sβ (αHL-oligonucleotide adduct formation) was achieved with POC1 and nanoreactors 115 or 143, POC2 and nanoreactor 119, and POC4 and nanoreactor 121. In these cases, the nucleophilic thiolate can only reach the lower part of the disulfide. Reactive region ( Fig.18A ), thereby allowing only the transition state leading to the αHL-oligonucleotide adduct to form.

[0589] The fully Sβ regioselective thiol-disulfide exchange between POC1 and nanoreactor 115 indicated that the thiolate was located below the Sβ in the barrel. Assuming a fully stretched linker ( Fig. 17B )extend And Cys-115 is located about 1.5 m from the outlet of the barrel. At , about 4 phosphodiester bonds of POC1 must be located within the electric field of the barrel. The peptide moves one phosphodiester bond out of the electric field with each stretch of two amino acids (in a stretched single-stranded DNA, the distance between nucleotides is 21). Controlled positioning of the disulfide requires at least one charged residue within the barrel to perform the pulling, making POC4 the longest workable conformation in this assembly. Over 80% regioselectivity for Sα (αHL-peptide adduct formation) was achieved with POC4 and nanoreactor 113. We propose that complete Sα regioselectivity can be achieved by using longer peptides and longer pores.

[0590] Kinetics of thiol-disulfide interchange. After the substrate penetrates the nanoreactor, one of three possible reactions occurs: formation of αHL-peptide adducts, formation of αHL-oligonucleotide adducts, or cleavage by DTT. At the single-molecule level, the rate constant is related to the average residence time of the state 22a For the formation of unimolecular adducts, the rate constants—kα for the formation of αHL-peptide adducts; kβ for the formation of αHL-oligonucleotide adducts—are the inverse of the corresponding average residence time (of only one reaction to be carried out) (τ α or τ β , for example τ α =1 / k α ). For bimolecular DTT cleavage at a fixed DTT concentration (e.g., 5 mM), <τ DTT >=1 / k' DTT =1 / k DTT [RS - ] DTT , where k' DTT is incorporated into [RS - ] DTT The pseudo-first-order rate constant for the electrochemical reaction is the concentration of DTT thiolate (at pH 8.5, the concentration of doubly ionized DTT is negligible). 23a ).

[0591] After many flips, the average waiting time before reacting (<τ>) is given by:

[0592]

[0593]

[0594] Depending on the arrangement, one of the three pathways may dominate, such as <τ α >,<τ DTT >>><τ β >, in this case, <τ> = <τ β >. In cases where more than one pathway competed, residence time analysis was performed using the maximum interval likelihood (MIL) algorithm of QuB software to derive independent rate constants (k α ,k β ,k DTT ') 24a (Table 1).

[0595] Assuming that the thiol-disulfide exchange involves the thiolate anion, the rate constant k α and k β depends not only on the arrangement but also on the pH and pKa value of the nanoreactor thiol. The latter is estimated to be 8.5 ± 1.5 in the wide β barrel. 25aHowever, when comparing different substrates reacted with the same nanoreactor (i.e., cysteine ​​pKa fixed) at a fixed pH (e.g., pH 8.5), k α and k β Compared with S α and S β The thiolate arrangement is directly related to the β / k α =n β / n α gives the regioselectivity, where n β is the number of αHL-oligonucleotide adducts formed in the time interval, n α is the number of αHL-peptide adducts formed in the same interval. The spatial relationship between the substrate and a particular nanoreactor can be inferred from the obtained rate constants (Table 1A). For each nanoreactor, a substrate S β The atoms are optimally arranged to obtain the highest rate of αHL-oligonucleotide adduct formation (k β The highest k is between nanoreactor 113 and POC1, between nanoreactor 115 and POC3, between nanoreactor 117 and POC3, and between nanoreactor 119 and POC4. β For example, with nanoreactor 117, k β The value range is about 300 times, which is 5.3 × 10 -4 s -1 , using POC3 gives 0.17s -1 This changes the overall results from the control by using DTT cleavage of POC1 (k' DTT =9.4×10-3s -1 ) is transformed into S β The effects of arrangement on reaction kinetics and site selectivity were demonstrated by controlling the thiol-disulfide exchange of the substrates. As expected, the reaction rates of various substrates with DTT were similar and did not vary significantly with the placement of cysteine ​​residues in the nanoreactor. Therefore, as shown in this paper ( Fig.18B ) and the rates of adduct formation (kα and kβ, Table 1) are correlated. Furthermore, the kinetic data are consistent with the arrangements obtained from modeling studies in which the substrate chain is fully extended ( Fig.18C ).

[0596] Site- and regio-selective tandem thiol-disulfide interchanges. To recapitulate the common tandem thiol-disulfide interchanges of protein dithiols 26a,27aWe constructed two bicysteine ​​nanoreactors based on nanoreactor 115, which has an additional cysteine ​​on the antiparallel strands (115 / 143) or on the same strand (115 / 117) within a single αHL subunit. β Regioselectively reacting three single-cysteine ​​nanoreactors (115, 143, and 117), we demonstrated that the dual-cysteine ​​nanoreactors underwent sequential thiol-disulfide interchanges.

