nanopore

Abstract

This paper describes nanopores that can be selectively converted between open and closed states using light, methods for manufacturing such nanopores, methods for regulating the flow of one or more substances through nanopores, the use of nanopores in regulating the flow of ions across amphiphilic membranes, devices containing nanopores, the use of nanopores as iontronic elements, the use of nanopores in biocomputing and / or bioiontronics, methods for transmitting information using nanopores, methods for receiving information containing nanopores, and iontronic elements containing nanopores.

Description

[Technical Field]

[0001] field This disclosure relates to nanopores that can be selectively converted between open and closed states using light, methods for manufacturing such nanopores, methods for regulating the flow of one or more substances through nanopores, the use of nanopores in regulating the flow of ions across amphiphilic membranes, devices comprising nanopores, the use of nanopores as iontronic elements, the use of nanopores in biocomputing and / or bioiontronics, methods for transmitting information using nanopores, methods for receiving information comprising nanopores, and iontronic elements comprising nanopores. This disclosure also provides kits suitable for providing the nanopores of the present invention, particularly proteins that are easily functionalized (e.g., having one or more photoisomerizable groups), the use of nanopores in drug delivery, transmitters comprising nanopores, and receivers comprising nanopores. [Background technology]

[0002] background Molecular tools for remote control of transmembrane ion communications are widely used in biomedical science. Among these, optically active tools are attracting attention due to their excellent properties such as spatiotemporal control, reversibility, minimal invasiveness, and multiplexing capabilities.

[0003] Light-controlled ion flow across membranes is typically mediated by photoswitchable chromophores embedded in the membrane or bound to proteins. Protein-dependent ion transporters, such as microbial rhodopsins, generate or dissipate electrochemical gradients when their chromophores (e.g., all-trans retinal) are activated by light. This allows for the control of the electrical activity of rhodopsin-expressing cells in vitro and in vivo, enabling the investigation or manipulation of fundamental biological activities such as calcium signaling and neuronal firing. These optogenetic tools also function as integrated components of bioelectronic systems with potential therapeutic applications, such as treating cardiac arrhythmias and restoring vision. Simultaneously, synthetic alternatives have been devised to directly or indirectly replicate photoactivated passive ion transport. High molecular weight ion transporters activated by built-in photoswitches (e.g., azobenzene) transport ions along gradients, offering potential therapeutic applications for channelopathy. Similarly, synthetic photochromic binders are being developed as photopharmacological agents that target ligand-dependent ionotropic receptors such as potassium channels, TRPVs, and GABA, and modulate ion signaling.

[0004] In bottom-up synthetic biology, natural ion transporters (e.g., rhodopsin) are being reconstituted into artificial membranes for optically controlling ionic flow. Achieving sustained ion conduction effects in diverse applications requires careful selection of activatable protein structures at desired wavelengths, promotion of directional insertion into bilayers, and provision of continuous irradiation. Photoactivatable protein nanopores represent an alternative class of optical tools, characterized by robust channel formation capabilities at design, ease of handling of soluble monomers, and the potential for controlled molecular transport. Previous research has focused on designing α-hemolysin (αHL) and fragaceatoxin C (FraC) monomers in photocages. These are released upon prolonged UV irradiation (~10-30 minutes), inducing irreversible channel formation and permanent activation of ion signaling.

[0005] Therefore, there remains a need to provide nanopores that can be reversibly activated by light. In particular, there is a need to provide photoreversible ON-OFF nanopores (such as protein nanopores) that can control ion flow with low background current in the OFF state and with a long channel lifetime in the ON state. [Overview of the project]

[0006] overview This disclosure relates to nanopores that can be selectively converted between open and closed states using light. Such nanopores are sometimes referred to as “photopores.” Preferably, the nanopore has one or more photoisomerizable groups (“photoswitches”) within its lumen. The photoisomerizable groups have desirable photosteady properties, thereby allowing the nanopore (or “photopore”) to exhibit diode-like or resistor-like behavior under photocontrol.

[0007] In summary, the inventors have developed a nanopore that can be selectively converted between an open and closed state using light. The nanopore of the present invention provides the following: - Excellent optical reversibility (shows robust switching over tens of cycles) - Useful spectral operating range (Switch ON: 365nm; Switch OFF: 455nm or 530nm) - Excellent photofade resistance (photoswitching lasts for several hours even under irradiation) - Good lifetime characteristics (for example, the preferred nanopore of the present invention having a diazene photoisomerizable group provides a lifetime of more than 1 day for the E isomer relative to (7E,0Z)). - Good switching response time (e.g., 30 mW / cm²) 2 (Irradiation takes about 1 second) - Excellent current control (Under reverse bias, the photopore can gate 95% of the current). Current gating = 1 - (I530nm - I365nm)

[0008] Further beneficial properties of the specific nanopore according to the present invention are as follows: • At 530nm, a photopore is a diode, while at 365nm, it is a resistor. Photopore is compatible with ion flow based on concentration gradients. Photoswitching is compatible with synthetic tissues. Photopore can be used as a biotronic logic element.

[0009] Therefore, the present invention relates to a nanopore that can be selectively converted between an open state and a closed state using light. Thus, the nanopore of the present invention can be converted using light between a state that allows the flow of one or more substances through the nanopore (open state) and a state that restricts the movement of one or more substances through the nanopore (closed state). Preferably, the closed state (i.e., the state in which the movement of one or more substances through the nanopore is restricted) restricts the flow of one or more substances by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, compared to the flow of one or more substances through the open state. In a preferred embodiment, when the nanopore is in the closed state, substantially no substance can move through the nanopore. This means that the closed state (i.e., the state in which the movement of one or more substances through the nanopore is restricted) restricts the flow of one or more substances by 80% or more, preferably 90% or more, preferably 95% or more, and more preferably 99% or more, compared to the flow of one or more substances through the open state. Most preferably, a closed configuration (i.e., a configuration in which the movement of one or more substances through the nanopore is restricted) completely restricts the flow of one or more substances compared to the flow of one or more substances through an open configuration. In other words, most preferably, when the nanopore is in a closed configuration, no substance can move through the nanopore.

[0010] Accordingly, this specification provides photoactivatable nanopores or ion channels that can be switched between an active state and an inactive state by the application of light. In one embodiment, the active state is an open state. In the open state, the nanopore or ion channel is completely or substantially open. In one embodiment, the inactive state is a closed state. In the closed state, the nanopore or ion channel is completely or substantially closed. An open pore can typically bridging two compartments in a maneuverable manner, allowing one or more chemical species to pass between compartments through the pore. A closed pore can be used to insulate two compartments, typically preventing one or more chemical species from passing between compartments through the pore.

[0011] Typically, a nanopore has one or more photoisomerizable groups within its lumen, and each of these photoisomerizable groups is independently a group of formula (I):

[0012] [ka]

[0013] (In the formula: * indicates a connection point to a nanopore; L is a linker; and Ar 1 and Ar 2 These are selected from aryl groups and heteroaryl groups that may be independently substituted, respectively. Preferably, Ar 1 and Ar 2 (At least one of them is a heteroaryl group which may be substituted.)

[0014] Most preferably, one or more photoisomerizable groups are independently of each other, with chemical formula (IC):

[0015] [ka]

[0016] This is the basis of (where * indicates a connection point to a nanopore)

[0017] Preferably, the nanopore is a transmembrane protein pore which is an alpha-hemolysin (αHL) pore, preferably an αHL pore formed from seven αHL monomers. Most preferably, the αHL pore contains an E111C substitution.

[0018] The present invention also provides a method for producing nanopores according to the present invention, the method comprising introducing one or more photoisomerizable groups into the nanopores.

[0019] The present invention also provides a method for regulating the flow of one or more substances through a nanopore according to the present invention, the method comprising irradiating the nanopore with light. Most preferably, the substances are ions.

[0020] Furthermore, the present invention provides the use of nanopores according to the present invention in regulating the flow of ions across an amphiphilic membrane.

[0021] The present invention also provides a device comprising a vesicle surrounded by an amphiphilic membrane, wherein the vesicle contains one or more drugs within its lumen, and the amphiphilic membrane comprises a nanopore according to the present invention.

[0022] Furthermore, the present invention provides the use of the nanopore according to the present invention as an iontronic element that can be photoswitched between a resistor and a diode.

[0023] Furthermore, the present invention provides for the use of nanopores according to the present invention in biocomputing and / or bioiontronics.

[0024] Furthermore, this invention: Irradiating the nanopore according to the present invention with multiple light pulses The present invention provides a method for transmitting information including, wherein each pulse includes light having one of a predetermined group of wavelengths.

[0025] Furthermore, this invention: The method involves measuring the ion current passing through the nanopore according to the present invention while irradiating the nanopore with multiple light pulses. The present invention provides a method for receiving information including, wherein each pulse includes light having one of a predetermined group of wavelengths.

[0026] Furthermore, the present invention provides an iontronic element containing a nanopore according to the present invention. Simple description of the drawing [Brief explanation of the drawing]