[0597] After threading the POC1 molecule into the dual-cysteine ​​nanoreactor, the substrate disulfide was first reacted with one of the two available cysteine ​​thiolates to form a covalent αHL-oligonucleotide adduct. The site selectivity of the initial thiol-disulfide interchange was determined. In the case of nanoreactor 115 / 117, the oligonucleotide adducts at positions 115 and 117 could be distinguished by their residual currents (ΔIres% = Ires% (117) - Ires% (115) = +2.0% ± 0.5%, n = 3) ( Fig. 20A ), which is consistent with the results obtained using single cysteine ​​nanoreactors (Ires%(115)=45%±1%, n=9; Ires%(117)=47%±1%, n=12). In the case of nanoreactor 115 / 143, the αHL-oligonucleotide adducts formed at positions 115 and 143 can also be distinguished by their residual currents (ΔIres%=Ires%(115)-Ires%(143)=+0.68%±0.03%, n=3) ( Fig. 20B and Figure S23), which is again consistent with the results obtained using single-cysteine ​​nanoreactors (Ires%(115)=45%±1%, n=9; Ires%(143)=46%±1%, n=6). In the case of nanoreactors 115 / 117, we found that the substrate disulfide reacted selectively with the Cys-115 site (100%) to form αHL-oligonucleotide adducts (n=30), which is consistent with the kinetics of the single-cysteine ​​nanoreactors: k β (115)>>k β (117) In contrast, we observed an 81% reaction with Cys-115 and a 19% reaction with Cys-143 using nanoreactor 115 / 143, which is also consistent with the kinetics of the single-cysteine ​​nanoreactor (Table 1A, Fig. 20B , S23).

[0598] Once the adduct is formed, the free cysteine ​​on the nanoreactor 115 / 117 or nanoreactor 115 / 143 attacks the nanoreactor cysteine ​​(S γ ) contributed by sulfur or oligonucleotides (S β) to initiate the second thiol-disulfide exchange (Figure 20). Interestingly, the regioselectivity of these two intramolecular reactions is different. When the free cysteine ​​is on the same chain (115 / 117), the complete S β Regioselectivity causes the oligonucleotide to move back and forth from one site to another ( Fig. 20A In the presence of DTT, before the adduct is released by DTT ( Fig. 20A The average number of transfers per cycle was 11 (n=40 cycles), and up to 29 consecutive transfers were recorded in a single cycle. When the free cysteine ​​was located on the antiparallel strand (115 / 143), we observed that thiolates activated S in the absence of DTT. β and S γ , which results in the irreversible release of the oligonucleotide from the nanoreactor and the formation of cross-linked disulfide bonds ( Fig. 20B Subsequent addition of DTT regenerates free cysteine ​​via the transient αHL-DTT intermediate 28 ( Fig. 20B , see SI Section 3). The average number of transfers before intramolecular release depends on which site the αHL-oligonucleotide adduct is initially formed: for αHL-oligonucleotide adducts initially formed on Cys-115 (n=11 cycles), there are 0.54 transfers per cycle (n=48 cycles) and for those initially formed on Cys-143, there are 1.36 transfers per cycle (n=11 cycles). The difference of about 1 suggests that the release is only from Cys-115, which is indeed the case. Up to 6 transfers have been seen within a single cycle (Figure 23). Of the αHL-oligonucleotide adducts formed on Cys-115, 85% are immediately released by Cys-143 through intramolecular Sγ attack in the absence of transfer.

[0599] The regioselectivity of the second thiol-disulfide exchange was attributed to the Cα-Cα distance between the two cysteines involved (Figures 20 and 23). Only Sβ attack occurs (Figure 20). For nanoreactor 115 / 143, where When the adduct is on Cys-115 ( FIG. 20 , FIG. 23 ), Sγ attack is dominant and more favorable.

[0600] in conclusion

[0601] In biology, thiol-disulfide interchanges in proteins are highly site- and regio-selective due to the accessibility or altered chemical nature of reactive groups. 26a,29a-31aUntil now, no artificial systems have been constructed to mediate precise site-selective control of this important chemistry. In the present work, we examine the effects of spatial arrangement on thiol-disulfide interchange between nanoreactor cysteines and disulfides composed of chemically equivalent sulfur atoms (Sα and Sβ). Our results demonstrate that thiol-disulfide interchange is sensitive to arrangement and can in fact be controlled with angstrom precision. By manipulating the relative positions of substrate disulfides and thiolates in single-cysteine ​​nanoreactors, we control complete Sβ regioselectivity and >80% S α Interchanges between regioselective sites. Serial thiol-disulfide interchanges using dual-cysteine ​​nanoreactors simultaneously exhibit remarkable site- and regioselectivity, which is present in biological systems but unprecedented in synthetic systems. The ability of αHL-oligonucleotide adducts to move back and forth between Cys-115 and Cys-117 has been further exploited to construct molecular “hoppers” that can advance up and down the 5- or 6-cysteine ​​track. 32a .

[0602] To aid in the description, we have so far assumed that our reaction system is static. However, we anticipate significant molecular motion of the components, especially the confined polymer substrate. 33a In static systems, site- and regio-selectivity can be achieved by precise alignment, which will then result in the fastest reaction rate. However, as in host-client systems, strict organization is difficult to achieve, and a degree of conformational flexibility may be advantageous. In fact, we find that in the reaction with the lowest k β In the basis of values, we can always reach the complete S β Regioselectivity (Table 1, Fig.19B , C). In these cases, it appears that the reactive thiols in the nanoreactor are on average positioned below the substrate disulfide (i.e., toward the trans side). The rare encounters in the colinear arrangement caused by conformational flexibility then lead to S β Regioselectivity, while S α Inaccessible Fig.19C ).

[0603] Based on this study, we designed transmembrane communication based on substrate arrangement and site- and regio-selective chemistry. 34a and organization 35a Signals based on thiol-disulfide exchange have not been used in bottom-up manufacturing of , presumably because they are difficult to control. The results described here show that selective vectorial cargo movement across membranes is achievable, which would involve the controlled transmission of multiple chemical signals ( Fig.21 ). The system is catalytic and allows multiple turnovers ( Fig. 17C) will increase the sensitivity of the signal. 36a,37a ) or using synthetic film sensors or transmitters 38a–42a The system reported here can be directly incorporated into droplet-based synthetic tissues with lipid bilayers as the backbone. 35a , to achieve internal (inter-compartment) or external communication.

[0604] The spatial arrangement and electronic properties of the functional groups flanking the disulfide in the substrate can be manipulated during synthesis to produce intrinsic regioselectivity. Furthermore, the spatial arrangement of reactants within the narrow 'tube' can be generalized for site- and regio-selective control of other chemistries. In fact, when the reactive azide is properly spaced within the porous structure of the metal-organic framework, the click reaction with the diyne becomes site-selective. 6a .

[0605] method

[0606] Single molecule monitoring of thiol-disulfide exchange. The substrate (μL MilliQ aqueous solution of 400μM1) was added to a solution of traptavidin (Kerafast) (40μM, 10μL phosphate buffered saline, pH 7.4) and incubated at room temperature (20°C ± 1°C) for 15 minutes to form a substrate labeled with traptavidin. The device containing the cis compartment of the recording planar double layer of the αHL nanoreactor was stirred until a single hole in the double layer was inserted. Then the substrate (3μl, 36μm) labeled with traptavidin- was added to the cis compartment, which contained 500μL recording buffer (2M KCl, 20mM HEPBS, 20mMEDTA, pH 8.5). The trans compartment contained the same buffer supplemented with 5mM DTT. In order to drive the macromolecular substrate into the nanopore, a potential of +150mV was applied to the trans side of the double layer. The substrate passed through reduced the current to an Ires% of about 33% (see SI Section 4 for each substrate-nanoreactor combination). After the thiol-disulfide exchange between the substrate and the nanoreactor, the current increased as a segment of the substrate left the nanoreactor (see SI Section 4 for Ires% for each substrate-nanoreactor combination). The subsequent reaction with DTT released the adduct and returned the nanoreactor to its initial state, which was ready for another round of thiol-disulfide exchange.

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[0650] Supplementary information for Example 2

[0651] 1. Identification of covalent adducts by current model

[0652] To confirm the identity of the adduct formed upon thiol-disulfide interchange, we probed its behavior in response to a voltage step ( FIG. 22 ).

[0653] For αHL-oligonucleotide adducts ( Fig.22A ), once the applied potential is switched between high positive and negative values ​​(e.g., ±150 mV), the negative charge on the oligonucleotide in the barrel redirects the chain. At low potentials (e.g., +50 mV), the pulling force is insufficient to overcome the energy barrier to redirection. Therefore, due to the condensation of the oligonucleotide, the current level recorded at low potentials (e.g., +50 mV) is lower if the potential just applied has the opposite polarity.

[0654] For αHL-peptide adducts ( Fig. 22B ), the peptide is locked within the upper vestibule of the αHL pore due to traptavidin acting as a barrier. In this case, the current pattern at low applied potentials (e.g., -50 mV) is the same as that at high potentials of opposite polarity (e.g., ±150 mV), indicating that the conformation is not affected.

[0655] See Figure 22.

[0656] 2. Site- and regio-selectivity of thiol-disulfide exchange between nanoreactors 115 / 143 and POC1

[0657] See Figure 23.

[0658] 3. Regioselectivity of thiol-disulfide exchange between DTT and αHL adducts

[0659] Previously, the transition intermediate was detected upon DTT reduction of the mixed disulfide formed between the αHL pore with a single Cys-117 and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). 1b .

[0660] In this work, DTT was used to release covalent disulfide adducts—αHL-oligonucleotide adducts or αHL-peptide adducts—from the nanoreactor. DTT thiolate is able to attack the nanoreactor cysteine ​​sulfur (Sγ) or the sulfur atom donated by the polymer: oligonucleotide or peptide (Sδ) ( Fig.24A ). When Sδ is attacked, an αHL-DTT adduct with a unique failure signature is formed ( Fig.25 ). The formation of αHL-DTT adducts was monitored (Table S1, Fig.24A ), we investigated the regioselectivity of αHL-polymer adduct release by DTT. The Sγ regioselectivity was defined as the number of αHL-DTT adducts detected per the total number of events:

[0661]

[0662] In general, DTT attacked Sδ more frequently than Sγ (Table S1). When mixed disulfide bonds were formed between nanoreactor 113 or nanoreactor 143 and polymer (oligonucleotide or peptide), complete Sδ regioselectivity was seen. In contrast, when mixed disulfide bonds were formed between nanoreactor 113 and DTNB (Table S1, Fig. 24B ), and over 80% of the reaction was seen in Sγ when mixed disulfides were formed between nanoreactor 143 and DTNB. This ruled out the possibility that the αHL-DTT adduct was too short-lived to be detected.