[0027] [Figure 1] Figure 1 | Construction and characterization of an example of a non-restrictive photopore with seven ON-OFF switches. a. Bromoacetylazopyrazole (Br-pzMe) photoswitches are shown as either the E isomer (green, top) or the Z isomer (purple, bottom). b. UV / Vis spectra of the E or Z isomers of Br-pzMe in DMSO at room temperature. c. Photosteady state (PSS) of Br-pzMe determined by the 1H-NMR chemical shift of aromatic protons in DMSO. d. α-hemolysin (αHL) monomers containing cysteine ​​at position 111 were functionalized with an azopyrazole group via a bromoacetamide handle before heptamerization to form (E111C-pzMe)7 photopores. e. Total ion chromatogram confirming the complete modification of the αHL monomer using Br-PzMe. f. The remote control of the ionic current passing through one or more photopores was measured using voltage clamp electrical recording, with a transmembrane potential (Vm) applied to the cis side of the planar lipid bilayer and the transformer side grounded. LED light was made parallel and irradiated onto the bilayer from the transformer side. Irradiation at 530 nm or 365 nm isomerized the azopyrazole groups in the individual pores, causing all seven photoswitches to assemble in either the E configuration (7E, 0Z) or Z configuration (0E, 7Z). [Figure 2]Figure 2 | Reversible ON-OFF switching of photopores. a. Optical control of ion flow passing through a single photopore. Irradiation at 365 nm (violet) converted the photopore to the (0E, 7Z) state, and irradiation at 530 nm (green) converted it to the (7E, 0Z) state. b. ON-OFF switching of a single photopore by a UV (365 nm)-dark-green (530 nm)-dark irradiation cycle. The moving average of the ion current (gray) is shown in black, which was generated by a Savitzky-Golay filter. c. Magnified view of the gray box in b. The decrease in current at each step is due to isomerization of the photoswitch. de. In the dark state, the ion current was stable. Under continuous irradiation at 530 nm, the current fluctuated because the photopore frequently switched between the (7E, 0Z) state and the adjacent (6E, 1Z) state. f. Optical control of ion flow passing through a collection of photopores. g. ON-OFF switching of >2000 photopores at -100mV. The number of pores was estimated assuming each pore contributes 50pA in PSS365. h. ON-OFF switching of photopore clusters by Nernst potential generated by asymmetric KCl concentrations across the membrane, without external transmembrane potential application. i. Gating efficiency of single, double, and ensemble pores with and without 355nm and 532nm inline filters (n=3, standard deviation shown as error bars). Current traces were recorded at -100mV and filtered using a 20Hz Bessel filter. Recording conditions: 2MKCl, 10mMTris-HCl, 0.1mMEDTA, pH 8.5, 24±1℃. [Figure 3]Figure 3 | Diode characteristics of photopores. a. Current-voltage (IV) characterization of a single (E111C-pzMe)7 photopore under 365 nm or 530 nm irradiation revealed diode behavior at negative potentials (0 to -200 mV) and resistor behavior at positive potentials (0 to +200 mV). 95% confidence intervals (n=3) are shown in the shaded area. b. Wavelength-dependent IV characteristics of the (E111C-pzMe)7 photopore group are due to the seven pzMe photoswitches within each photopore taking on different dominant combination states. Currents in each IV graph are normalized assuming each pore contributes 50 pA at +100 mV. The number of pores ranges from 60 to 904 (n=7). c. Percentage of pzMe photoswitches present in the Z configuration in the photosteady state, calculated at wavelengths of 365 nm, 405 nm, 455 nm, or 530 nm. d. Voltage and wavelength dependence of gating efficiency (GP = 1 - IOFF / ION). e. Rectification was hypothesized to be due to the reverse orientation of the pzMe photoswitch along the pore axis in the reverse electroosmotic flow determined by the polarity of the through-film potential (see Supplementary Figure XX). f. Voltage dependence of the rectification ratio of one or more (E111C-pzMe)7 photopores. The rectification ratio was defined as the ratio of the absolute currents at +100mV and -100mV (n=3, standard deviation shown as error bars). g. Current passing through the (E111C-pzMe)7 photopore group in response to the applied AC potential. h. Truth table of photopore logic. [Figure 4]Figure 4 | Effects of light intensity and wavelength on photopore conductance. a. The transition rate from the low-conductance state to the high-conductance state of (E111C-pzMe)7 pores decreased as the light intensity (Q%) decreased. The drifted baseline resulted from the gradual depletion of AgCl on the electrodes when the current was recorded on an nA scale for several minutes. b. After 228 photopores reached a fully ON (ION=-11.4nA) or OFF (IOFF=-1.8nA) equilibrium state under continuous irradiation at 530nm (green) or 365nm (purple), no further change in conductance levels occurred even when the light intensity was reduced from the maximum LED output (Q%=100%). c. The equilibrium current level was determined by the irradiation wavelength; irradiation at 530 nm (green) resulted in a completely OFF state, irradiation at 365 nm (purple) resulted in a completely ON state, and intermediate current levels were achieved at 405 nm (purple) and 455 nm (blue). d. By irradiating with mixed wavelengths of 365 nm and 455 nm light at various ratios at 3-minute intervals, the PSS of the photopore group could be fine-tuned, and an intermediate equilibrium current state could be accessed. Current traces were recorded at -100 mV and filtered using a 20 Hz Bessel filter. Recording conditions: 2 MKCl, 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.5, 24 ± 1 °C. [Figure 5] Figure 5 | Remote signal transmission using photopores. a. An image or text message was coded as an optical sequence, and an ion current output was generated through a (E111C-pzMe)7 photopore. Using a provided key, the ion current data was deconvoluted and the original message was reconstructed. b. Transmission of a binary pixel pattern. A representative segment of the output trace is shown along with the binary current levels. c. Transmission of a 3-state pixel art converted to a 1D optical sequence. The pixel diagram was generated from PDB:7AHL. d. Transmission of a 3-state Morse code. The blue boxes are the Morse code decoded from the current trace, and the purple boxes are the translated text. The optical sequence was input at a rate of 20s per bit at wavelengths of 365nm, 455nm, or a mixed wavelength of 365 / 455nm, and a 3-level current pattern was generated in the photopore group. [Figure 6] Figure 6 | What is a photopore? A photopore is a photoreversible nanopore that switches between two states: an iontronic resistor and a diode, depending on the wavelength of incident UV-Vis light. When a reverse bias is applied, the current is reduced by 95% due to photoswitching. By using a photopore that exhibits robust and reversible photoswitching, optical control of through-film ion communications can be realized. [Figure 7] Figure 7 | Application of photopore. Ion flow due to asymmetric KCl concentration across a lipid bilayer is controlled by photopore (neuron science). Photoswitching is observed in 3D bioprinted droplet networks with droplet interface bilayers (smart drugs or photopharmacology). Information encrypted as optical pulses is converted into ionic signals and deconvoluted (biocomputing and bioiontronics). [Figure 8] Figure 8 | Analysis of the photoswitching pathway. The photopore has seven azobenzene photoswitches that isomerize in a stepwise manner. The contribution of each photoswitch to the conductance was a question. Furthermore, due to the complexity of the isomers (configurational isomers, conformational isomers, axial enantiomers), the isomerization pathway is not clear. In this specification, we addressed this problem using a simple graph theory analysis. [Figure 9]Figure 9 | Example 2 Figure 1 - Construction and characterization of (E111C-pzMe)7 photopore. a. α-hemolysin (αHL) monomer containing cysteine ​​at position 111 was modified with (E)-bromoacetylarylazopyrazole (Br-pzMe) before oligomerization to form (E111C-pzMe)7 photopore. b. Br-pzMe exists as either the E isomer (green) or the Z isomer (purple). c. The deconvoluted masses of E111C and E111C-pzMe monomers confirm that the αHL monomer was quantitatively modified with pzMe. d. UV / Vis spectra of the E isomer (green) or Z isomer (purple) of Br-pzMe in DMSO at room temperature. e. Photosteady state (PSS) of Br-pzMe investigated by the 1H NMR chemical shift of aromatic protons in DMSO-d6. The regulation of the ionic current through one or more photopores was evaluated using voltage-clamp electrical recording, and the transmembrane potential (Vm) was defined as the potential at which a positive charge moves from the transformer side to the cis side of the pore across the bilayer. The LED light was made parallel and projected onto the bilayer from the transformer side. Irradiation at 530 nm or 365 nm isomerized the arylazopyrazole groups within individual pores, filling the photopores in either the E or Z state. If all seven arylazopyrazoles adopted either the E or Z configuration, such configurations were named (7E,0Z) or (0E,7Z). [Figure 10]Figure 10 | Example 2 Figure 2 - Reversible ON-OFF switching of (E111C-pzMe)7 photopores. a. Optical control of ion flow through an ensemble of approximately 1839 (~1839) photopores. Irradiation with 365 nm (violet) converted the photopores to the ON state, and irradiation with 530 nm (green) produced the OFF state. Current traces demonstrate that the ON-OFF switching of the photopore ensemble was performed by a UV (365 nm) → dark → green (530 nm) → dark cycle. b. Optical control through a single photopore switching between (0E, 7Z) and (7E, 0Z) states. The same optical cycle as in a was applied. The moving average of the ion current (gray) is shown in black and smoothed with a Savitzky-Golay filter (window length 400 ms). During 365 nm irradiation, the photopores were constantly switching, and a black downward event occurred. In contrast, these events ceased in the dark. c, Magnified view of the box enclosed by the dashed lines in b. Each stepwise decrease in current is due to isomerization of one or more pzMe photoswitches. de, Ion current under continuous illumination. The current fluctuated with switching between the (7E,0Z) state and the adjacent (6E,1Z) state at 530nm in panel d, or between the (0E,7Z) state and the adjacent (1E,6Z) state at 365nm in panel e. f, Ion current in the dark. The current remained stable, with occasional upward bursts lasting several seconds. Such dark-field events were unrelated to photoisomerization. g, Percentage of gate current (Igate%) in bilayers containing one, two, or more pores (n=4, standard deviation shown by error bars). In-line filters (355nm filter for 365nm LED, 532nm filter for 530nm LED) were applied to multiple pores, but had little effect on Igate%. h, ON-OFF switching of a photopore ensemble under an electrochemical driving force generated by the asymmetric KCl concentration across the membrane, without the application of an external transmembrane potential. Current traces were recorded at +100mV and a sampling frequency of 25kHz, and filtered using a 5kHz inline Bessel filter and a 20Hz digital Bessel filter.Recording conditions: 2M KCl(af), 0.2M KCl(cis) / 2M KCl(trans)(h), 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. The number of pores in the ensemble experiment was evaluated assuming that each (0E,7Z) pore contributed +64pA at +100mV. [Figure 11] Figure 11 | Example 2 Figure 3 - Diode characteristics of (E111C-pzMe)7 photopores. a. Current-voltage (IV) curves of a single (E111C-pzMe)7 after irradiation at 365 nm or 530 nm represent the behavior of the photoswitchable resistor and diode. 95% confidence intervals (N=3 photopores) are shaded. b. IV response of an ensemble of (E111C-pzMe)7 recorded after irradiation at four wavelengths (365 nm, 405 nm, 455 nm, and 530 nm) and normalized according to the number of pores. Each pore was assumed to contribute +64 pA at +100 mV after 365 nm irradiation. The number of pores in each experiment ranged from 60 to 904 (N=7 experiments). 95% confidence intervals are shaded. c. Voltage and wavelength dependence of the gate current ratio [Igate%=(1-IOFF / ION)×100%], where ION is the current after 365nm irradiation and IOFF is the current at the target wavelength. The schematic diagram on the right shows the estimated distribution of intermediate states in PSS at four wavelengths. d. Voltage-dependent rectification ratio (I- / I+) of a single or multiple (E111C-pzMe)7 photopore (N=3 experiments, standard deviation shown by error bars). e. Current passing through an ensemble of (E111C-pzMe)7 photopores in response to an AC potential. The curves from top to bottom show the applied voltage and current response at 365nm and 530nm. f. Truth table of NAND logic realized using photopores. High and low current levels are realized by combinations of input wavelength (λ) and applied potential. Current-IV curves were recorded using a 5kHz inline Bessel filter at a sampling frequency of 25kHz, and a 20Hz digital Bessel filter was used for data analysis. Recording conditions: 2M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. [Figure 12]Figure 12 | Example 2 Figure 4 - Effects of light intensity and wavelength on the conductance of the (E111C-pzMe)7 ensemble. a. Decreased irradiation intensity at 365 nm (purple) reduced the transition rate from the low-conductance state (IOFF) to the high-conductance state (ION). Irradiance (Q) is shown above the optical sequence. Baseline drift is caused by degradation of the Ag / AgCl electrode when recorded at nA levels for several minutes. b. Insensitivity of output current to light intensity after reaching equilibrium. When the photopore reached equilibrium at ION = 11.4 nA (530 nm, green) or IOFF = 1.8 nA (365 nm, purple) (purple), no further change in current occurred even when the light intensity decreased from high Q to low Q. c. In equilibrium, the continuous irradiation wavelength determines the state: ON at 365 nm (violet), partially OFF at 405 nm (violet) and 455 nm (blue), and completely OFF at 530 nm (green). d. Intermediate current levels can be accessed with multicolor light. By irradiating with mixed wavelengths of 365 nm and 455 nm light at various ratios at 3-minute intervals, the PSS ratio could be fine-tuned with over 200 (>200) photopores. Current traces were recorded at +100 mV using a 5 kHz in-line Bessel filter at a sampling frequency of 25 kHz, and a 20 Hz digital Bessel filter was used for data analysis. Recording conditions: 2 M KCl, 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.5, 24 ± 1 °C. [Figure 13]Figure 13 | Example 2 Figure 5 - Conversion from light to current signal. a. Overview. Image or text data was coded as an optical sequence to generate an ionic current response from (E111C-pzMe)7. The ionic current was decoded and the original data reconstructed by assigning current levels using predetermined keys. b. Transmission of binary pixel patterns with monochromatic 365nm and 455nm illumination. c. Transmission of 3-state pixel art in optical sequence. Images of αHL pores were generated from PDB:7AHL and converted to 3-color pixel art. Each pixel was assigned 0, 1, or 2 based on its color. The pixel matrix was flattened to 1D to generate the optical sequence. d. Transmission of 3-state Morse code. The blue boxes are Morse code from the decoded current traces, and the purple boxes are the translated text. The optical sequence was input at a rate of 20s per bit at 365nm, 455nm, or a mixed wavelength of 365 / 455nm to generate a current pattern consisting of three levels. Current traces were recorded at +100mV using a 5kHz inline Bessel filter at a sampling frequency of 25kHz, and a 20Hz digital Bessel filter was used for data analysis. Currents from the (E111C-pzMe)7 ensemble were recorded at 150mM KCl, 20mM Tris-HCl, 0.1mM EDTA, pH 8.5, and 24±1℃. [Figure 14]Figure 14 | Example 2 Supplementary Figure 1 - (E111C-fAzo)7 and (E111C-pzH)7 photopores. a, the fAzo or pzH photoswitch was covalently attached to the cysteine ​​residue (E111C) at position 111 via thioether bond formation. The photoswitch is shown as the E isomer. Electrical traces using an ensemble of wild-type (WT) α-hemolysin (αHL)(b), unmodified (E111C)7(c), (E111C-fAzo)7(d), and (E111C-pzH)7(e) were recorded under alternating light cycles. Irradiation wavelengths are color-coded: 365 nm, burgundy; 405 nm, violet; 530 nm, green; dark, gray. WT αHL and (E111C)7 did not show photoresponsiveness. The ensemble of (E111C-fAzo)7 and (E111C-pzH)7 responded to irradiation by E / Z isomerization. The gate current percentage (Igate%) was approximately 20% (~20%). For (E111C-fAzo)7, the pore closed at 405 nm irradiation and opened at 530 nm irradiation. In contrast, for (E111C-pzH)7, the pore closed at 530 nm irradiation and opened at 365 nm irradiation. Current traces were recorded at +100 mV using a 5 kHz in-line Bessel filter at a sampling frequency of 25 kHz, and a 20 Hz digital Bessel filter was used for data analysis. Recording conditions: 2 M KCl, 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.5, 24 ± 1 °C. [Figure 15]Figure 15 | Example 2 Supplementary Figure 2 - Photopore with pzMe modification in αHL β barrel. a, Representation of residues in αHL pore and β barrel. The gray translucent structure of the αHL heptamer was generated from PDB:7AHL using PyMOL. The gray solid dumbbell model showing the volume of the protein channel was obtained from ChannelsDB