[0663] For the thiol-disulfide interchange between DTT and the covalent adduct, the constituent sulfurs are chemically and spatially dissimilar, and DTT diffuses freely in solution. Thus, no steric control over the thiolate attacked and innate regioselectivity were observed (Table S1).

[0664] The lifetime of the αHL-DTT adduct (<ταHL-DTT>)—the average waiting time before cyclization (Table S1)—varied between nanoreactors. This means that the nanoreactor cysteine ​​thiols, which are leaving groups upon attack by DTT thiolate, have different pKa values. 2b .

[0665] See Figure 24.

[0666]

[0667] a. Unless otherwise stated, residence time analysis was performed by using QuB's maximum margin likelihood algorithm.

[0668] b. / , cannot be measured

[0669] c. 〈τDTT-αHL〉 is calculated as the arithmetic mean due to insufficient events with nanoreactors 119 or 121 .

[0670] d. We speculate that once nanoreactors 119 or 121 are reacted with DTNB, the current level of the adduct formed is indistinguishable from that of the αHL-DTT adduct. Additional experiments using other reducing agents (e.g., dihydrolipoic acid) may give identifiable current steps for transient intermediates.

[0671] 4. Current levels for each substrate / nanoreactor combination

[0672] Table S2 Residual current levels (Ires%) of individual substrate / nanoreactor combinations

[0673]

[0674]

[0675] a. All measurements in the table are made at +150mV.

[0676] b. Standard deviation of Ires% is less than 0.5% (from n>3 separate experiments for each substrate / nanoreactor combination).

[0677] c. For each adduct, three discrete levels of exchange were recorded. However, adduct-to-adduct variation in Ires% was seen. The observed Ires% ranges for all experiments are reported herein.

[0678] d. Three discrete interchange levels are recorded during the pass-through phase.

[0679] 5. Conformational flexibility of macromolecular substrates during penetration

[0680] Interchange current levels were observed during the substrate crossing phase and were more prominent on substrates containing longer peptides (i.e., POC3 and POC4) ( Fig.25 ).

[0681] The conformational switch during the pass-through phase is associated with fewer phosphodiester units remaining within the electric field of POC3 and POC4. Therefore, less pulling force is applied to the peptide, resulting in greater conformational freedom.

[0682] See also Fig.25 .

[0683] 6. Synthesis and Characterization of Macromolecular Substrates

[0684] 6.1. Chemicals

[0685] Acetic anhydride (Ac2O), acetonitrile (HPLC grade), 5-azidopentanoic acid, N,N-diisopropylethylamine (DIPEA), diethyl ether, dimethylformamide (DMF), hexafluoro-2-propanol (HFIP), N,N-hydroxybenzotriazole (HOBt), 1-methyl-2-pyrrolidone (NMP), Nα-Fmoc-Nε-biotinyl-L-lysine, piperidine, triethylamine (TEA), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), Fmoc-Phe-OH, and Rink amide MBHA resin LL (100-200 mesh) were from Novabiochem. Fmoc-NH-polyethylene glycol (PEG)(12)-COOH and Fmoc-Gly-Gly-OH were purchased from Iris Biotech GmbH.

[0686] 6.2. Peptide preparation using solid phase peptide synthesis

[0687] Peptides of POC1 and POC2 were synthesized by manual solid-phase peptide synthesis using a protocol adapted from a previously reported protocol. 3b ( Fig.26A , 27A ). The peptides of POC3 and POC4 were purchased from Peptide Protein Research Ltd.

[0688] The Fmoc method was used using HBTU and DIPEA as coupling reagents on 0.25 mmol Rink amide MBHA resin LL (0.37 mmol g -1). The resin was washed and swollen in DMF overnight and Fmoc was removed at room temperature by treatment with 20% (v / v) piperidine in NMP. The peptides were assembled by standard chain extension using reactants Nα-Fmoc-Nε-biotinyl-L-lysine, Fmoc-Phe-OH, Fmoc-NH-PEG(12)-COOH, Fmoc-Gly-Gly-OH (e.g., POC2 peptide) and 5-azidopentanoic acid. For each coupling reaction, a solution of 1.0 mmol reactant, 0.95 mmol HBTU and 2.0 mmol DIPEA in 1.9 mL DMF was added to the resin, which was then shaken for 12 minutes. After each coupling, the resin was washed with NMP and capped with 1.5 mmol Ac2O solution, 0.044 mmol HOBt and 0.39 mmol DIPEA in 3 mL DMF for 2 minutes. Then before the next coupling, the blocked resin was washed again with NMP. After completing the chain assembly, the resin was washed successively with DMF and ethanol and dried in a vacuum. By treating with TFA for 2 hours, the peptide was cut from the resin. TFA was evaporated in a nitrogen stream, and the peptide was precipitated with cold ether, centrifuged subsequently, and ground several times with ether.