[43] . Electrical traces from an ensemble of (E111C-pzMe)7(b), (T115C-pzMe)7(c), (T125C-pzMe)7(d) and (T129C-pzMe)7(e) were recorded under alternating light cycles. Irradiation wavelengths are color-coded: 365 nm, burgundy; 530 nm, green; dark, gray. (E111C-pzMe)7 showed a maximum gate current percentage (Igate%) of 82% in the illustrated trace. All pzMe photopores exhibited the following correlations: Z, ON; E, OFF. Current traces were recorded at +100mV using a 5kHz inline Bessel filter at a sampling frequency of 25kHz, and a 20Hz digital Bessel filter was used for data analysis. Recording conditions: 2M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. [Figure 16] Figure 16 | Example 2 Supplementary Figure 3 - Structural model of αHL modified with pzMe at position 111. a. Top view of a single pzMe group covalently attached to position E111C and extending into the central lumen of the αHL pore in either the Z or E state. b. Side view of a single pzMe group in the E state, facing either the cis antrum or trans opening of the β barrel. c. Top view of seven pzMe groups inside the αHL pore, positioned toward the trans opening of the β barrel. The model was manually created and modified from PDB:7AHL. [Figure 17]Figure 17 | Example 2 Supplementary Figure 4 - LC-MS characterization of E111C monomer before and after chemical modification. The left panel is the total ion current chromatogram. The right panel is the deconvolution ESI-MS spectrum in the range of 30-40 kDa. a. E111C-D8H6 monomer: mass = 34966 (calculated) and 34966 (measured). b. E111C-pzMe-D8H6 monomer: mass = 35249 (calculated) and 35249 (measured). c. E111C-fAzo-D8H6 monomer: mass = 35289 (calculated) and 35289 (measured). d. E111C-pzH-D8H6 monomer: mass = 35221 (calculated) and 35221 (measured). [Figure 18] Figure 18 | Example 2 Supplementary Figure 5 - LC-MS characterization of photopores obtained by modification with Br-pzMe. The left panel is the total ion current chromatogram. The right panel is the deconvolution ESI-MS spectrum in the range of 30-40 kDa. a. T115C-pzMe-D8H6 monomer: mass = 35277 (calculated) and 35277 (measured). b. T125C-pzMe-D8H6 monomer: mass = 35277 (calculated) and 35277 (measured). c. T129C-pzMe-D8H6 monomer: mass = 35277 (calculated) and 35277 (measured). [Figure 19]Figure 19 | Example 2 Supplementary Figure 6 - Quantitative photoswitching of Br-pzMe. a. UV / Vis absorption spectrum of Br-pzMe in photosteady state (PSS) in DMSO. b. Ratio of extinction coefficients (ε) of Br-pzMe between 365 nm and 530 nm calculated from the data in a. The high ε of the E isomer of Br-pzMe at 365 nm shifted the equilibrium towards the Z isomer at PSS 365 nm. Similarly, the Z isomer of pzMe showed a higher ε at 530 nm, promoting isomerization to the E isomer at PSS 530 nm. c. 1H NMR signal of aryl protons (green) of Br-pzMe in DMSO-d6. d. UV / Vis absorption spectra of Br-pzH after heating, in the dark and at PSS 365 nm. The right panel shows portions of the 1H NMR spectra of Br-pzH at PSS 365 nm and 530 nm. e. UV / Vis absorption spectra of Br-fAzo in the dark after heating and at PSS 530 nm. The right panel shows portions of the 1H NMR spectra of Br-fAzo at PSS 405 nm and 530 nm. UV / Vis spectra and 1H NMR were recorded in DMSO-d6 at 298 K. [Figure 20]Figure 20 | Example 2 Supplementary Figure 7 - Custom recording chamber for planar lipid bilayer (PLB) experiment under irradiation. a. Recording chamber for photopore characterization. A potential was applied across the PLB using an Ag / AgCl electrode (yellow wire). The cis electrode was grounded, and the compartment with the transformer electrode was connected to an optical fiber (1000 μm diameter, NA=0.39). b. Technical schematic diagram of the chamber. The holes on the front were fitted with a collimator and an optical window (not shown). The electrodes were inserted into buffer-containing compartments, each containing 1 mL of buffer. c. Components of the chamber. A fiber-coupled LED irradiated light onto the Teflon film between the compartments. After adjustment, a collimated beam irradiated the opening (~100 μm diameter). d. Measurement of light intensity. The transformer portion of the chamber was replaced, and the light irradiance was measured with a power meter. Parallel light was shielded by a 300 μm diameter pinhole. The beam profile through the pinhole was almost identical to the beam profile through the Teflon aperture. Irradiance values ​​of the LED were measured at 365 nm, 405 nm, 455 nm, and 530 nm. Irradiance was calculated from the intensity through the pinhole. Irradiance (Q) = Intensity / Pinhole Area. [Figure 21]Figure 21 | Example 2 Supplementary Figure 8 - Photopore preparation and characterization. (E111C-pzMe)7 is used as an example, but the procedure is applicable to other photopores. a. To obtain homoheptamers, monomers were incubated in sodium deoxycholate (DOC) buffer after irradiation at 530 nm. The remaining monomers were removed by anion exchange chromatography (IEX). The purified homoheptamers were examined by electrorecording at the single-molecule level. The ON-state current (ION) cluster was examined by observing the stepwise insertion of homoheptamers generated with DOC and irradiated at 365 nm. The OFF-state current was not pursued because each pore contributed only a few pA and could not be distinguished from noise. For ensemble characterization, monomers irradiated at 530 nm were added directly to the recording chamber. b. Monitoring of IEX purification by gel electrophoresis. Each monomer was tagged with D8H6. Homoheptamers from lanes 5 and 6 were used for recording. Anion IEX conditions: 20 mM Tris-HCl, pH 7.5, NaCl gradient 0.2 → 0.6 M. Gel: 10% SDS-PAGE using Tris-glycine SDS buffer. [Figure 22] Figure 22 | Example 2 Supplementary Figure 9 - Stepwise insertion of (E111C-pzMe)7 irradiated at 365 nm. a. Insertion of (E111C-pzMe)7 in the (0E,7Z) state in the dark. (E111C-pzMe)7 was irradiated at 365 nm for 30 minutes and then introduced into the syscompartment. b. Histogram of the current process generated by pore insertion in a. Current units (pA) were converted to conductance units (nS) to match the recording conditions. (WT-H6)7 showed a monodisperse distribution of conductance values. In contrast, the distribution of conductance values ​​for (E111C-pzMe)7 was broader. c. Conductance values ​​of the pore. After 365 nm irradiation, the average unit conductance of (WT-H6)7 was three times that of (E111C-pzMe)7. Current traces were recorded at +100mV using a 5kHz inline Bessel filter at a sampling frequency of 25kHz, and a 20Hz digital Bessel filter was used for data analysis. Recording conditions: 2M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. [Figure 23] Figure 23 | Example 2 Supplementary Figure 10 - ON-OFF switching of (E111C-pzMe)7. a. Schematic scheme of single-channel recording of a pre-heptamerized (E111C-pzMe)7 pore. The ON-state current (ION), OFF-state current (IOFF), and gate current ratio (Igate%) were determined by current traces recorded with the pre-formed homoheptamer. b. ON-OFF switching performance of individual pre-formed homoheptamers. Each repeat points to a new pore, and the data is sorted in descending order of ION. The ION values ​​are varied (0.51 ± 0.31 nS, n = 21 repeats), which is consistent with the results of stepwise insertion, although the average Igate% is more strictly defined. The 7th repeat was performed on the pore used to collect a 40-minute recording of ON-OFF switching (Figure 10b / Example 2 Figure 2b). c. Schematic scheme for the (E111C-pzMe)7 ensemble produced by monomer insertion. The ensemble was characterized with a pair of inline filters that shifted the LED irradiation wavelength from 365 / 530 nm to 355 / 532 nm. The filters also narrowed the spectral bandwidth from 9 / 30 nm to 10 / 4 nm while reducing the output intensity. de. ON-OFF switching of the ensemble with and without filters. Current and Igate% values ​​were calculated from traces recorded at +100 mV using a 5 kHz inline Bessel filter at a sampling frequency of 25 kHz. Recording conditions: 2 M KCl, 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.5, 24 ± 1 °C. [Figure 24]Figure 24 | Example 2 Supplementary Figure 11 - Representative determination of Igate% value for a single (E111C-pzMe)7-pore. Moving average of ion current (gray traces) was generated using a Savitzky-Golay filter and is shown in black. The top bar shows the irradiation cycle of dark → 365 nm → dark → 530 nm → dark. Igate% for all traces is above 90% (>90%), and Igate% appears to be consistent regardless of absolute current (ION and IOFF). Current traces were recorded at +100 mV using a 5 kHz inline Bessel filter at a sampling frequency of 25 kHz, and a 20 Hz digital Bessel filter was used for data analysis. Recording conditions: 2 M KCl, 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.5, 24 ± 1 °C. [Figure 25] Figure 25 | Example 2 Supplementary Figure 12 - Long-term trace of ON-OFF cycles recorded in a single (E111C-pzMe) 7-pore. 10 cycles were recorded over 40 minutes. Figure 10d / Example 2 Figure 2d was acquired from the region between the two gray dashed lines at 6.5 minutes and 18.5 minutes. Current traces were recorded at +100mV using a 5kHz inline Bessel filter at a sampling frequency of 25kHz, and a 20Hz digital Bessel filter was used for data analysis. Recording conditions: 2M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. [Figure 26]Figure 26 | Example 2 Supplementary Figure 13 - Multiple current levels arise from a single (E111C-pzMe)7-pore (1E,6Z) combination. a. Superposition of 10 Z-to-E transitions recorded with a single (E111C-pzMe)7-pore. The transitions consistently started at a 64pA level (ON) and ended at 1.6pA (OFF). The inserted histogram summarizes all current levels observed during the 10 transitions from ON to OFF. Green and purple dots represent the OFF and ON states. bc. Heatmap of all transitions observed with a single (E111C-pzMe)7-pore under continuous 530nm (b) or 365nm (c) irradiation. The color bar on the right indicates the number of events (N). d. Superposition of transitions between current levels above 30pA. There are four dominant transitions between (0E,7Z) and (1E,6Z). According to the substitutional isomer model (e), only one (1E,6Z) state is expected. Therefore, it is not possible to construct a compelling model based simply on E / Z isomerization. [Figure 27] Figure 27 | Example 2 Supplementary Figure 14 - Long-term recording of a single ON-state (E111C-pzMe)7 pore in the dark. A single (E111C-pzMe)7 photopore was switched on by 365 nm irradiation for 1 minute. The current level in the ON state was 98 pA at +100 mV. The light was then turned off, and an electrical recording of the ON-state (E111C-pzMe)7 photopore with a Z-state photoswitch was performed for 1 hour. The current level remained stable in the dark, with occasional reversible events (purple asterisks) up to approximately 110 pA (~110 pA) and rarely gating events (blue arrows) to nearly 0 pA. Gating was reversed automatically or by briefly applying -100 mV. No pore closure due to thermal relaxation from Z to E was observed. The photopore was switched off by 530 nm irradiation after 59 minutes. Current traces were recorded at a sampling frequency of 25kHz with +100mV using a 5kHz inline Bessel filter and a 20Hz digital Bessel filter. Recording conditions: 2.0M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. [Figure 28]Figure 28 | Example 2 Supplementary Figure 15 - Estimation of Igate% value of (E111C-pzMe)7 ensemble based on single-channel recording. a. Under continuous irradiation at 530 nm, the photopore remained in a completely OFF state (green line) for 74% of the time (transitions between OFF and partially ON states were N=84 times). For 26% of the time, the photopore was partially ON (black line), and the average residual current rate (Ires%=I / ION) was 21%. b. Under continuous irradiation at 365 nm, the photopore remained in a completely ON state (purple line) for 70% of the time (transitions between ON and partially ON states were N=349 times). For 30% of the time, the photopore was partially ON (black line), and the average Ires% was 64%. c. Incomplete closure at 530 nm and incomplete opening at 365 nm reduced the macroscopic gating efficiency. Based on single-channel observations, the ensemble Igate% is estimated to be 100% - 12% / 89% = 86%, which is consistent with the experimental average Igate% of 85% (Figure 10g / Example 2, Figure 2g). Recording conditions: 2.0M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH 8.5, 24±1℃. [Figure 29] Figure 29 | Example 2 Supplementary Figure 16 - Charge selectivity of (E111C-pzMe)7. a. Transmembrane potential generated by asymmetric KCl concentration across the lipid bilayer. The inversion potential (Vr) is defined as the potential required to zero out the current flow. b. IV characterization of the ensemble of (E111C-pzMe)7. Inversion potentials were measured after irradiation at 530 nm and 365 nm. Shading around the curves represents the 95% confidence interval after 3 repeats. The measured Vr suggests anion selectivity. cd. Current driven by the KCl concentration gradient. The Igate% values ​​for c and d were consistent with the voltage dependence of Igate% reported in Figure 11c / Example 2 Figure 3c. Experiments were recorded at a sampling frequency of 25 kHz and filtered using a 5 kHz inline Bessel filter. Recording conditions: (b,c) 2M / 0.2M KCl, (d) 0.2M / 2M KCl, 10mM Tris-HCl, 0.1mM EDTA, pH8.5, 24±1°C. [Figure 30]Figure 30 | S2 1H NMR spectrum (chloroform-d, 298K). [Figure 31] Figure 31|S3 1H NMR spectrum (chloroform-d, 298K). [Figure 32] Figure 32|S5 1H NMR spectrum (chloroform-d, 298K). [Figure 33] Figure 33|S6 1H NMR spectrum (chloroform-d, 298K). [Figure 34] Figure 34|S7 1H NMR spectrum (chloroform-d, 298K). [Figure 35] Figure 35|S8 1H NMR spectrum (chloroform-d, 298K). [Figure 36] Figure 36 | S10 1H NMR spectrum (chloroform-d, 298K). [Figure 37] Figure 37|S10 13C NMR spectrum (chloroform-d, 298K). [Figure 38] Figure 38|S10 HSQC NMR spectrum (chloroform-d, 298K). [Figure 39] Figure 39|S10 HMBC NMR spectrum (chloroform-d, 298K). [Figure 40] Figure 40 shows the 1H NMR spectrum of |S11 (MeOH-d4, 298K). [Figure 41] Figure 41|S11 13C NMR spectrum (MeOH-d4, 298K). [Figure 42] Figure 42|S11 HSQC NMR spectrum (MeOH-d4, 298K). [Figure 43] Figure 43|S11 HMBC NMR spectrum (MeOH-d4, 298K). [Figure 44] Figure 44 | 1H NMR spectrum of Br-pzMe (DMSO-d6, 298K). [Figure 45] Figure 45 | 13C NMR spectrum of Br-pzMe (DMSO-d6, 298K). [Figure 46]Figure 46 | HSQC NMR spectrum of pzMe (DMSO-d6, 298K). [Figure 47] Figure 47 | HSQC NMR spectrum of pzMe (DMSO-d6, 298K). [Figure 48] Figure 48|S13 1H NMR spectrum (chloroform-d, 298K). [Figure 49] Figure 49|S13 13C NMR spectrum (chloroform-d, 298K). [Figure 50] Figure 50 shows the 1H NMR spectrum of |S14 (MeOD-d4, 298K). [Figure 51] Figure 51|S14 13C NMR spectrum (MeOD-d4, 298K). [Figure 52] Figure 52 | 1H NMR spectrum of Br-pzH (DMSO-d6, 298K). [Figure 53] Figure 53 | 13C NMR spectrum of Br-pzH (DMSO-d6, 298K). [Figure 54] Figure 54|S20 1H NMR spectrum (chloroform-d, 298K). [Figure 55] Figure 55|S20 13C NMR spectrum (chloroform-d, 298K). [Figure 56] Figure 56 | 1H NMR spectrum of Br-fAzo. The (Z)-Br-fAzo signal is marked with an asterisk (*). (Chloroform-d, 298K). [Figure 57] Figure 57 | 13C NMR spectrum of Br-fAzo. The (Z)-Br-fAzo signal is marked with an asterisk (*). (Chloroform-d, 298K). [Figure 58] Figure 58|HRMS spectrum of S10. Calculated value of HRMS-EI(m / z)C18H26N5O2[M+H]+: 344.2081; Measured value: 344.2084. [Figure 59] Figure 59|HRMS spectrum of S10. Calculated value of HRMS-EI(m / z)C13H18N5[M+H]+: 244.1557; Measured value: 244.1557. [Figure 60]Figure 60 | HRMS spectrum of Br-pzMe. Calculated value of HRMS-EI(m / z)C15H19N5OBr[M+H]+: 364.0767; Measured value: 364.0763. [Figure 61] Figure 61|HRMS spectrum of S14. Calculated value of HRMS-EI(m / z)C11H14N4[M+H]+: 216.1244; Measured value: 216.1245. [Figure 62] Figure 62 | HRMS spectrum of Br-pzH. Calculated value of HRMS-EI(m / z)C13H15N5OBr[M+H]+: 336.0454; Measured value: 336.0457. [Figure 63] Figure 63|HRMS spectrum of S20. Calculated value of HRMS-EI(m / z)C28H20N3O2F4[M+H]+: 506.1486; Measured value: 506.1488. [Figure 64] Figure 64 | HRMS spectrum of Br-fAzo. Calculated value of HRMS-ESI(m / z)C15H11ON3BrF4[M+H]+: 404.0016; Measured value: 404.0017 [Figure 65] Figure 65 | Partial 1H NMR spectra of pzMe in the dark (bottom), at 530 nm and in the 365 PSS state (DMSO-d6, 298 K). Using an NMR sample (4 mM). [Figure 66] Figure 66 | UV-vis spectrum of pzMe (DMSO-d6, 298K). [Figure 67] Figure 67 shows the deconvoluted spectrum of |(E)-pzMe (DMSO-d6, 298K). [Figure 68] Figure 68 shows the deconvoluted spectrum of |(Z)-pzMe (DMSO-d6, 298K). [Modes for carrying out the invention]

[0028] Detailed explanation The present invention is described with reference to specific embodiments and specific drawings, but the present invention is not limited thereto and is limited only by the claims. Reference numerals in the claims should not be construed as limiting the scope of the present invention. Of course, it should be understood that not all aspects or advantages are necessarily achieved according to specific embodiments of the present invention. Therefore, for example, a person skilled in the art will understand that the present invention can be carried out or performed in a manner that achieves or optimizes one advantage or group of advantages taught herein, but without necessarily achieving other aspects or advantages taught or suggested herein.

[0029] The present invention, both in terms of its configuration and operation, as well as its features and advantages, can be best understood by referring to the following detailed description in conjunction with the accompanying drawings. The aspects and advantages of the present invention will become apparent and will be described by referring to the embodiments described below. Throughout this specification, the expression “one embodiment” or “embodiment” means that certain features, structures, or characteristics described in relation to that embodiment are included in at least one embodiment of the present invention. Therefore, where the expression “one embodiment” or “embodiment” is used in various places throughout this specification, it does not necessarily refer to the same embodiment, although it may. Similarly, in the description of exemplary embodiments of the present invention, it should be understood that various features of the present invention may be summarized in a single embodiment, figure, or description for the purpose of simplifying the disclosure and aiding in the understanding of one or more of the various embodiments of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires features beyond those explicitly described in each claim. Rather, as the following claims show, the aspects of the present invention lie in fewer features than all the features of the single disclosed embodiment described above.

[0030] It should be understood that the “embodiments” of this disclosure may be specifically combined unless otherwise indicated in the context. Any specific combination of all disclosed embodiments (unless otherwise indicated in the context) is further a disclosed embodiment of the invention described in the claims.

[0031] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise. Thus, for example, a reference to "photoisomerisable group" includes two or more photoisomerisable groups.

[0032] All publications, patents, and patent applications (whether above or below) cited herein are incorporated herein by reference.

[0033] definition Where an indefinite or definite article is used to refer to a singular noun (e.g., "a" or "an," "the"), the plural form of that noun is also included unless otherwise specified. Where the term "comprising" is used in this specification and in the claims, it does not exclude other elements or steps. Furthermore, terms such as "first," "second," and "third" in this specification and in the claims are used to distinguish similar elements and are not necessarily used to describe an order or chronological sequence. These terms are interchangeable under appropriate circumstances, and it should be understood that embodiments of the invention described herein may operate in an order other than that described or illustrated herein. The following terms or definitions are provided solely to aid in understanding the invention. Unless otherwise specifically defined herein, all terms used herein have the same meaning as those to those skilled in the art.

[0034] As used herein, “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, which are either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes double-stranded DNA, single-stranded DNA, and RNA. As used herein, “nucleic acid” is a single-stranded or double-stranded covalent nucleotide sequence in which the 3' and 5' ends of each nucleotide are joined by a phosphodiester bond. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized in vitro or isolated from natural resources. Nucleic acids may further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modifications, such as 5' capping with 7-methylguanosine, 3' processing such as cleavage and polyadenylation, and splicing. Nucleic acids may also include synthetic nucleic acids (XNAs) such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), roq nucleic acid (LNA), and peptide nucleic acid (PNA). The size of nucleic acids (also called “polynucleotides” herein) is typically expressed in terms of the number of base pairs (bp) for double-stranded polynucleotides and the number of nucleotides (nt) for single-stranded polynucleotides. 1000 bp or nt is equivalent to 1 kilobase (kb). Polynucleotides with a length of less than approximately 40 nucleotides are typically called “oligonucleotides” and may include primers used for DNA manipulation, such as via polymerase chain reaction (PCR).

[0035] In the context of this disclosure, the term “amino acid” is used in its broadest sense to mean an organic compound comprising an amine functional group (NH2) and a carboxyl functional group (COOH), as well as a side chain specific to each amino acid (e.g., an R group). In some embodiments, an amino acid refers to a naturally occurring L-α-amino acid or residue. Common one- and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" also includes chemically modified amino acids such as D-amino acids, retro-inversoamino acids, and amino acid analogs, naturally occurring amino acids not typically incorporated into proteins such as norleucine, and chemically synthesized compounds known in this field to possess the characteristic properties of amino acids, such as β-amino acids. For example, analogs or mimetic forms of phenylalanine or proline, which, like natural Phe or Pro, restrict the structure of peptide compounds, are included in the definition of an amino acid. Such analogs and mimetic forms are referred to herein as "functional equivalents" of their respective amino acids. Further examples of amino acids are enumerated by Roberts and Vellaccio, *The Peptides: Analysis, Synthesis, Biology*, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, and are incorporated herein by reference.

[0036] As used herein, the terms “polypeptide” and “peptide” are interchangeable to refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers, where one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogs of corresponding natural amino acids, as well as natural amino acid polymers. Polypeptides may undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like. Peptides can be prepared through recombinant techniques, such as recombinant expression or synthetic polynucleotides. Peptides produced by recombinants typically contain substantially no culture medium; for example, the culture medium constitutes less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0037] The term "protein" is used to describe folded polypeptides that have a secondary or tertiary structure. A protein may consist of a single polypeptide or may contain multiple polypeptides that assemble to form a multimer. The multimer may be a homooligomer or a heterooligomer. A protein may be a naturally occurring, or wild-type, protein, or a modified, or non-natural protein. For example, a protein may differ from a wild-type protein by the addition, substitution, or deletion of one or more amino acids.

[0038] A "mutant" of a protein includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the original or wild-type protein in question, and that possess similar biological and functional activity to the original protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical amino acid-wise within a comparison window. Therefore, the "percentage of sequence identity" is calculated by comparing two sequences that are optimally aligned across the entire comparison window, determining the number of positions where identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) occur in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0039] In all aspects and embodiments of the present invention, a “mutant” has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with respect to the amino acid sequence of the corresponding wild-type protein. Sequence identity can be established even for fragments or parts of a full-length polynucleotide or polypeptide. Therefore, even if a sequence has only 50% overall sequence identity with the full-length reference sequence, the sequence of a particular region, domain, or subunit may share as much as 80%, 90%, or 99% sequence identity with the reference sequence.

[0040] The term "wild-type" refers to a gene or gene product isolated from a natural resource. A wild-type gene is the gene most frequently observed in a population and is therefore arbitrarily designed as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, cleavage, or insertions), post-translational modifications, and / or functional properties (e.g., altered properties) compared to a wild-type gene or gene product. Spontaneously occurring mutants can be isolated and are identified by the fact that they have altered properties compared to a wild-type gene or gene product. Methods for introducing or substituting natural amino acids are well known in the art. For example, methionine (M) can be substituted for arginine (R) by substituting the methionine codon (ATG) with the arginine codon (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-natural amino acids are also well known in the art. For example, non-natural amino acids can be introduced by incorporating synthetic aminoacyl-tRNA into the IVTT system used for mutant monomer expression. Alternatively, a nutrient-dependent mutant monomer for a specific amino acid can be introduced by expressing it in Escherichia coli (E. coli) in the presence of a synthetic (i.e., non-natural) analog of that specific amino acid. Furthermore, if the mutant monomer is produced using a partial peptide synthesis method, non-natural amino acids can also be produced by bare ligation. Conservative substitution involves replacing an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the substituted amino acid. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acids are well known in the art and can be selected according to the properties of the 20 major amino acids defined in Table 1 below. If the amino acids have similar polarity, this can be determined by referring to the amino acid side-chain hydropathic scale in Table 2.

[0041]

Table 1

[0042]

Table 2

[0043] Mutants or modified proteins, monomers or peptides can also be chemically modified by any method and at any site. Mutants or modified monomers or peptides are preferably chemically modified by connection of a molecule to one or more cysteines (cysteine conjugation), connection of a molecule to one or more lysines, connection of a molecule to one or more unnatural amino acids, enzymatic modification of an epitope, or terminal modification. Suitable methods for carrying out such modifications are well known in the art. Mutants of modified proteins, monomers or peptides can be chemically modified by connection of any molecule. For example, mutants of modified proteins, monomers or peptides can be chemically modified by connection of a dye or a fluorophore.

[0044] A photoisomerizable group (known herein as a photoswitch) is a group that undergoes isomerization (E- / Z-isomerization herein) under light irradiation. Preferred photoisomerization groups will be described later.

[0045] The phrase "optionally substituted" means that one or more hydrogen atoms present in the group indicated as "optionally substituted" may be substituted, for example, with R described below 1 and R 2 that can be replaced with another chemical group selected from the list. However, for example, one or more optionally substituted substituents may be COO - , SO3 - , or NR 3 3 + optionally substituted C 1-6 alkyl; C 1-6 alkyl, halogen, COO- , SO3 - , or NR 3 3 + C may be replaced by 5-10 Cycloalkyl; C 1-6 Alkyl, halogen, COO - , SO3 - , or NR 3 3 + C may be replaced by 6-10 Aryl; having 1 to 4 ring heteroatoms independently selected from N, O, and S, C 1-6 Alkyl, halogen, COO - , SO3 - , or NR 3 3 + A 5-10 membered heteroaryl or heterocyclic ring which may be substituted with; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-6 Alkenil; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-6 Alkynyl; Halogen; OH; CN; NO2; C 1-6 Haloalkyl; COO - , SO3 - , or NR 3 3 + C may be replaced by 1-6 Alkoxy; C 1-6 Haloalkoxy; COO - , SO3 - , or NR 3 3 + COOC may be replaced with 1-6 Alkyl;CONH2;COO - , SO3 - , or NR 3 3 + CONR may be replaced with 4 C 1-6 Alkyl;CSNH2;COO - , SO3 - , or NR 3 3 +CSNR optionally substituted with 4 C 1-6 alkyl; COO - , SO3 - , or NR 3 3 + NR optionally substituted with 4 COC 1-6 alkyl; SO2NH2; COO - , SO3 - , or NR 3 3 + SO2NR optionally substituted with 4 C 1-6 alkyl; COO - , SO3 - , or NR 3 3 + NR optionally substituted with 4 SO2C 1-6 alkyl; COO - , SO3 - , or NR 3 3 + ; (wherein each R3 is independently H or C 1-6 alkyl, and each R 4 is independently H or C 1-6 alkyl). More typically, the one or more optional substituents may be selected from the group consisting of C 1-6 alkyl; C 5-10 cycloalkyl; C 6-10 aryl; 5- to 10-membered cyclic heteroaryl or heterocycle having 1 to 4 ring heteroatoms independently selected from N, O and S; C 2-6 alkenyl; C 2-6 alkynyl; halogen; OH; CN; NO2; C 1-6 haloalkyl; C 1-6 alkoxy; C 1-6 haloalkoxy; COOC 1-6 alkyl; CONH2; CONR 4 C 1-6 alkyl; CSNH2; CSNR 4 C 1-6 alkyl; NR 4 COC 1-6 alkyl; SO2NH2; SO2NR 4 C 1-6Alkyl;NR 4 SO2C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + ;(In the formula, each R 4 H or C 1-6 It can be selected from the group consisting of alkyl groups. More typically, one or more substituted substituents are C 1-3 Alkyl; Halogen; OH; CN; NO2; C 1-3 Haloalkyl; C 1-3 Alkoxy; C 1-3 The substituents may be selected from the group consisting of haloalkoxys. More typically, one or more substituted substituents may be selected from the group consisting of methyl, F, Cl, Br, OH, CN, CF3OCH3, and OCF3.

[0046] Alkyl is a general formula C n H 2n+1 This means a linear or branched monovalent hydrocarbon group having a heteroatom (i.e., O or S, or NR (where R is H or C)) in the chain, wherein the group optionally contains one or two heteroatoms (i.e., O or S, or NR) in the formula. 1-6 It contains alkyl. Preferably, the alkyl is of general formula C n H n+1 This refers to a linear or branched hydrocarbon group having C. Unless otherwise defined, alkyl groups are C 1-20 It is an alkyl group. Preferably, any alkyl group is C 1-6 It is an alkyl group, more preferably C 1-3 Alkyl alkyl groups, most preferably methyl groups, are used.

[0047] Alkylenes have the general formula C n H 2n This means a linear or branched divalent hydrocarbon group having one or two heteroatoms (i.e., O or S, or NR (where R is H or C)) in the chain. 1-6 It optionally contains an alkyl group. Preferably, the alkyl group is of the general formula C n H 2nThis refers to a linear or branched hydrocarbon group having C. Unless otherwise defined, the alkylene group is C 1-20 It is an alkylene group. Preferably, any alkylene is C 1-6 It is an alkylene group, and more C 1-3 It is an alkylene group, most preferably a methylene group.

[0048] An alkenyl refers to a carbon-carbon double bond attached to an alkyl group as defined above, and this alkenyl group is a C3 or greater alkenyl group. Unless otherwise specified, an alkylene group is C 2-20 It is the alkenyl group.

[0049] Alkynyl refers to a carbon-carbon triple bond attached to an alkyl group as defined above, and this alkenyl group is an alkenyl group with three or more carbon atoms. Unless otherwise defined, an alkylene group is C 2-20 It is the alkenyl group.

[0050] The "substances" flowing through nanopores are chemical substances and can be independently selected from ions, water, sugars, inorganic salts, lipids, amino acids, peptides, nucleotides, nucleic acids, metabolites, neurotransmitters, labels, and drugs (i.e., medicines). Most typically, the substances traveling through nanopores are ions. Furthermore, or alternatively, the substance may be one or more substances that are taken into or expelled from cells.

[0051] One or more substances may contain ions. Ions can be cations such as metal cations. Cations include potassium (K + ), sodium (Na + ) and / or calcium (Ca 2+ ) can be selected from. Ions can be anions. Anions are chlorides (Cl - ) is possible.

[0052] One or more substances may contain nucleic acids, i.e., polynucleotides. Polynucleotides can be double-stranded or single-stranded. Typically, when one or more substances contain nucleic acids, the nucleic acids are single-stranded, such as cDNA or RNA. Polynucleotides may contain DNA and / or RNA. Nucleic acids are negatively charged. Nucleic acids are macromolecules containing two or more nucleotides. Nucleic acids may be naturally occurring or artificial. Nucleic acids typically contain a nucleic acid base, a sugar, and at least one phosphate group. Nucleic acid bases are typically heterocyclic. Examples of nucleic acid bases include purines and pyrimidines, more specifically adenine, guanine, thymine, uracil, and cytosine. Sugars are typically pentose sugars. Examples of nucleotide sugars include ribose and deoxyribose. Nucleic acids are typically ribonucleotides or deoxyribonucleotides. Nucleic acids typically contain a monophosphate, diphosphate, or tripphosphate. The phosphate can be attached to the 5' or 3' end of the nucleotide.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.

[0053] One or more substances may contain peptides, such as polypeptides. Polypeptides may be proteins or fragments thereof. Polypeptides may be of natural or non-natural origin. Polypeptides may contain synthetic or modified amino acids. Various types of modifications to amino acids are well known in the art. Peptides may be polymers consisting of about 2 to about 50 amino acids, or polymers of longer amino acids. Proteins are typically polypeptides that are folded into a functional structure or form part of a functional complex. For example, polypeptides may consist of about 2 to about 1000 amino acids, such as about 5 to about 500 amino acids, for example, about 20 to about 60 amino acids, for example, about 30 to about 50 amino acids, for example, about 10 to about 100 amino acids. Suitable polypeptides include, but are not limited to, proteins such as enzymes, antibodies, hormones, growth factors, cytokines and other growth regulatory proteins, or fragments of such proteins. Polypeptides may be derived from bacteria, archaea, fungi, viruses, or parasites. Polypeptides may be derived from plants. Polypeptides are typically mammalian, and more commonly human.

[0054] One or more substances may comprise saccharides such as polysaccharides. A polysaccharide is a macromolecular carbohydrate molecule consisting of a chain of monosaccharide units linked by glycosidic bonds. Polysaccharides may be linear or branched. Polysaccharides may be homogeneous (containing only a single repeating unit) or heterogeneous (containing modified repeating units). Examples of polysaccharides include callose or laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, and galactomannan. Polysaccharides may be produced by bacteria such as pathogenic bacteria. The polysaccharide may be a capsular polysaccharide with a molecular weight of 100 to 2000 kDa. Polysaccharides may be synthesized from nucleotide-activated precursors (referred to as 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 derived from, for example, bacteria or plants. The polysaccharide may be useful as an antibiotic such as streptomycin, neomycin, paromomycin, kanamycin, chalcomycin, erythromycin, magnamycin, spiramycin, oleandomycin, synerbin and amicetin, or a derivative of any one of these compounds. The polysaccharide may comprise from about 2 to about 1000 monosaccharide units, for example from about 5 to about 500 monosaccharide units, such as from about 10 to about 100 monosaccharide units, from about 20 to about 60 monosaccharide units, such as from about 30 to about 50 monosaccharide units.

[0055] One or more substances may comprise a neurotransmitter. The neurotransmitter may be selected from glutamate, glycine, serotonin, epinephrine, dopamine, opioids, ATP, GBP, nitric oxide, carbon monoxide, γ-aminobutyric acid (GABA), acetylcholine, and norepinephrine.

[0056] One or more substances may further include substances with a large charge, such as sulfo-Cy5 and FMN.

[0057] nanopore As described above, the nanopore of the present invention is a nanopore that can be selectively converted between an open state and a closed state using light. Therefore, the nanopore of the present invention can be converted using light between a state that allows the flow of one or more substances through the nanopore (open state) and a state that restricts the movement of one or more substances through the nanopore (closed state). Preferably, the closed state (i.e., the state in which the movement of one or more substances through the nanopore is restricted) restricts the flow of one or more substances through the open state by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more. Preferably, the closed state (i.e., the state in which the movement of one or more substances through the nanopore is restricted) restricts the flow of one or more substances through the open state by 90% or more, preferably 95% or more, more preferably 99% or more. Most preferably, a closed configuration (i.e., a configuration in which the movement of one or more substances through the nanopore is restricted) completely restricts the flow of one or more substances compared to the flow of one or more substances through an open configuration. In other words, most preferably, when the nanopore is in a closed configuration, no substance can move through the nanopore.

[0058] Definition of nanopore for use in the present invention A nanopore typically contains a channel through which a substance (preferably an ion) can pass. The channel is also known as the lumen of the nanopore. As will be described later, typically, the nanopore of the present invention has one or more photoisomerizable groups within the lumen of the nanopore.

[0059] Any suitable nanopore can be used. The nanopore can be a transmembrane nanopore. A transmembrane pore is a structure that penetrates the membrane to some extent. This allows hydrated ions, driven by the applied potential, to flow across or within the membrane. Because transmembrane pores typically penetrate the entire membrane, hydrated ions can flow from one side of the membrane to the other. However, transmembrane pores do not necessarily have to penetrate the membrane; they can be closed at one end. For example, a pore can be a depression, gap, passage, groove, or crack within the membrane, through which hydrated ions can flow or toward it.

[0060] Nanopores can be protein pores, DNA origami pores, solid-state pores, or polymer-based pores. For example, a nanopore can be a transmembrane protein pore, a transmembrane DNA origami pore, or a transmembrane solid-state pore. Preferably, the nanopore is a transmembrane protein pore.

[0061] A pore can be a monomer or an oligomer, i.e., a monomer or oligomer containing two or more monomers. For example, a transmembrane protein pore can be a monomer or an oligomer, i.e., an oligomer that may contain two or more protein subunits / monomers. A pore can be a homooligomer (all monomer units are identical) or a heterooligomer (two or more different monomers). A pore can contain linked monomers, such as dimers that aggregate to form the oligomeric structure of the pore. Monomers can be linked on the same polypeptide chain, i.e., genetically fused.

[0062] A pore may contain at least one dimer and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 monomers. A pore may contain 2, 3, 4, or more dimers. Such a pore may contain enough monomers to form further pores. Further pores may contain only dimers; for example, a pore may contain 4, 5, 6, 7, or 8 dimers. A particular pore for use according to the present invention contains 4 dimers. Dimers can be oligomerized into pores having a structure in which only one monomer of the dimer contributes to the barrel or antler portion of the pore. Typically, the other monomer of the construct is located outside the barrel or antler portion of the pore. For example, if the barrel or antler portion contains 8 monomers, the pore may contain 5, 6, 7, or 8 dimers.

[0063] Each monomer in the nanopore may contain at least one photoisomerizable group. For example, if the nanopore is an αHL pore containing seven αHL monomers, preferably each αHL monomer contains at least one photoisomerizable group. Each monomer in the nanopore may contain two or more photoisomerizable groups.

[0064] Suitable transmembrane pores for use in the present invention may be solid-state pores. Solid-state nanopores are typically nanometer-sized holes formed in synthetic films. Suitable solid-state pores include, but are not limited to, silicon nitride pores, silicon dioxide pores, and graphene pores. Solid-state nanopores can be fabricated, for example, by focused ion beams or electron beams, thereby allowing for free adjustment of the pore size. Suitable solid-state pores and methods for manufacturing them are described in US Patent No. 6,464,842, International Publication No. 03 / 003446, International Publication No. 2005 / 061373, US Patent No. 7,258,838, US Patent No. 7,466,069, US Patent No. 7,468,271, and US Patent No. 7,253,434, each incorporated by reference as a whole.

[0065] Transmembrane pores can be DNA origami pores, as disclosed in Langecker et al., Science, 2012; 338: 932-936 and International Publication No. 2013 / 083983, each incorporated by reference as a whole. Transmembrane pores can also be scaffold-based pores, such as DNA scaffold protein nanopores, as disclosed in E. Spruijt, Nat. Nanotechnol. 2018 (incorporated herein by reference). Transmembrane protein pores are polypeptides, or one or more polypeptides, that allow material to move within, along, or through the pore.

[0066] Transmembrane pores can be polymer-based pores. Suitable pores can be formed from polymer-based plastics such as polyester (e.g., polyethylene terephthalate (PET)) by track etching.

[0067] A transmembrane pore suitable for use in the present invention may be a transmembrane protein pore. A transmembrane protein pore is a polypeptide or a polypeptide aggregate that allows ions driven by an applied potential to flow from one side of a membrane to the other. Transmembrane protein pores are particularly suitable for use in the present invention.

[0068] Transmembrane protein pores 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 containing less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as lipids or other pores. Pores are typically found in membranes (e.g., synthetic membranes such as lipid bilayers or block copolymer membranes).

[0069] Transmembrane protein pores can be monomers or oligomers. Transmembrane protein pores are often composed of several repeating subunits (also known as monomers), 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. These pores are typically hexamers, heptamers, octamers, nonamers, or tetradecamer pores.