[0689] 6.3. Peptide purification and characterization

[0690] The crude peptide fragments of POC1 or POC2 were dissolved in DMF and analyzed by preparative RP-HPLC (Dionex UltiMate 3000, Vydac C18 column: 250 × 22 mm, 10-15 μm (particle size), linear gradient: 5% to 95% of eluent B in eluent A over 45 min, flow rate: 15 mL min -1 The product was purified by analytical LC-MS (Waters LCT accurate mass time-of-flight instrument (ESI-positive), Chromolith RP-18e column: 50 mm × 2 mm, linear gradient: 5% to 100% of eluent B in eluent A over 8 min, flow rate: 1 mL min -1 The identity and purity of the fractions were determined by HPLC (Agilent 1260 Infinity HPLC; Polaris C18 column: 150×4.6 mm, 5 μm (particle size), linear gradient: 5% to 95% of eluent B in eluent A over 10 min, flow rate: 1 mL min -1, eluent A: 0.1% TFA in water, eluent B: 0.1% TFA in acetonitrile) characterization ( Fig.26B , 26C , 27B, 27C).

[0691] See Figures 26 and 27.

[0692] 6.4. Generation and characterization of macromolecular substrates

[0693] Oligonucleotides modified with dibenzocyclooctyne-disulfide (DBCO-SS) at the 5' end were purchased from biomers.net.

[0694]

[0695] 5'DBCO-SS-A40 oligonucleotide (10 μL, 1 mM MilliQ water) was added to each peptide segment (1 μL, 10 mM MilliQ water). The mixture was placed at room temperature for 30 minutes to form peptide-oligonucleotide conjugates (POC1-4) by copper-free click reaction ( Fig.28 ).

[0696] The conjugate was purified by semi-preparative HPLC (Agilent 1260 Infinity; Supelco Discovery BIO wide-pore C18 column: 250×10 mm, 10 μm; linear gradient: 10% to 90% of eluent B in eluent A over 30 min, flow rate: 4.5 mL min -1 ; eluent A: 0.1% TFA in water; eluent B: 0.1% TFA in acetonitrile) and by LC-MS (UPLC-MS Waters XEVO G2-QTOF (ESI-negative); ACQUITY UPLC Oligonucleotide BEH C18 column: 2.1×50 mm, 1.7 μm (particle size); linear gradient: 0% to 70% of eluent B in eluent A over 8 min, flow rate: 0.2 mL min -1 ; Eluent A: 8.6 mM TEA, 200 mM HFIP in 5% methanol / water (v / v) solution; Eluent B: 20% methanol solution of eluent A).

[0697] 7. Preparation of αHL Monomers Containing Cysteine

[0698] Single-cysteine ​​nanoreactors based on pT7-αHL-D8H6, encoding wild-type αHL with an octa-aspartic acid tail and a histidine tag at the C-terminus, have been reported previously113,115,117,119,121 and dual-cysteine ​​nanoreactors115 / 117. 4b.

[0699] Plasmid pT7-αHL-143C-D8H6 encodes an αHL mutant containing a cysteine ​​at position 143 and an octa-aspartic acid tail and a histidine tag at the C-terminus. It was generated from pT7-αHL-D8H6 by in vivo homologous recombination. Two sets of PCR reactions were performed. For the first reaction, the template was first linearized by NdeI (New England Biolabs) before PCR using a forward mutant primer: 5'-CAAATGTTTCGATTTGTCATACACTGAAATATGTTC-3' and a reverse mutant primer (SC47): 5'-CAGAAGTGGTCCTGCAACTTTAT-3'. For the second reaction, the template was linearized by HindIII (New England Biolabs) before PCR using a reverse mutant primer: 5'-GAACATATTTCAGTGTATGACAAATCGAAACATTTG-3' and a forward non-mutant primer (SC46): 5'-ATAAAGTTGCAGGACCACTTCTG-3'. The linearized template and primers are mixed with 1X Phusion Flash HF Mastermix (New England Biolabs) and placed in the following cycle program: 94°C for 5 minutes, then 94°C (30 seconds), 50°C (30 seconds), 72°C (30 seconds) for 30 cycles, then 50°C for 5 minutes. After PCR, 5 μL of each reaction is mixed and transformed into E. coli XL10-Gold cells (Agilent). The transformed cells are cultured overnight at 37°C on LB (Luria Broth)-carbenicillin plates. Plasmid DNA is isolated from bacterium colonies by using QIAprep Spin Miniprep kit (QIAGEN). Successful mutation is confirmed by DNA sequencing.

[0700] Similarly, pT7-αHL-115C143C-D8H6 was made from pT7-αHL-115C-D8H6 using the same two mutant primers. pT7-αHL-123C-D8H6 was made from pT7-αHL-WT-D8H6 using two mutant primers: 5'-CGGATTCAACGGTTGTGTTACTGGTGATGATACAGG-3' (forward), 5'-CCTGTATCATCACCAGTAACACAACCGTTGAATCCG-3' (reverse).

[0701] 8. Preparation of αHL heptamer containing cysteine ​​on one of the seven subunits

[0702] The engineered αHL polypeptides were expressed using a commercially available in vitro transcription and translation (IVTT) kit: E. coli T7 S30 Extract System for Circular DNA (Promega). To inhibit transcription by E. coli RNA polymerase, the T7 S30 extract provided in the kit was treated with rifampicin (1 μg mL -1 , final concentration). The standard reaction includes: DNA template (3.2 μg), amino acid mixture without methionine (provided by the kit, 5 μL), S30 premix without amino acids (provided by the kit, 20 μL), [ 35 S]Methionine (2 μL, 1,200 Ci mmol -1 ,15mCi mL -1 ,MP Biomedicals), T7 S30 extract (provided by the kit, 15 μL), and nuclease-free water to a final volume of 50 μL. To prepare heteroheptamers, a mixture of plasmids encoding WT αHL and mutant monomers was mixed in a ratio of 6:1 (WT: mutant). The IVTT mixture was incubated at 37°C for 1 hour.