[0070] Transmembrane protein pores typically contain barrels or channels through which ions can flow. The pore's subunits typically surround a central axis and contribute chains to transmembrane β-barrels or channels, or transmembrane α-helix bundles or channels. Transmembrane protein pores can originate from β-barrel pores or α-helix bundle pores.

[0071] Suitable transmembrane pores for use according to the present invention may be β-barrel pores, α-helix bundle pores, or solid-state pores. β-barrel pores include barrels or channels formed from β-chains. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin, and leucosidine, as well as bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porins (Msp) (e.g., MspA, MspB, MspC, or MspD), CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP), and other pores such as lysenin. α-helix bundle pores include barrels or channels formed from α-helices. Suitable α-helix bundle pores include, but are not limited to, inner and outer membrane proteins such as Wza (see, e.g., KR Mahendran, Nat. Chem. 2016, incorporated by reference) and ClyA toxin. For example, transmembrane pores may be derived from or based on Msp, α-hemolysin (α-HL), lysenin, Phi29, CsgG, CgsF, ClyA, Sp1, aerolysin, and the hemolytic protein fragaceatoxin C (FraC).

[0072] Transmembrane protein pores, for example, one or more subunits of a transmembrane protein pore, for example, all subunits of a transmembrane protein pore, typically contain nucleophilic amino acids. Nucleophilic amino acids can, or form, covalent bonds with photoisomerizable groups. The nucleophilic amino acids are preferably amino acids facing the lumen of the nanopore, i.e., amino acids on the luminal surface of the nanopore. The nucleophilic amino acids may be cysteine, lysine, histidine, tyrosine, or other native or non-native nucleophilic amino acids. Preferably, the nucleophilic amino acid is cysteine.

[0073] Transmembrane protein nanopores may include porins, pore-forming proteins, or channel proteins.

[0074] The transmembrane protein pore may be a porin. Porins are typically β-barrel proteins that function as pores across a membrane. The porin may be a porin selected from Porin Superfamily I, Porin Superfamily II (MspA Superfamily), Protein Superfamily III, Protein Superfamily IV and Porin Superfamily V. The porin may be a pore of Mycobacterium smegmatis porin (Msp) (e.g., MspA, MspB, or MspC), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), or a pore of the Omp family (e.g., OmpF, OmpG, etc.).

[0075] The transmembrane protein pore may be a pore-forming protein. Pore-forming proteins are typically produced by bacteria and other organisms, and typically induce lysis of target cells. The pore-forming protein may be alpha-pore-forming toxins (proteins of the hemolysin E family, actinoporins (e.g., fragaceatoxin C), Corynebacterial porin B, E. coli cytolysin A, etc.), β-barrel pore-forming toxins (α-hemolysin (αHL), Pantone-Valentine leukocidin, etc.), large β-barrel pore-forming toxins (MACPF and cholesterol-dependent cytolysins (CDC), gasdermin, etc.), binary toxins (anthrax toxin, pleurotolysin, etc.), or small pore-forming toxins (gramicidin A, etc.). The pore-forming protein is preferably α-hemolysin (αHL).

[0076] The transmembrane protein pore may be a channel protein. The channel protein may be an ion channel. Ion channels are pore-forming proteins that allow the passage of ions through channel pores. Ion channels may be voltage-gated ion channels, ligand-gated ion channels, or lipid-gated ion channels. The ion channel may be a chloride channel, potassium channel, sodium channel, calcium channel, proton channel, or non-selective cation channel.

[0077] Voltage-gated ion channels may be voltage-gated sodium channels, voltage-gated calcium channels, voltage-gated potassium channels, voltage-gated chloride channels, transient receptor potential (TRP) channels, hyperpolar cyclic nucleotide-gated channels, or voltage-gated proton channels. Voltage-gated potassium channels may be shaker potassium channels.

[0078] Ligand-dependent ion channels can be cis-loop receptors. Cis-loop receptors can be acetylcholine receptors, serotonin receptors, glycine receptors, glutamate receptors, or γ-aminobutyric acid (GABA) receptors. Ligand-dependent ion channels can be cationic cis-loop receptors (such as cationic serotonin receptors, cationic nicotinic acetylcholine receptors (nAChRs), or cationic zinc-activated ion channels). Ligand-dependent ion channels can be anionic cis-loop receptors (such as GABA receptors or glycine receptors). Ligand-dependent ion channels can be ionotropic glutamate receptors (such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kynate receptors, N-methyl-D-aspartate (NMDA) receptors, or orphan ionotropic glutamate receptors). Ligand-dependent ion channels can be ATP-dependent channels (such as P2X channels).

[0079] Lipid-dependent ion channels may be inwardly rectifying potassium channels or 2-pore domain potassium channels. Lipid-dependent ion channels may be phosphatidylinositol 4,5-bisphosphate (PIP2)-dependent channels (K ir Channel, K v 7 channels (such as TRP channels). Lipid-dependent ion channels include phosphatidic acid (PA)-dependent channels (K 2pThese may be lipid-dependent ion channels (such as nAChR or Kv channels). Lipid-dependent ion channels may be phosphatidylglycerol (PG)-dependent channels.

[0080] The transmembrane protein pore is preferably derived from α-hemolysin (αHL). The wild-type αHL pore is formed from seven identical monomers or subunits (i.e., it is a heptamer). The transmembrane protein pore preferably contains seven monomers derived from αHL. The sequence of the wild-type monomer or subunit of αHL (WT αHL) is shown in SEQ ID NO: 1.

[0081] Sequence ID 1 ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTL KYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0082] 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 barrel or channel constriction of α-HL.

[0083] Preferably, the pore contains seven proteins or monomers, each containing the sequence shown in SEQ ID NO: 1 or a variant thereof. The transmembrane protein may be (a) formed from seven identical subunits shown in SEQ ID NO: 1, or (b) one or more or all of the seven subunits may be variants of SEQ ID NO: 1 and retain pore activity. One, two, three, four, five, six, or seven of the subunits may be variants. The variants within the pore may be identical or different. The seven subunits may be identical (homoheptamers) or different (heteroheptamers).

[0084] The mutant of SEQ ID NO: 1 is a protein having a different amino acid sequence from SEQ ID NO: 1, while retaining its pore-forming ability. The pore-forming ability of the mutant can be assayed using any method known to those skilled in the art. For example, the mutant can be inserted into an amphiphilic layer, such as a lipid bilayer, along with other suitable subunits, and its ability to oligomerize and form pores can be measured. Methods for inserting subunits into amphiphilic layers, such as lipid bilayers, are well known in the art. For example, the subunit can be inserted by suspending it in a purified form in a solution containing a lipid bilayer, diffusing it into the lipid bilayer, binding it to the lipid bilayer, and assembling it into a functional state. Alternatively, the subunit 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 International Publication No. 2006 / 100484).

[0085] αHL may contain one or more nucleophilic amino acids exposed in the lumen of the αHL pore. αHL may contain one or more nucleophilic amino acids in the constriction of the αHL pore. αHL may contain a nucleophilic amino acid at positions 111, 113 and / or 147 of SEQ ID NO: 1, preferably at position 111 of SEQ ID NO: 1. αHL may contain a nucleophilic amino acid at positions corresponding to positions 111, 113 and / or 147 of SEQ ID NO: 1, preferably at position 111 of SEQ ID NO: 1. The nucleophilic amino acid may be histidine, serine, threonine, tyrosine, or cysteine, preferably cysteine. αHL may contain cysteine ​​at positions 111, 113 and / or 147 of SEQ ID NO: 1. αHL may contain E111C, M113C, or K147C substitutions. Preferably, αHL contains the E111C substitution. αHL may contain cysteine ​​at positions corresponding to positions 111, 113, and / or 147 of SEQ ID NO: 1. αHL may contain the E111C, M113C, or K147C substitutions of SEQ ID NO: 1. Preferably, αHL contains the substitution corresponding to the E111C substitution of SEQ ID NO: 1.

[0086] In another embodiment, αHL may contain a nucleophilic amino acid at positions 111, 115, 125 and / or 129 of SEQ ID NO: 1, preferably at positions 111 and / or 115, and most preferably at position 111 of SEQ ID NO: 1. αHL may contain a nucleophilic amino acid at positions corresponding to positions 111, 115, 125 and / or 129 of SEQ ID NO: 1, preferably at positions 111 and / or 115, and most preferably at position 111 of SEQ ID NO: 1. The nucleophilic amino acid may be histidine, serine, threonine, tyrosine, or cysteine, preferably cysteine. αHL may contain cysteine ​​at positions 111, 115, 125 and / or 129 of SEQ ID NO: 1. αHL may contain E111C, T115C, T125C, or T129C substitutions. Preferably, αHL includes the E111C or T115C substitution, most preferably the E111C substitution. αHL may include cysteine ​​at positions 111, 115, 125 and / or 129 of SEQ ID NO: 1. αHL may include substitutions corresponding to the E111C, T115C, T125C or T129C substitutions of SEQ ID NO: 1. Preferably, αHL includes substitutions corresponding to the E111C or T115C substitution of SEQ ID NO: 1, most preferably the E111C substitution of SEQ ID NO: 1.

[0087] Mutants can be naturally occurring mutants expressed spontaneously by organisms, such as Staphylococcus bacteria. Alternatively, mutants can be expressed in vitro or by recombination by bacteria such as Escherichia coli. Mutants also include non-natural mutants produced by recombinant technology. Mutants may include non-natural amino acids and other molecules that can be introduced by native or non-native chemical ligation. Mutants may also involve non-covalent modifications, such as using cyclodextrins as adapters; these modifications may include molecules that strongly bind to the pore.

[0088] The variant is preferably at least 50% homologous to the amino acid sequence of SEQ ID NO: 1, based on amino acid identity, across its entire length. More preferably, the variant may 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 even more preferably at least 95%, 97%, or 99% homologous to the amino acid sequence of SEQ ID NO: 1, based on amino acid identity, across its entire length. At least 80%, for example at least 85%, 90%, or 95% amino acid identity may exist across 200 or more consecutive amino acids, for example 230, 250, 270, or 280 or more ("hard homology").

[0089] Standard methods in this field can be used to determine homology. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology with default settings (Devereux et al (1984) Nucleic Acids Research 12, pp. 387-395). The PILEUP and BLAST algorithms can be used to calculate homology and sort sequences (such as identifying equivalent residues or corresponding sequences, typically with default settings), as described, for example, in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0090] In addition to the above, the amino acid sequence of Sequence ID No. 1 may undergo up to 1, 2, 3, 4, 5, 10, 20, or 30 amino acid substitutions. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid being replaced. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acids are well known in the art and can be selected according to the properties of 20 major amino acids as defined in Table 1 below. If the amino acids have similar polarity, this can also be determined by referring to the hydroxyl scale of the amino acid side chains in Table 2. Non-conservative substitutions can also be performed while the protein pore maintains its structure and function.

[0091] One or more amino acids from the amino acid sequence of SEQ ID NO: 1 may be further deleted from the polypeptide. Up to 1, 2, 3, 4, 5, 10, 20, or 30 residues, or more, may be deleted.

[0092] The mutant may contain a fragment of SEQ ID NO: 1. Such a fragment retains pore-forming activity. The length of the fragment may be at least 50, 100, 200, or 250 amino acids. Such a fragment can be used to generate pores. The fragment preferably contains the pore-forming domain of SEQ ID NO: 1. The fragment typically contains residues 111, 119, 121, 135, 113, and 139 of SEQ ID NO: 1.

[0093] One or more amino acids may be added to the polypeptide described above, either by substitution (insertion) or additionally. An elongation may be provided at the amino or carboxyl terminus of the amino acid sequence of SEQ ID NO: 1 or its polypeptide variant or fragment. This elongation may be very short, for example, about 1 to about 10 amino acids in length. Alternatively, the elongation may be longer, for example, about 50 or about 100 amino acids.

[0094] As described above, the mutants are polypeptides that have a different amino acid sequence from SEQ ID NO: 1 and retain pore-forming ability. The mutants typically contain the region of SEQ ID NO: 1 involved in pore formation. The pore-forming ability of α-HL containing β-barrels is provided by the β-chains within each subunit. These regions can be shortened, and the ability to form pores in the membrane is retained even with shortened β-barrels. Mutants of SEQ ID NO: 1 typically contain the region in SEQ ID NO: 1 that forms the β-chain. One or more modifications can be made to the region of SEQ ID NO: 1 that forms the β-chain, as long as the mutant retains pore-forming ability. Specific modifications that can be made to the β-chain region of SEQ ID NO: 1 are discussed above.

[0095] The variant of Sequence ID No. 1 preferably contains one or more modifications, such as substitutions, additions, or deletions, within its α-helix, β-chain, and / or loop region. The amino acids forming the α-helix and loop are discussed above.

[0096] As is evident from the above discussion, the nanopores for use in the disclosed method typically have a first opening, a second opening, and a channel accessible to the solvent present between them.

[0097] Furthermore, the nanopore arrays described herein are also provided, for example, nanopore arrays having one or more photoisomerizable groups in the lumen of the nanopores described herein. The methods and uses described herein can be carried out using nanopore arrays. The use of pore arrays may allow for monitoring of the method by monitoring signals such as electrical signals or optical signals. Optical detection of analytes using nanopore arrays can be carried out using techniques well known in the art, such as those described by Huang et al, Nature Nanotechnology (2015) 10: 986-992.

[0098] Photoisomerizable group In a preferred embodiment, the nanopore has one or more photoisomerizable groups within the lumen of the nanopore. Preferably, the nanopore of the present invention is converted between an open state and a closed state using light by photoisomerization of the photoisomerizable groups. Thus, the nanopore of the present invention can be converted using light between a state that allows the flow of one or more substances through the nanopore (open state) and a state that restricts the movement of one or more substances through the nanopore (closed state). Preferably, the closed state (i.e., the state that restricts the movement of one or more substances through the nanopore) restricts the flow of one or more substances by 10% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more, compared to the flow of one or more substances through the open state. In a preferred embodiment, when the nanopore is in a closed configuration, substantially no substance can move through the nanopore; that is, the closed configuration (i.e., a configuration in which the movement of one or more substances through the nanopore is restricted) restricts the flow of one or more substances by 80% or more, preferably 90% or more, preferably 95% or more, and more preferably 99% or more, compared to the flow of one or more substances through an open configuration. Most preferably, the closed configuration (i.e., a configuration in which the movement of one or more substances through the nanopore is restricted) restricts the flow of one or more substances by 100% compared to the flow of one or more substances through an open configuration. In other words, most preferably, when the nanopore is in a closed configuration, no substance can move through the nanopore.

[0099] In the present invention, preferably, one or more photoisomerizable groups are independently of formula (I):

[0100] [ka]

[0101] (In the formula: * indicates a connection point to a nanopore; L is a linker; and Ar 1 and Ar 2The groups are (each independently selected from an aryl group that may be substituted and an heteroaryl group that may be substituted).

[0102] When the photoisomerizable group is a group of formula (I), the conversion of the nanopore of the present invention from the open form to the closed form is isomerization from the Z type to the E type. Correspondingly, the conversion of the nanopore of the present invention from the closed form to the open form is isomerization from the E type to the Z type.

[0103] In a preferred embodiment, the base of formula (I) is further defined as follows: -The substituted aryl groups consist of 1 to 4 R 1 A 6-10 membered aryl group which may be substituted with; and - The substituted heteroaryl group is a 5-10 membered ring heteroaryl group having 1-4 ring heteroatoms independently selected from the group consisting of N, O, and S; the 5-10 membered ring heteroaryl group has 1-4 R 2 It may be replaced with; and -R 1 and R 2 Each of them is independent of the COO - , SO3 - , or NR 3 3 + C may be replaced by 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-6 Alkenil; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-6 Alkynyl; Halogen; CN; NO2; C 1-6 Haloalkyl; OH; COO - , SO3 - , or NR 3 3 + C may be replaced by 1-6 Alkoxy; C 1-6 Haloalkoxy; COO- , SO3 - , or NR 3 3 + COOC may be replaced with 1-6 Alkyl;CONH2;COO - , SO3 - , or NR 3 3 + CONR may be replaced with 4 C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + Replace with GoodNR 4 COC 1-6 Alkyl;SO2NH2;COO - , SO3 - , or NR 3 3 + SO2NR may be substituted with 4 C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + Replace with GoodNR 4 SO2C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + ;(In the formula, each R 3 H or C 1-6 It is alkyl, and each R 4 H or C 1-6 Selected from the group consisting of alkyl groups.

[0104] Typically, substituted aryl groups consist of 1 to 3 R groups. 1 It may be substituted with 1 or 2 R groups. More preferably, the substituted aryl group is 1 or 2 R groups. 1 It may be substituted with one R. More preferably, the aryl group that may be substituted is one R 1 It may be substituted with another group. Most preferably, the aryl group is unsubstituted.

[0105] Typically, a heteroaryl group that may be substituted has 1 to 3 R 2 It may be substituted with. More preferably, the heteroaryl group that may be substituted is 1 to 3 R 2 It is substituted with. More preferably, the heteroaryl group that may be substituted is 2 or 3 R 2 It is substituted with. Most preferably, the heteroaryl group has 3 R 2 It will be replaced by this.

[0106] Typically, the aryl group is a naphthyl group or a phenyl group. Most preferably, the aryl group is a phenyl group.

[0107] Typically, a heteroaryl group is a 5- or 6-membered heteroaryl group having 1 to 4 ring heteroatoms independently selected from the group consisting of N, O, and S. Preferably, the heteroaryl group is a 5- or 6-membered heteroaryl group having 1 to 4 ring heteroatoms independently selected from the group consisting of N and O. More preferably, the heteroaryl group is a 5-membered ring heteroaryl group having 1 to 4 ring heteroatoms independently selected from the group consisting of N and O. More preferably, the heteroaryl group is a 5-membered ring heteroaryl group having 1 to 3 ring heteroatoms independently selected from the group consisting of N and O. Even more preferably, the heteroaryl group is a group of the following formula:

[0108] [ka]

[0109] (In the formula: *** indicates the connection point to the azo group; R 2 This is as defined above (and below); p is an integer from 0 to 3, preferably 1 to 3, more preferably 2 or 3, and most preferably 3.

[0110] More preferably, the heteroaryl group is a group of the following formula:

[0111] [ka]

[0112] (In the formula: *** indicates the connection point to the azo group; R 2 This is as defined above (and below); p is an integer from 0 to 3, preferably 1 to 3, more preferably 2 or 3, and most preferably 3.

[0113] More preferably, the heteroaryl group is a group of the following formula:

[0114] [ka]

[0115] (In the formula: *** indicates the connection point to the azo group; R 2 This is as defined above (and below); p is an integer from 0 to 3, preferably 1 to 3, more preferably 2 or 3, and most preferably 3.

[0116] More preferably, the heteroaryl group is a group of the following formula:

[0117] [ka]

[0118] (In the formula: *** indicates the connection point to the azo group; R 2 This is as defined above (and below); p is an integer from 0 to 3, preferably 1 to 3, more preferably 2 or 3, and most preferably 3.

[0119] More preferably, the heteroaryl group is a group of the following formula:

[0120] [ka]

[0121] (In the formula: *** indicates the connection point to the azo group; R 2 (As defined above (and below)).

[0122] R 1 and R 2 Each of them is independent of the COO - , SO3 - , or NR 3 3 + C may be replaced by 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-6 Alkenil; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-6 Alkinyl; halogen; C 1-6 Haloalkyl; OH; COO - , SO3 - , or NR 3 3 + C may be replaced by 1-6 Alkoxy; C 1-6 Haloalkoxy; CONH2; COO - , SO3 - , or NR 3 3 + CONR may be replaced with 4 C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + Replace with GoodNR 4 COC 1-6 Alkyl;SO2NH2;COO- , SO3 - , or NR 3 3 + SO2NR may be substituted with 4 C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + Replace with GoodNR 4 SO2C 1-6 Alkyl; COO - , SO3 - , or NR 3 3 + ;(In the formula, each R 3 H or C 1-6 It is alkyl, and each R 4 H or C 1-6 Selected from the group consisting of alkyl groups.

[0123] Comfortable, R 1 and R 2 Each of them is independent of the COO - , SO3 - , or NR 3 3 + C may be replaced by 1-3 Alkyl; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-3 Alkenil; COO - , SO3 - , or NR 3 3 + C may be replaced by 2-3 Alkinyl; halogen; C 1-6 Haloalkyl; OH; COO - , SO3 - , or NR 3 3 + C may be replaced by 1-3 Alkoxy; C 1-3 Haloalkoxy; CONH2; COO - , SO3 - , or NR 3 3 +CONR may be replaced with 4 C 1-3 Alkyl; COO - , SO3 - , or NR 3 3 + Replace with GoodNR 4 COC 1-3 Alkyl;SO2NH2;COO - , SO3 - , or NR 3 3 + SO2NR may be substituted with 4 C 1-3 Alkyl; COO - , SO3 - , or NR 3 3 + Replace with GoodNR 4 SO2C 1-3 Alkyl; COO - , SO3 - , or NR 3 3 + ;(In the formula, each R 3 H or C 1-3 It is alkyl, and each R 4 H or C 1-3 Selected from the group consisting of alkyl groups.

[0124] Comfortable, R 1 and R 2 Each of them is independent of C 1-3 Alkyl; Halogen; C 1-3 Haloalkyl; COO - , SO3 - , or NR 3 3 + NHCOC may be replaced with 1-3 Alkyl; COO - , SO3 - , or NR 3 3 + NHSO2C may be replaced with 1-3 Alkyl, COO - , SO3 - , or NR 3 3 + ;(In the formula, each R3 is independently C 1-3Selected from the group consisting of alkyl groups.

[0125] Furthermore, R 1 and R 2 Each of them is independent of C 1-3 A group consisting of alkyl;F;Cl;Br; and CF3 (for example, C 1-3 Selected from the group consisting of alkyl, Cl, Br, and CF3. More preferably, R 1 and R 2 Each of these is methyl. In one embodiment, Ar 1 is a substituted aryl group (as described above). More preferably, the group of formula (I) is of formula (IA'):

[0126] [ka]

[0127] (In the formula: L is synonymous with the above (and below), Ar 2 This may be a substituted aryl group or a substituted heteroaryl group (as described above), R 1 and R 2 This is synonymous with the above, n is an integer from 0 to 4, preferably from 0 to 3, more preferably from 0 to 2; and p is a base of integers from 0 to 4, and preferably the same as above.

[0128] In one specific example of such an embodiment, the base of formula (I) is the base of formula (IA''):

[0129] [ka]

[0130] However, preferably, Ar 1 and Ar 2At least one of them is a substituted heteroaryl group (as described above). More preferably, Ar 1 and Ar 2 One of them is a heteroaryl group that may be substituted (as described above). More preferably, the group of formula (I) is of formula (IA):

[0131] [ka]

[0132] (In the formula: L is synonymous with the above (and below), Ar 2 This may be a heteroaryl group (same as above), R 1 and R 2 This is synonymous with the above, n is an integer from 0 to 4, preferably from 0 to 3, more preferably from 0 to 2, more preferably 0 or 1, most preferably 0; and p is a base of integers from 0 to 4, and preferably the same as above.

[0133] Therefore, in a preferred embodiment, the base of formula (I) is formula (IA):

[0134] [ka]

[0135] (In the formula: L is synonymous with the above (and below), Ar 2 is a heteroaryl group, a five-membered ring heteroaryl group having 1 to 3 ring heteroatoms independently selected from the group consisting of N and O; and R 1 and R 2 Each of them is independently C 1-3 Alkyl; Halogen; C 1-3 Haloalkyl; COO - , SO3 - , or NR3+ NHCOC may be replaced with 1-3 Alkyl;SO2NH2;COO - , SO3 - , or NR3 + NHSO2C may be replaced with 1-3 Alkyl, COO - , SO3 - , or NR3 + (In the formula, each R3 is independently either H or C 1-3 It is a group selected from the group consisting of alkyl groups.

[0136] In one specific example of such an embodiment, the base of formula (I) is formula (IB'):

[0137] [ka]

[0138] It is the base of; however, more preferably, the base of formula (I) is formula (IB):

[0139] [ka]

[0140] It is the basis of. The linker can be any divalent group capable of connecting the diarylazo moiety to the nanopore. Appropriate linker chemistry is well known to those skilled in the art, especially those proficient in the preparation of antibody-drug conjugates (for example).

[0141] Preferably, the linker is structure (L1);

[0142] [ka]

[0143] (In the formula: * indicates a connection point to a nanopore. ** is ar 1 This indicates the connection point. LL is a substituted 5- to 10-membered carbocyclic ring or a substituted 5- to 10-membered heterocyclic ring; Y is independently a halogen, OH, or C 1-6 C may be substituted with alkyl 1-20 Alkylene group; amide group (-CONR 5 - or NR 5 CO-; sulfonamide group (-SO2NR) 5 - or NR 5 SO2-); ester group; thioester group; ether group; thioether group; and amine group (-NR) 5 -), (where each R 5 H or C 1-6 It is alkyl; q is 0 or 1; and m, o, and r are linkers (each independently an integer from 0 to 5).

[0144] If q is 0, the linker is structured (L2):

[0145] [ka]

[0146] (wherein *, **, Y, o, m, and r are synonymous with those described above (or below)).

[0147] When q is 1, LL is preferably a substituted 5 or 6-membered carbon ring (i.e., a 5 or 6-membered cycloalkyl or phenyl, preferably phenyl) or a substituted 5 or 6-membered heteroring having 1 to 4 (preferably 1 to 3, more preferably 1 or 2, most preferably 1) heteroatoms (selected from O, N, and S, preferably selected from O and N, more preferably N). The heteroring may be aromatic or aromatic, but is preferably aromatic. 1 to 3 arbitrary substituents may be present, any substituent being R 1 and R 2This is synonymous with the above. However, if the carbocyclic or heterocyclic ring is non-aromatic, it is preferable that the ring is substituted with at least one oxo group. Most preferably, LL is a succinamide ring. Therefore, in one embodiment, the linker is structure (L3)

[0148] [ka]

[0149] (In the formula, *, **, Y, o, m, and r may have the same meaning as described above (or below).)

[0150] In each of L1 to L3, Y is preferably C which may be independently substituted with OH. 1-6 Alkylene; amide group (-CONR 5 - or NR 5 CO-), sulfonamide group (-SO2NR 5 - or NR 5 SO2-), (where each R 5 H or C 1-6 Y is selected from alkyl groups. Y is more preferably an amide group (-CONR 5 - or NR 5 CO-), sulfonamide group (-SO2NR5- or NR5SO2-) (wherein each R, 5 (is independently H or methyl). Y is more preferably an amide group (-CONH- or NHCO-), a sulfonamide group (-SO2NH- or NHSO2-); more preferably an amide group (-CONH- or NHCO-), and most preferably an amide group (-CONH-).

[0151] In one embodiment, m, o, and r are each independently integers from 0 to 3. Preferably, m, o, and r are each independently integers from 0 to 2. Preferably, m, o, and r are each 1 or 0. Typically, at least one, preferably both, m and o are 1, and r is 1.

[0152] In a particularly preferred embodiment, the linker has structure (L4):

[0153] [ka]

[0154] (In the formula: * indicates a connection point to a nanopore; ** is ar 1 This is the linker (which indicates the connection point to...).

[0155] Therefore, in embodiments of the present invention, one or more photoisomerizable groups are each independently a group of chemical formula (IC') or each independently a group of chemical formula (IC''):

[0156] [ka]

[0157] However, in a particularly preferred embodiment of the present invention, one or more photoisomerizable groups are each independently of the chemical formula (IC):

[0158] [ka]

[0159] (In the formula: * indicates the base of the nanopore (the symbol indicates the connection point to the nanopore).

[0160] Photoisomerizable groups of chemical formula (IC) are particularly advantageous because the photosteady state (PSS) results in a higher proportion of one isomer than the other.

[0161] Advantageously, one or more photoisomerizable groups are covalently attached to the nanopore. Therefore, preferably, one or more photoisomerizable groups are covalently attached to a nucleophilic amino acid exposed in the lumen of the nanopore (as further defined above). Most preferably, in the formula, more preferably: - The nucleophilic amino acid is cysteine. Photoisomerizable group. That is, most preferably, one or more photoisomerizable groups are covalently bonded to a nucleophilic amino acid exposed in the lumen of the nanopore (wherein the formula is cysteine).

[0162] film Typically, nanopores are typically located within a membrane. Any suitable membrane can be used in the system. In one embodiment, multiple nanopores (i.e., an ensemble / array of nanopores) are present within the membrane.

[0163] Suitable membranes are well known in the art. The membrane is typically an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, having at least one hydrophilic moiety and at least one lipophilic or hydrophobic moiety. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphilic substances and amphiphilic substances forming monolayers are well known in the art, such as block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450).

[0164] In some embodiments, the membrane contains one or more archaeal bipolar tetraether lipids or mimics thereof. Such lipids are commonly found in extremophilic microorganisms such as thermophiles, halophiles, and acidophiles that inhabit harsh biological environments. Their stability is thought to derive from the fused properties of the final bilayer. It is easy to construct block copolymer materials that mimic these biological entities by creating triblock polymers with common motif hydrophilic-hydrophobic-hydrophilic properties. These materials can form monomer membranes that behave similarly to lipid bilayers, encompassing a variety of phase behaviors from vesicles to layered membranes.

[0165] Block copolymers are polymer materials in which two or more monomer subunits polymerized together form a single polymer chain. Block copolymers typically possess properties derived from each individual monomer subunit. However, block copolymers can possess unique properties not found in polymers formed from individual subunits. Block copolymers can be manipulated so that, while other subunits are hydrophilic in an aqueous medium, one monomer subunit is hydrophobic (i.e., lipophilic). In this case, the block copolymer can possess amphiphilic properties and form structures that mimic biological membranes. Block copolymers can be diblocks (consisting of two monomer subunits), but they can also be constructed from three or more monomer subunits to form more complex arrangements that behave as amphiphilic substances. Copolymers can be triblocks, tetrablocks, or pentablock copolymers. Typically, a copolymer is a triblock copolymer containing two monomer subunits A and B in an ABA pattern; typically, monomer A is hydrophilic and subunit B is hydrophobic.

[0166] The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer.

[0167] The amphiphilic layer is typically a lipid bilayer. Lipid bilayers serve as a model of the cell membrane and function as an excellent platform for various experimental studies. For example, lipid bilayers can be used for in vitro investigations of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of various substances. The lipid bilayer can be any type of lipid bilayer. Preferred lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes. The lipid bilayer is typically a planar lipid bilayer. Preferred lipid bilayers are disclosed in International Publications 2008 / 102121, 2009 / 077734, and 2006 / 100484.

[0168] Any lipid composition may be used to form a lipid bilayer. Lipids typically comprise a head group, an interface, and two hydrophobic tail groups, which may be the same or different. Preferred head groups include, but are not limited to, neutral head groups, e.g., diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups, e.g., phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin (CA); and positively charged head groups, e.g., trimethylammonium-propane (TAP). Preferred interface groups include, but are not limited to, naturally occurring interface groups, e.g., glycerol-based or ceramide-based portions. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic acid), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid); unsaturated hydrocarbon chains such as oleic acid (cis-9-octadecanoic acid); and branched hydrocarbon chains such as phytanoyl. The length of the chain and the position and number of double bonds in the unsaturated hydrocarbon chains may vary. The length of the chain and the position and number of branches such as methyl groups in the branched hydrocarbon chains may also vary. The hydrophobic tail group may be linked to the interface as an ether or ester. The lipid may be mycolic acid.

[0169] Lipids may be chemically modified. The head group or tail group of a lipid may be chemically modified. Preferred lipids whose head group is chemically modified include, but are not limited to, PEG-modified lipids, e.g., 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; functionalized PEG lipids, e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000]; and lipids modified for conjugation, e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids whose tail groups are chemically modified include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine; fluorinated lipids such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine; deuterated lipids such as 1,2-dipalmitoyl-D62-sn-glycero-3-phosphocholine; and ether-linked lipids such as 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine. Lipids may be chemically modified or functionally linked to facilitate polynucleotide coupling.

[0170] Other components that affect the properties of the amphiphilic layer may be incorporated, such as fatty acids, e.g., palmitic acid, myristic acid, and oleic acid; fatty alcohols, e.g., palmitic alcohol, myristic alcohol, and oleic alcohol; sterols, e.g., cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids, e.g., 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

[0171] Methods for forming lipid bilayers are known in the art. Preferred 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), according to which a lipid monolayer is delivered to the aqueous solution / air interface by passing either side of the opening perpendicular to the interface. The Montal & Mueller method is popular because it is cost-effective and relatively straightforward in forming good quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip dipping, bilayer painting, and patch clamping of liposome bilayers. Lipid bilayers can be formed as described in International Publication No. 2009 / 077734. The lipid bilayer may be a droplet interface bilayer formed between two or more aqueous droplets, each containing a lipid shell, such that the lipid bilayer is formed at the droplet interface when the droplets come into contact.