[0703] Rabbit erythrocyte membranes (3 μL, about 1 mg protein mL -1 ) was added to the IVTT reaction mixture (50 μL), followed by an additional incubation at 37°C for 1 h for heptamerization. The mixture was then centrifuged at 25,000 × g for 10 min. The supernatant was removed and the pellet was resuspended in MBSA buffer (200 μL, 10 mM 3-morpholinopropane-1-sulfonic acid (MOPS), 150 mM NaCl, 1 mg mL -1 Bovine serum albumin, pH 7.4). The MBSA wash was repeated before the pellet was resuspended in 2X Laemmli sample buffer (50 μL) and electrophoresed in a 5% SDS polyacrylamide gel at 70 V for 15 h.

[0704] αHL heteroheptamers containing different numbers of mutant subunits were separated in gels according to their different electrophoretic mobilities, which were determined by the number of octaaspartic acid (D8) tails. 5b The top and bottom bands correspond to WT7 and (mutant-D8H6)7, respectively. The second band from the top is the desired heteroheptamer containing a single mutant subunit.

[0705] To extract the heptamer pores, the gel was first vacuum dried at room temperature for 5 hours without being fixed on Whatman 3M filter paper. After visualization by autoradiography using Kodak Biomax MR film, the desired bands were cut from the gel with a scalpel. Each excised band was rehydrated in TE buffer (300 μL, 10 mM Tris·HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) at room temperature for 1 hour. The filter paper was then removed and the rehydrated gel was impregnated with a pestle. The resulting slurry was filtered through a 0.2 μm hydrophilic membrane filter (Proteus mini clarification spin column, Generon). The filtrate was stored in 10 μL aliquots at -80°C.

[0706] 9. Single channel recording

[0707] 9.1. Overview

[0708] 1,2-Diphytanoyl-sn-glycero-3-phosphatidylcholine (dPhPC) was purchased from Avanti Polar Lipids. All other chemicals were purchased from Sigma-Aldrich unless otherwise stated.

[0709] Planar two-layer recording according to the method established by Montal and Muller 6b The two Delrin compartments were separated by a 25 μm thick Teflon membrane containing a pore (60 μm in diameter). The pores were treated with a 1% (v / v) hexadecane pre-dissolved pentane solution. Each compartment was then filled with a buffer solution (500 μL, 2M KCl, 20 mM N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), 20 μM EDTA, pH 8.5). The trans compartment contained an additional 5 mM DTT. A pentane solution of DPhPC (5 mg mL -1 ) was added dropwise to both compartments. Repeated up and down pipetting of the buffer solution resulted in the formation of a lipid bilayer across the pore. The transbilayer potential was applied using two Ag / AgCl electrodes, each contained within a salt bridge formed by 3% (w / v) low melting point agarose in 3M KCl.

[0710] Ionic currents were recorded using a patch clamp amplifier (Axopatch 200B, Axon Instruments) at room temperature (20°C ± 1°C) with a 4-pole low-pass Bessel filter (80 dB / decade). Signals were digitized using a Digidata 1320A digitizer (Molecular Devices) connected to a computer running the pCLAMP 10.3 software suite (Molecular Devices). Unless otherwise stated, signals were filtered with a corner frequency of 5 kHz and sampled at 25 kHz.

[0711] 9.2. Monitoring Flip

[0712] Substrate (1 μL 400 μM MilliQ water) was added to a solution of traptavidin (Kerafast) (40 μM, 10 μL phosphate buffered saline, pH 7.4) and incubated at room temperature (20°C ± 1°C) for 15 minutes to form a traptavidin-labeled substrate. The cis compartment of the device containing the recording planar double layer of the αHL nanoreactor was stirred until a single hole was inserted into the double layer. Then the traptavidin-labeled substrate (3 μl, 36 μm) was added to the cis compartment, which contained 500 μL recording buffer (2M KCl, 20mM HEPBS, 20mM EDTA, pH 8.5). The trans compartment contained an additional 5 mM DTT. In order to drive the macromolecular substrate into the nanopore, a potential of +150 mV was applied to the trans side. The substrate that passed through reduced the current to about 33% of Ires% (see Table S2 for each substrate-nanoreactor combination). After the thiol-disulfide exchange between the substrate and the nanoreactor, the current increases as a segment of the substrate leaves the nanoreactor (Ires% 39% to 53% for αHL-oligonucleotide adducts and about 24 to 56% for αHL-peptide adducts, see Part 3, Table S2). DTT then releases the adduct and returns the nanoreactor to its initial state, which is ready for another round of thiol-disulfide exchange.

[0713] 9.3. Single channel data analysis

[0714] The current traces were idealized by using Clampfit 10.3 (Molecular Devices). The idealized data were analyzed using QUB 2.0 software (www.qub.buffalo.edu). 7b Residence time analysis and rate constant estimation were performed by using QuB's Maximum Interval Likelihood (MIL) algorithm. 8b .

[0715] 10. References

[0716] 1b. Luchian, T., Shin, S.-H. & Bayley, H. Single-molecule covalent chemistry with spatially separated reactants. Angew. Chem. Int. Ed. 42, 3766–71 (2003).

[0717] 2b. Singh, R. & Whitesides, GMThiol-disulfide interchange. in Sulphur-Containing Functional Groups 633–658 (John Wiley & Sons, Inc., 1993).