[0172] In another preferred embodiment, the film is a solid layer. The solid layer is not of biological origin. In other words, the solid layer does not originate from or is not isolated from a biological environment, e.g., an organism or cell, or a synthetically produced version of a biologically available structure. The solid layer may be formed from both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials, e.g., Si3N4, A12O3, and SiO2, organic and inorganic polymers, e.g., polyamides, plastics, e.g., Teflon® or elastomers, e.g., two-component cured silicone rubber, and glass. The solid layer may be formed from a single-atom layer, e.g., graphene, or a layer that is only a few atoms thick. A suitable graphene layer is disclosed in International Publication No. 2009 / 035647. Nanopores may, in some embodiments, be present in amphiphilic films or layers contained within the solid layer, e.g., holes, wells, gaps, channels, grooves, or slits within the solid layer. Preferred systems are disclosed in International Publication Nos. 2009 / 020682 and International Publication Nos. 2012 / 005857. Any of the amphiphilic membranes or layers described above may be used.

[0173] Advantageously, the photoisomerizable groups can potentially be oriented to a preferred structure by electroosmotic flow present within the charge-selective protein scaffold (i.e., the nanopore). Therefore, the present invention further provides the use of oriented forces in future photopore preparation or application stages. Such oriented forces may be osmotic flow, electrophoretic force (in the case of high-charge photoswitches), or by using facilitating molecules.

[0174] Nanopore manufacturing method As described above, the present invention also provides a method for producing a nanopore according to the present invention, the method comprising introducing one or more photoisomerizable groups into the nanopore. To avoid any doubt, a nanopore into which photoisomerizable groups are introduced is synonymous with the nanopore of the present invention as described above.

[0175] Typically, in a method for producing nanopores, one or more photoisomerizable groups are of formula (II):

[0176] [ka]

[0177] (In the formula: Ar 1 and Ar 2 This is as defined above in relation to formula (I), and includes all desirable features: and L elec The nanopore is introduced by treating it with a compound that is the same as described above, except that the atom connected to the nanopore is an electrophile that can react with a nucleophile (preferably corresponding to the linker as defined below).

[0178] Therefore, one or more photoisomerizable groups are given by formula (IIA'):

[0179] [ka]

[0180] (In the formula, R 1 Ar 2 , R 1 , R 2 n or p are as defined above in relation to formula (IA') and include all desirable features; and L elec This can be introduced into the nanopore by treating the nanopore with a compound that is the same as described above, except that the atom connected to the nanopore is an electrophile that can react with a nucleophile (preferably corresponding to a linker as defined below).

[0181] In one specific example, one or more photoisomerizable groups form a nanopore of formula (IIA''):

[0182] [ka]

[0183] It is introduced into the nanopore by treatment with the compound. However, more preferably, one or more photoisomerizable groups form a nanopore of formula (IIA):

[0184] [ka]

[0185] (In the formula, R 1 Ar 2 , R 1 , R 2 n or p are as defined above in relation to formula (IA) and include all desirable features; and L elec The nanopore is introduced by treating it with a compound (which corresponds to a linker as described above, preferably as described below, except that the atom connected to the nanopore is an electrophile capable of reacting with a nucleophile).

[0186] Therefore, in one specific example, one or more photoisomerizable groups form a nanopore with formula (IIB'):

[0187] [ka]

[0188] It is introduced into the nanopore by treatment with the compound. However, more preferably, one or more photoisomerizable groups form a nanopore of formula (IIB):

[0189] [ka]

[0190] (In the formula, L elecThe nanopore is introduced by treating it with a compound (which corresponds to a linker as described above, preferably as described below, except that the atom connected to the nanopore is an electrophile capable of reacting with a nucleophile).

[0191] Typically, L elec is, (L elec 1) or (L elec 1');

[0192] [ka]

[0193] (In the formula: ** is ar 1 This indicates the connection point. LL is as defined above in relation to (L1) and includes desirable features; Y is as defined above in relation to (L1) and includes desirable characteristics; q, m, o, and r are as defined above in relation to (L1) and include preferred characteristics; X is a leaving group as understood by those skilled in the art, and is typically a halogen (such as F, Cl, Br, or I, most preferably Br), or a sulfonate (preferably tosylate or trifluoromethanesulfonate), most preferably Br; and LL elec It is an analog of LL as defined above in relation to (L1), which can undergo nucleophilic addition reactions (i.e., has a double bond conjugated to an electron-withdrawing group such as a carbonyl group). Most preferably, LL elec This is the structure of maleimide.

[0194] If q is 0, L elec Preferably, the structure (L elec 2):

[0195] [ka]

[0196] (In the formula, ** represents Ar 1 This indicates the connection point. Y is as defined above in relation to (L2) and includes desirable characteristics; m, o, and r are as defined above in relation to (L2) and include preferred characteristics; X has a leaving group as defined above.

[0197] If q is 1, L elec Preferably, the structure (L elec 3)

[0198] [ka]

[0199] (In the formula, ** represents Ar 1 This indicates the connection point. Y is as defined above in relation to (L3) and includes desirable characteristics; m, o, and r are as defined above in relation to (L3) and have the following desirable characteristics.

[0200] In a particularly preferred embodiment, the structure (L elec 4):

[0201] [ka]

[0202] (In the formula: ** is ar 1 It indicates the connection point, and X is the leaving group as defined above. elec .

[0203] Therefore, in embodiments of the present invention, the compound is of formula (IIC') or (IIC''):

[0204] [ka]

[0205] (wherein X is a leaving group, preferably a group selected from halogens (preferably F, Cl, Br or I) and sulfonate groups (preferably tosylate or trifluoromethanesulfonate), most preferably Br) and a compound of 2-bromo-N-(4-((2,6-difluorophenyl)diazenyl)-3,5-difluorobenzyl)acetamide or 2-bromo-N-(4-((1-methyl-1H-pyrazole-3-yl)diazenyl)benzyl)acetamide.

[0206] However, in the most preferred embodiment, the compound is of formula (IIC):

[0207] [ka]

[0208] (wherein X is a leaving group, preferably a group selected from halogens (preferably F, Cl, Br or I) and sulfonate groups (preferably tosylate or trifluoromethanesulfonate), most preferably Br) is a compound of the above. Therefore, most preferably the compound is 2-bromo-N-(4-((1,3,5-trimethyl-1H-pyrazole-4-yl)diazenyl)benzyl)acetamide.

[0209] In the method of the present invention, it is preferable to introduce one or more photoisomerizable groups into the nanopore by treating the monomer of the nanopore with a compound of formula (II), (IIA), (IIA'), (IIA''), (IIB), (IIB'), (IIC), (IIC'), or (IIC'') as previously defined (preferably (II), (IIA), (IIB), or (IIC)). More preferably, one or more photoisomerizable groups are introduced into the nanopore by treating the monomer of the nanopore with a compound of formula (II), (IIA), (IIA'), (IIA''), (IIB), (IIB'), (IIC), (IIC'), or (IIC'') as previously defined (preferably (II), (IIA), (IIB), or (IIC)), and then oligomerizing the monomer to provide the nanopore.

[0210] Typically, photoisomerizable groups are introduced into the nanopore by treating the nanopore (preferably the monomer of the nanopore) with a compound of formula (II), (IIA), (IIA'), (IIA''), (IIB), (IIB'), (IIC), (IIC'), or (IIC'') (preferably (II), (IIA), (IIB), or (IIC)) which exists in amounts of 1 to 100 equivalents of the compound per monomer to be functionalized (e.g., 10 to 90, 20 to 80, 30 to 70, 40 to 60, most preferably about 50 equivalents of the compound).

[0211] Typically, the treatment is carried out at the location where the nanopores are retained in the membrane. Typically, the treatment is carried out under buffering conditions, preferably with a buffer that buffers the solution to pH 6-9, preferably pH 7 or 8, more preferably pH 8. The buffer is preferably Tris-HCl.

[0212] Typically, monomers are assembled in situ to provide nanopores.

[0213] Alternatively, the treatment is carried out in vitro in a detergent (e.g., deoxycholate), and the nanopores are separated by chromatography (e.g., ion exchange chromatography).

[0214] compound The present invention also provides compounds of formulas (IIA), (IIA'), (IIA''), (IIB), (IIB'), (IIC), (IIC'), or (IIC'') as previously defined (preferably compounds of formulas (IIA), (IIB), and (IIC) as previously defined), and 2-bromo-N-(4-((1,3,5-trimethyl-1H-pyrazole-4-yl)diazenyl)benzyl)acetamide, 2-bromo-N-(4-((2,6-difluorophenyl)diazenyl)-3,5-difluorobenzyl)acetamide, and 2-bromo-N-(4-((1-methyl-1H-pyrazole-3-yl)diazenyl)benzyl)acetamide (preferably 2-bromo-N-(4-((1,3,5-trimethyl-1H-pyrazole-4-yl)diazenyl)benzyl)acetamide itself.

[0215] kit This disclosure also provides a kit of parts including: (a) Nanopores to be functionalized, and (b) Any one compound of formula (IIA), (IIA'), (IIA''), (IIB), (IIB'), (IIC), (IIC'), or (IIC'') as defined previously (preferably any one compound of formula (IIA), (IIB), or (IIC) as defined previously), or 2-bromo-N-(4-((1,3,5-trimethyl-1H-pyrazole-4-yl)diazenyl)benzyl)acetamide, 2-bromo-N-(4-((2,6-difluorophenyl)diazenyl)-3,5-difluorobenzyl)acetamide, or 2-bromo-N-(4-((1-methyl-1H-pyrazole-3-yl)diazenyl)benzyl)acetamide (preferably 2-bromo-N-(4-((1,3,5-trimethyl-1H-pyrazole-4-yl)diazenyl)benzyl)acetamide).

[0216] To avoid any doubt, the functionalized nanopore may be the nanopore as previously defined in the above section describing the nanopore of the present invention.

[0217] A method for regulating the flow of one or more substances through a nanopore. As described above, the present invention also provides a method for regulating the flow of one or more substances through a nanopore according to the present invention, the method comprising irradiating the nanopore with light.

[0218] In a preferred embodiment of the method, the nanopore of the present invention is a nanopore containing a photoisomerizable group, as described above. Therefore, when the photoisomerizable group is a group of formula (I) (or (IA), (IA'), (IA''), (IB), (IB'), (IC), (IC'), (IC''), etc., preferably (IA), (IB), (IC), etc.), the conversion of the nanopore of the present invention from open to closed is an isomerization from Z-type to E-type (which prevents the flow of one or more substances through the nanopore). Correspondingly, the conversion of the nanopore of the present invention from closed to open is an isomerization from E-type to Z-type (which allows the flow of one or more substances).

[0219] Preferably, the light is light with a wavelength between 355 and 550 nm.

[0220] In a preferred embodiment, the method includes irradiating the nanopore with a first wavelength of light and a second wavelength of light. Preferably, the first wavelength of light isomerizes the photoisomerizable group to the Z-type, and the second wavelength of light isomerizes the photoisomerizable group to the E-type. However, the order of the first and second wavelengths of light may be reversed.

[0221] Therefore, the wavelength of light is preferably selected to selectively induce the isomerization of the photoisomerizing group to the E-type or Z-type.

[0222] Therefore, in the present invention, the first wavelength of light is preferably a wavelength of light that can isomerize the photoisomerizable group to the Z type. Similarly, the second wavelength of light is preferably a wavelength of light that can isomerize the photoisomerizable group to the E type.

[0223] Preferably, the first wavelength of light is the wavelength in the UV / Vis spectrum where the ratio of εE / εZ is maximized, and the second wavelength of light is the wavelength in the UV / Vis spectrum where the ratio of εZ / εE is maximized, where: εE is the molar extinction coefficient of the E-type of the photoisomerizable group, and εZ is the molar quenching coefficient of the Z-type of the photoisomerizable group.

[0224] Preferably, εE and εZ are determined using a UV / Vis spectrophotometer.

[0225] Therefore, typically, the photoisomerizable group is a group of formula (I) (or (IA), (IA'), (IA''), (IB), (IB'), (IC), (IC'), (IC''), etc., preferably (IA), (IB), (IC), etc.), the first wavelength of light is approximately 320-400 nm (preferably 350-380 nm, more preferably 360-370 nm, and most preferably approximately 365 nm), and the second wavelength of light is 420-550 nm, typically 440-540 nm, preferably approximately 455 nm or approximately 530 nm, most preferably approximately 530 nm.

[0226] To obtain a moderate flow of one or more substances, other wavelengths of light can be conveniently selected. For example, to adjust the flow to 50%, wavelengths of light can be selected such that the εZ / εE ratio is approximately 1.

[0227] To induce photoisomerization, any light intensity can be selected. For example, a light intensity of 1 mW / cm² is possible. 2 For example, anything exceeding 5 mW / cm² 2 For example, exceeding 10 mW / cm². 2 For example, anything exceeding 20 mW / cm². 2 It may exceed this. Preferably, the light intensity is approximately 30 mW / cm². 2 It is possible.

[0228] The irradiation can last for any duration, for example, from 1 ms to 1 day, from 10 ms to 1 hour, from 50 ms to 10 minutes, from 500 ms to 1 minute, or for example, approximately 1 second.

[0229] The one or more substances may be any one or more of the substances described in the definition section above. Therefore, the one or more substances may be independently selected from ions, water, sugars, inorganic salts, lipids, amino acids, peptides, nucleotides, nucleic acids, metabolites, neurotransmitters, and drugs. Most preferably, the one or more substances are ions, preferably cations such as metal cations. The cations are potassium (K + ), sodium (Na + ) and / or calcium (Ca 2+ ) can be selected from. The ion can be an ion. The anion is a chloride ion (Cl - ) may be. Preferably, the ion is both an anion and a cation, typically K + and Cl - That is the case.

[0230] Use in regulating ion flow As described above, the present invention also provides use in regulating the flow of ions across membranes such as amphiphilic membranes. To avoid any doubt, the membrane / amphiphilic membrane may be as defined in the section relating to nanopores of the present invention. Preferably, ions are as defined above.

[0231] In one embodiment, the ion flow is from the high-concentration side of the membrane to the low-concentration side. By opening and closing the nanopores, the ion flow from the high-concentration side to the low-concentration side is regulated, resulting in a change in the ionic current. Advantageously, the regulation of the ion flow is between the ON state (when the nanopore is open) and the OFF state (when the nanopore is closed).

[0232] Advantageously, since ion flow is permitted in only one direction (ON / open state), the open nanopore functions as a diode. In the OFF / closed state, no ion flow occurs in either direction, so the nanopore functions as a resistor.

[0233] Advantageously, the use of the present invention in regulating ion flow can be applied in neuron science, for example, by using light to induce action potentials (i.e., by enabling a rapid influx of ions) in neurons having the nanopores of the present invention in the cell membrane.

[0234] Use in regulating the flow of one or more substances Furthermore, this disclosure also provides the use of the nanopores of the present invention in regulating the flow of one or more substances across a membrane, such as an amphiphilic membrane. To avoid doubt, the membrane / amphiphilic membrane may be as defined in the section relating to the nanopores of the present invention. The substances are preferably as defined previously.

[0235] In one embodiment, the flow is the flow of a substance from the high-concentration side to the low-concentration side of the membrane. By opening and closing the nanopores, the flow of the substance from the high-concentration side to the low-concentration side is regulated, resulting in a change in the delivery rate of the substance. Advantageously, the flow regulation is between the ON state (when the nanopore is open) and the OFF state (when the nanopore is closed).

[0236] In one embodiment, when one or more substances are oligomers or polymers (e.g., polynucleotides, polypeptides, and polysaccharides), their use in regulating the flow of one or more substances may be in the context of characterizing one or more substances as analytes. Accordingly, in one embodiment, the present invention provides the use of the nanopore of the present invention in characterizing one or more analytes, where one or more analytes are preferably one or more substances as defined herein, such as polynucleotides, polypeptides, and polysaccharides (more preferably polynucleotides or polypeptides, most preferably polynucleotides).

[0237] device As described above, the present invention also provides comprising a vesicle surrounded by an amphiphilic membrane, wherein the vesicle contains one or more agents within its lumen, and the amphiphilic membrane contains the nanopore according to the present invention. To avoid any doubt, the amphiphilic membrane may be as previously defined with respect to the nanopore of the present invention.

[0238] The drug can be any drug, but advantageously it may be a drug that targets a pathological condition occurring in a specific part of the body. Particularly advantageous, the drug may be a chemotherapeutic agent. The device of the present invention can induce the release of a therapeutic agent using light on a target region. Advantageously, the release of the therapeutic agent can be prevented or stopped by irradiating a non-target region with a wavelength of light.