[0718] 3b. Lee, J. et al., Semisynthetic nanoreactor for reversible single-molecule covalent chemistry. ACS Nano 10, 8843–8850 (2016).

[0719] 4b. Pulcu, G.S., Mikhailova, E., Choi, L.-S. & Bayley, H. Continuous observation of the stochastic motion of an individual small-molecule walker. Nat. Nanotechnol. 10, 76–83 (2015).

[0720] 5b. Miles, G., Bayley, H. & Cheley, S. Properties of Bacillus cereushemolysin II: A heptameric transmembrane pore. Protein Sci. 11, 1813–1824 (2009).

[0721] 6b. Montal, M. & Mueller, P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc. Natl. Acad. Sci. USA 69, 3561–3566 (1972).

[0722] 7b. Nicolai, C. & Sachs, F. Solving ion channel kinetics with the QuB software. Biophys. Rev. Lett. 08, 191–211 (2013).

[0723] 8b. Qin, F., Auerbach, A. & Sachs, F. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys. J. 70, 264–280 (1996).

Claims

1. A method for moving a molecular hopper along a track, wherein: (a) the track comprises a plurality of primary functional groups arranged along a substrate; (b) the hopper comprises a secondary functional group capable of forming a chemical bond with each of the plurality of primary functional groups on the track; as well as (c) said hopper contains a polymer cargo portion, The method comprises the following steps: (i) contacting the hopper with the track so that the secondary functional group of the hopper is combined with the first primary functional group on the track; (ii) applying a driving force to directionally transfer the hopper from the first primary functional group to the second primary functional group on the track, thereby moving the hopper along the track; Wherein the driving force is a chemical potential and wherein the direction of the driving force relative to the track determines the direction of movement of the hopper along the track.

2. The method of claim 1, wherein the transfer of the hopper from the first primary functional group to the second primary functional group on the track is independent of the addition of exogenous fuel.

3. The method of claim 1, wherein the transfer of the hopper from the first primary functional group to the second primary functional group on the track is independent of the addition of chemical reagents.

4. The method of claim 1, wherein step (ii) comprises applying a driving force to the hopper so that the hopper is sequentially transferred between each of the plurality of primary functional groups on the track, thereby moving the hopper along the track.

5. The method of claim 1, wherein the substrate is an organic or inorganic surface comprising a plurality of primary functional groups. The method of claim 1 , wherein the substrate is the surface of a transmembrane pore.

7. The method of claim 6, wherein the transmembrane pore is a protein nanopore, a solid-state nanopore, a DNA nanopore, a polymer nanopore, or a synthetic or semi-synthetic nanopore.

8. The method of claim 6, wherein the transmembrane pore is a transmembrane β-barrel protein nanopore.

9. The method of claim 1, wherein the track comprises an array of natural and / or unnatural amino acid residues contained in the barrel and / or cavity of a transmembrane β-barrel protein nanopore, wherein each amino acid residue in the track comprises a primary functional group.

10. The method of claim 1, wherein the track comprises an array of amino acid residues evenly spaced along one or more beta strands in a barrel of a transmembrane beta barrel protein nanopore, wherein each amino acid residue in the track comprises a primary functional group.

11. The method of claim 1, wherein the hopper comprises a connecting portion between the secondary functional group and a cargo portion.

12. The method of claim 11, wherein the linking moiety comprises an unsubstituted or substituted alkylene, alkenylene, alkynylene, arylene, heteroarylene, carbocyclylene or heterocyclylene moiety, wherein the alkylene, alkenylene, alkynylene moiety may be uninterrupted or interrupted or terminated by one or more atoms or groups selected from O, N(R), S, C(O), C(O)NR, C(O)O, phosphate, thiophosphate, dithiophosphate, selenophosphate, diselenophosphate, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene, unsubstituted or substituted carbocyclylene and unsubstituted or substituted heterocyclylene, wherein R is selected from H, unsubstituted or substituted alkyl and unsubstituted or substituted aryl.

13. The method of claim 1, wherein the cargo moiety comprises one or more of a polynucleotide, a polypeptide and / or a polysaccharide.

14. The method of any one of the preceding claims, wherein the secondary functional group of the hopper is capable of forming a chemical bond with each of the primary functional groups on the track, wherein the chemical bond is (i) a covalent bond; (ii) a dynamic covalent bond; (iii) a coordinate bond; (iv) a hydrogen bond; or (v) a hydrophobic interaction.

15. The method of claim 14, wherein the covalent bond is a disulfide bond, a diselenide bond, or a sulfide-selenide bond.

16. The method of claim 14, wherein the covalent bond is a disulfide bond.

17. The method of claim 1, wherein the primary functional group and the secondary functional group are independently selected from thiol groups and selenol groups.

18. The method according to claim 17, wherein the thiol group is contained in a phosphorothioate or phosphorodithioate group and / or the selenol group is contained in a selenophosphorothioate or diselenophosphorothioate group.

19. The method of claim 9, wherein adjacent amino acid residues in the track are separated by about to about distance.

20. A method according to any of the preceding claims, wherein the hopper is connected to a positioning portion before contacting the hopper with the track, wherein the positioning portion positions the hopper relative to the track so that the first primary functional groups on the track combine with the secondary functional groups on the hopper.

21. The method of claim 20, wherein: - the substrate is the surface of a transmembrane pore; - before contacting the hopper with the track, connecting the secondary functional group of the hopper to a positioning portion; - the positioning portion comprises a blocking body for preventing the hopper from passing through the transmembrane hole; And wherein step (i) of the method includes contacting the hopper with the hole so that the blocking body prevents the hopper from passing through the hole, thereby maintaining the positioning portion in such a position that the first primary functional group on the track combines with the secondary functional group on the hopper, thereby releasing the positioning portion from the secondary functional group.

22. The method of claim 21, wherein the barrier comprises or consists of a protein, a nanoparticle or a polymer.

23. The method according to claim 1 further comprises, after the hopper moves along the track, a step (iii): contacting the primary functional groups of the track combined with the secondary functional groups of the hopper with the tertiary functional groups on the substrate, so that the tertiary functional groups combine with the primary functional groups, thereby replacing the secondary functional groups and thus releasing the hopper.

24. The method of claim 23, wherein the substrate is a surface of a protein nanopore, and the track comprises an array of amino acid residues contained in the protein nanopore; wherein each amino acid residue of the track comprises a reactive side chain having a primary functional group; and wherein the secondary functional group of the hopper is capable of forming a covalent bond with each of the primary functional groups of the reactive side chains of the amino acid residues of the track; And wherein the tertiary functional group is an additional amino acid residue of the protein nanopore comprising a reactive side chain capable of forming a covalent bond with the reactive side chain of the final amino acid residue of the track, thereby displacing the secondary functional group and thereby releasing the hopper.

25. The method of claim 23, wherein the substrate is a surface of a protein nanopore, and the track comprises an array of amino acid residues contained in the protein nanopore, wherein each amino acid residue in the track comprises a primary thiol functional group; and wherein the hopper comprises a secondary thiol functional group, such that the hopper forms a disulfide bond with the amino acid residue of the track; and wherein the protein nanopore comprises additional amino acid residues comprising a tertiary thiol functional group and positioned such that the tertiary thiol group is capable of forming a disulfide bond with a primary thiol group of the track, thereby displacing the secondary functional group from the primary thiol group of the track and thereby releasing the hopper.

26. The method of claim 24, wherein the amino acid residue comprising the tertiary functional group is separated from the primary functional group of the final amino acid residue in the track by a distance that is smaller than the distance between primary functional groups on the track.

27. The method of claim 24, wherein the amino acid residue comprising the tertiary functional group is separated from the primary functional group of the final amino acid residue in the track by less than about distance.

28. The method of claim 1 further comprising reversing the direction of the driving force relative to the track to reverse the direction of movement of the hopper along the track.

29. A method of characterizing a polymer analyte, the method comprising: (i) Provide (A) Detector; (B) a track comprising a plurality of primary functional groups arranged along a substrate; as well as (C) a molecular hopper attached to the polymer analyte, wherein the hopper comprises a secondary functional group capable of forming a chemical bond with each of the plurality of primary functional groups on the track; (ii) contacting the hopper with the track so that the secondary functional group of the hopper is combined with the first primary functional group on the track; (iii) applying a driving force to transfer the hopper directionally from the first primary functional group to the second primary functional group on the track, thereby causing the hopper to move along the track, wherein the driving force is a chemical potential; and wherein the direction of the driving force relative to the track determines the direction of movement of the hopper along the track; and Wherein the track is positioned such that movement of the hopper along the track causes the analyte to interact with the detector, thereby characterizing the analyte.

30. The method of claim 29, wherein: - the detector is a transmembrane protein pore; - the substrate is the surface of a transmembrane protein pore; - the track comprises an array of amino acid residues contained in the protein nanopore, wherein each amino acid residue of the track comprises a reactive side chain, and wherein the hopper comprises a functional group capable of forming a covalent bond, the covalent bond being bound to the reactive side chain of the amino acid residues of the track; and - The analyte comprises one or more of a polynucleotide, a polypeptide and / or a polysaccharide.

31. The method of claim 30, wherein the movement of the hopper cycles back and forth along the track a plurality of times, thereby causing the analyte to interact with the detector a plurality of times.

32. A kit for characterizing a polymer analyte, the kit comprising: (A) Detector; (B) a track comprising a plurality of primary functional groups arranged along a substrate; (C) a molecular hopper for coupling to the polymer analyte, wherein the hopper comprises a secondary functional group capable of binding to each of the plurality of primary functional groups on the track.

33. A kit according to claim 32, wherein the track is positioned relative to the detector so that application of a chemical potential causes movement of the hopper along the track thereby allowing the analyte to interact with the detector so that the analyte can be characterized by the detector, and wherein the direction of the driving force relative to the track determines the direction of movement of the hopper along the track.

34. The kit of claim 34, wherein the analyte comprises one or more of a polynucleotide, a polypeptide and / or a polysaccharide.

35. A molecular hopper comprising: -Secondary functional groups for chemical bonding to primary functional groups in orbit; - a cargo moiety comprising one or more of a polynucleotide, a polypeptide and / or a polysaccharide; and - a linking moiety between said secondary functional group and said cargo moiety.

36. The molecular hopper according to claim 35 further includes a positioning part, which is used to position the hopper relative to the track so that the first primary functional group on the track can combine with the secondary functional group on the hopper; or a blocking body, which is used to prevent the hopper from passing through the transmembrane pore.

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