[0239] Therefore, this application further provides the use of the nanopores of the present invention in drug delivery.

[0240] system The nanopore may be provided as part of a system including the nanopore and a light source. The light source may be configured to emit light that irradiates the nanopore and converts it between open and closed states. The light source may be a tunable wavelength light source. This allows the light source to be used to convert the nanopore from open to closed and from closed to open states by changing the wavelength of light used. The light source may be configured to emit light of at least a first wavelength and / or a second wavelength. The light source may be configured to emit light of multiple wavelengths simultaneously. The light source may be a pulsed light source configured to emit light pulses. This can be used to encode information, as described later. The light source may be configured to emit pulses of variable length. The pulse length may be at least 0.5 s, optionally at least 1 s, optionally at least 5 s, optionally at least 10 s. The pulse length may be up to 1 minute, optionally up to 30 s. The intensity of the light source is at least 5 mW / cm². 2 Optionally, at least 10 mW / cm² 2 Optionally, at least 20 mW / cm² 2 Optionally, at least 30 mW / cm² 2 It is possible.

[0241] The system may include, for example, multiple nanopores arranged in an array. The light source may be configured to irradiate multiple nanopores with light. The light source may be configured to irradiate multiple nanopores simultaneously with light of substantially the same wavelength and / or intensity.

[0242] Use as an iontronic device As described above, the present invention also provides the use of the aforementioned nanopore as an iontronic element. The nanopore can be selectively converted between open and closed states using light. This is achieved by irradiating the nanopore with light, as described above. The conductive properties of the nanopore differ between the open and closed states. This makes it possible to use the nanopore as a photoswitchable iontronic element. In this context, conductive properties refer to the resistance of the nanopore to the ionic current flowing through it.

[0243] In the closed configuration, the conductivity of a nanopore is asymmetric with respect to the direction of the applied voltage across the nanopore and / or the ionic current flowing through the nanopore. The ionic current flowing through a nanopore in response to a first applied voltage across the nanopore is different from the ionic current flowing through a nanopore in response to a second applied voltage across the nanopore, where the second applied voltage is of the same magnitude as the first but in the opposite direction. The resistance of the nanopore when the ionic current flows in the first direction is different from the resistance of the nanopore when the ionic current flows in the second direction, which is opposite to the first direction. Some asymmetry in conductivity may also exist in the open configuration, but the asymmetry in the closed configuration is greater than the asymmetry in the open configuration.

[0244] In the open configuration, the nanopore allows ionic current to flow through it in both the first and second directions. In the closed configuration, the nanopore allows ionic current to flow through it in the first direction, but blocks most, and potentially substantially all, of the ionic current flowing through it in the second direction. The ionic current flowing through a closed nanopore in the second direction for a first applied voltage across the nanopore is up to 30%, potentially up to 20%, potentially up to 10%, and potentially up to 5%, of the ionic current flowing through an open nanopore in the second direction for a first applied voltage. The ionic current flowing through a closed nanopore in the second direction for a first applied voltage across the nanopore is up to 30%, potentially up to 20%, potentially up to 10%, and potentially up to 5%, of the ionic current flowing through a closed nanopore in the first direction for a second applied voltage equal to and opposite to the first applied voltage.

[0245] The asymmetry of the conductive properties in this closed configuration means that the nanopore can be used as an iontronic resistor in the open configuration and as an iontronic diode in the closed configuration. The nanopore can be used as an iontronic element that is photoswitchable between resistor and diode. Furthermore, the nanopore can be used as a photoswitchable switch. Thus, the present invention also provides an iontronic element that includes a nanopore.

[0246] Iontronic elements can be resistors, diodes, or photoswitchable switches. Other iontronic elements similar to conventional electrical circuit components can also be constructed. By utilizing the diode properties of nanopores, the appropriate arrangement of nanopores can be used for rectification. As a result, iontronic elements can function as half-wave rectifiers.

[0247] Iontronic devices may include, for example, multiple nanopores arranged in an array. Using multiple nanopores can, for example, improve the iontronic device's ability to handle larger currents.

[0248] Because iontronic elements exhibit switching characteristics, they can be used as logic components, such as logic gates. Consequently, the present invention also provides for the use of nanopores in biocomputing and / or bioiontronics.

[0249] An iontronic element containing multiple nanopores can be used as a variable resistor. The variable resistor may be configured to be set to at least three different resistance values. Here, resistance refers to resistance to the ionic current flowing through the nanopores.

[0250] As described above, when a nanopore is irradiated with light having the first wavelength, the nanopore is converted to an open state. When a nanopore is irradiated with light having the second wavelength, the nanopore is converted to a closed state. When the nanopore is in a closed state, the ionic current flowing through the nanopore in the second direction experiences much higher resistance compared to when the nanopore is in an open state.

[0251] Multiple nanopores can be simultaneously irradiated with light of a first wavelength and light of a second wavelength. While we do not wish to be bound by theory, it is thought that this irradiation creates a dynamic equilibrium state as the nanopores switch between open and closed states. The equilibrium point can be adjusted by changing the relative intensity of the light of the first and second wavelengths. This means that by changing the relative intensity of the light of the first and second wavelengths, the overall resistance of multiple nanopores to ionic currents flowing in the second direction can be changed.

[0252] The resistance of an iontronic element containing multiple nanopores can also be changed by varying the wavelength of the incident light between a first and a second wavelength. Since the conversion rate from one form to the other changes, the relative rate at which the multiple nanopores convert from open to closed and vice versa also changes with wavelength.

[0253] How to send information As described above, the present invention also provides a method for transmitting information. As demonstrated in Figures 5 and 7, information can be transmitted by using incident light on a nanopore to regulate the flow of material (e.g., ion current) through the nanopore.

[0254] The method involves irradiating a nanopore with multiple light pulses. Each pulse contains light having one of a predetermined set of wavelengths. The predetermined set of wavelengths is selected such that pulses containing light with different wavelengths from the predetermined set of wavelengths convert the nanopore into either an open or closed state. Each pulse may consist of light having only a single wavelength from the predetermined set of wavelengths.

[0255] When a nanopore is irradiated with pulses of light having different wavelengths from a predetermined set of wavelengths, the resistance to the ionic current flowing through the nanopore in a second direction will change as the nanopore transforms between an open and closed state. Information can be encoded by utilizing this variation in current. The method may include encoding the information into the light pulse.

[0256] A predetermined set of wavelengths can consist of two different wavelengths, such as the first and second wavelengths described above. This makes it possible to encode information as binary data. Here, the two binary states are represented by the open and closed states of the nanopore and the corresponding resistance to the ionic current flowing through the nanopore. Figure 5b demonstrates that information is encoded as binary data.

[0257] To realize a method for transmitting information, the present invention provides a transmitter. This transmitter includes a nanopore of the present invention. The transmitter further includes a light source configured to emit light pulses and a controller configured to control the light source to irradiate the nanopore with light pulses. The controller may be configured to encode information into the light pulses.

[0258] Multiple nanopores can be used to transmit information. In this case, the method of transmitting information involves irradiating multiple nanopores with light pulses, and the transmitter includes multiple nanopores. As described above, multiple nanopores can be used as elements having variable resistors with three or more resistance values. These three or more resistance values ​​can be used to encode information as ternary data, quaternary data, etc., depending on the number of resistance values ​​that the multiple nanopores can provide.

[0259] When multiple nanopores are used, irradiation of multiple nanopores may involve simultaneously irradiating the nanopores with light pulses having different wavelengths from a predetermined set of wavelengths. That is, some of the light pulses may consist of light having only one wavelength from the predetermined set of wavelengths, while other light pulses may contain light having multiple wavelengths from the predetermined set of wavelengths. As described above, this can be used to generate further resistive states that can encode data into three or more states. Figures 5c and 5d demonstrate encoding information into three states using three types of light pulses: 1) a pulse consisting of light of a first wavelength, 2) a pulse consisting of light of a second wavelength, and 3) a pulse containing light having both the first and second wavelengths.

[0260] As described for variable registers, instead of using an optical pulse containing a mixture of light having a first wavelength and light having a second wavelength, it is also possible to encode three or more states by using an optical pulse with a wavelength intermediate between the first and second wavelengths. In this case, a given set of wavelengths may further include a third wavelength intermediate between the first and second wavelengths.

[0261] How to receive information As described above, the present invention also provides a method for receiving information. The method includes measuring the ion current passing through a nanopore while irradiating the nanopore with a plurality of light pulses. As described above, each pulse includes light having one of a predetermined set of wavelengths. Since the nanopore is converted between an open and a closed state, the ion current passing through the nanopore changes depending on the wavelength of the light irradiated onto the nanopore.

[0262] If multiple nanopores are used, then measuring the ionic current involves measuring the current flowing through multiple nanopores. The current is measured as the total current flowing through the multiple nanopores as a whole. The measurement is not necessarily performed for the ionic currents flowing through each individual nanopore among the multiple nanopores.

[0263] The measured ion current can be identified by decoding the information according to a predetermined key. The predetermined key indicates the level of ion current corresponding to each of multiple states. Multiple states may consist of two states, in which case the information is coded as binary data. When multiple nanopores are used, multiple states may include three or more states.

[0264] To realize a method for receiving information, the Disclosure also provides a receiver. The receiver comprises a nanopore of the present invention. The receiver further comprises a measuring circuit configured to measure an ion current passing through the nanopore, and a controller configured to decode information from the measured ion current according to a predetermined key.

[0265] The present invention also provides the use of the nanopore as a 2-in-1 sensor. This allows for the characterization of the same analyte with individual nanopores and the generation of two different signals under optical control. This is beneficial for nanopore sequencing and general biomolecular sensing, as snapshots taken from different angles provide more information. [Examples]

[0266] Example 1 Molecular tools for remote control of transmembrane ion communications are widely used in biomedical science. Among these, optically active tools are attracting attention due to their excellent properties such as spatiotemporal control, reversibility, minimal invasiveness, and multiplexing capabilities.

[0267] Light-controlled ion flow across membranes is typically mediated by photoswitchable chromophores embedded in the membrane or bound to proteins. Protein-dependent ion transporters, such as microbial rhodopsins, generate or dissipate electrochemical gradients when their chromophores (e.g., all-trans retinal) are activated by light. This allows for the control of the electrical activity of rhodopsin-expressing cells in vitro and in vivo, enabling the investigation or manipulation of fundamental biological activities such as calcium signaling and neuronal firing. These optogenetic tools also function as integrated components of bioelectronic systems with potential therapeutic applications, such as treating cardiac arrhythmias and restoring vision. Simultaneously, synthetic alternatives have been devised to directly or indirectly replicate photoactivated passive ion transport. High molecular weight ion transporters activated by built-in photoswitches (e.g., azobenzene) transport ions along gradients, offering potential therapeutic applications for channelopathy. Similarly, synthetic photochromic binders are being developed as photopharmacological agents that target ligand-dependent ionotropic receptors such as potassium channels, TRPVs, and GABA, thereby modulating ion signaling.

[0268] In bottom-up synthetic biology, natural ion transporters (e.g., rhodopsin) are being reconstituted into artificial membranes for optically controlling ionic flow. Achieving sustained ion conduction effects in diverse applications requires careful selection of activatable protein structures at desired wavelengths, promotion of directional insertion into bilayers, and provision of continuous irradiation. Photoactivatable protein nanopores represent an alternative class of optical tools, characterized by robust channel formation capabilities at design, ease of handling of soluble monomers, and the potential for controlled molecular transport. Previous research has focused on designing α-hemolysin (αHL) and fragaceatoxin C (FraC) monomers in photocages. These are released upon prolonged UV irradiation (~10-30 minutes), inducing irreversible channel formation and permanent activation of ion signaling.

[0269] This specification describes the construction of photoreversible ON-OFF protein nanopores to control ion flow with low background current in the OFF state and long-lived channels in the ON state. Our approach involves covalent modification of pore-forming monomers of αHL using photoswitches with desired photosteady properties. Individual photopores were characterized at both the single-molecule and ensemble levels, exhibiting diode-like or register-like behavior under photocontrol. This behavior has also been applied to signal processing.

[0270] Details of Example 1 are provided in Figures 1-5. A description of the figures is provided below.

[0271] Figure 1 | Construction and characterization of an example of a non-restrictive photopore with seven ON-OFF switches. a. Bromoacetylazopyrazole (Br-pzMe) photoswitches are shown as either the E isomer (green, top) or the Z isomer (purple, bottom). b. UV / Vis spectra of the E or Z isomers of Br-pzMe in DMSO at room temperature. c. Photosteady state (PSS) of Br-pzMe determined by the 1H-NMR chemical shift of aromatic protons in DMSO. d. α-hemolysin (αHL) monomers containing cysteine ​​at position 111 were functionalized with an azopyrazole group via a bromoacetamide handle before heptamerization to form (E111C-pzMe)7 photopores. e. Total ion chromatogram confirming the complete modification of the αHL monomer using Br-PzMe. f. The remote control of the ionic current passing through one or more photopores was measured using voltage clamp electrical recording, with a transmembrane potential (Vm) applied to the cis side of the planar lipid bilayer and the transformer side grounded. LED light was made parallel and irradiated onto the bilayer from the transformer side. Irradiation at 530 nm or 365 nm isomerized the azopyrazole groups in the individual pores, causing all seven photoswitches to assemble in either the E configuration (7E, 0Z) or Z configuration (0E, 7Z).

[0272] Figure 2 | Reversible ON-OFF switching of photopores. a. Optical control of ion flow passing through a single photopore. Irradiation at 365 nm (violet) converted the photopore to the (0E, 7Z) state, and irradiation at 530 nm (green) converted it to the (7E, 0Z) state. b. ON-OFF switching of a single photopore by a UV (365 nm)-dark-green (530 nm)-dark irradiation cycle. The moving average of the ion current (gray) is shown in black, which was generated by a Savitzky-Golay filter. c. Magnified view of the gray box in b. The decrease in current at each step is due to isomerization of the photoswitch. de. In the dark state, the ion current was stable. Under continuous irradiation at 530 nm, the current fluctuated because the photopore frequently switched between the (7E, 0Z) state and the adjacent (6E, 1Z) state. f. Optical control of ion flow passing through a collection of photopores. g. ON-OFF switching of >2000 photopores at -100mV. The number of pores was estimated assuming each pore contributes 50pA in PSS365. h. ON-OFF switching of photopore clusters by Nernst potential generated by asymmetric KCl concentration across the membrane, without external transmembrane potential application. i. Gating efficiency of single, double, and ensemble pores with and without 355nm and 532nm inline filters (n=3, standard deviation shown as error bars). Current traces were recorded at -100mV and filtered using a 20Hz Bessel filter. Recording conditions: 2MKCl, 10mMTris-HCl, 0.1mMEDTA, pH 8.5, 24±1℃.

[0273] Figure 3 | Diode characteristics of photopores. a. Current-voltage (IV) characterization of a single (E111C-pzMe)7 photopore under 365 nm or 530 nm irradiation revealed diode behavior at negative potentials (0 to -200 mV) and resistor behavior at positive potentials (0 to +200 mV). 95% confidence intervals (n=3) are shown in the shaded area. b. Wavelength-dependent IV characteristics of the (E111C-pzMe)7 photopore group are due to the seven pzMe photoswitches within each photopore taking on different dominant combination states. Currents in each IV graph are normalized assuming each pore contributes 50 pA at +100 mV. The number of pores ranges from 60 to 904 (n=7). c. Percentage of pzMe photoswitches present in the Z configuration in the photosteady state, calculated at wavelengths of 365 nm, 405 nm, 455 nm, or 530 nm. d. Voltage and wavelength dependence of gating efficiency (GP = 1 - IOFF / ION). e. Rectification was hypothesized to be due to the reverse orientation of the pzMe photoswitch along the pore axis in the reverse electroosmotic flow determined by the polarity of the through-film potential (see Supplementary Figure XX). f. Voltage dependence of the rectification ratio of one or more (E111C-pzMe)7 photopores. The rectification ratio was defined as the ratio of the absolute currents at +100mV and -100mV (n=3, standard deviation shown as error bars). g. Current passing through the (E111C-pzMe)7 photopore group in response to the applied AC potential. h. Truth table of photopore logic.

[0274] Figure 4 | Effects of light intensity and wavelength on photopore conductance. a. The transition rate from the low-conductance state to the high-conductance state of (E111C-pzMe)7 pores decreased as the light intensity (Q%) decreased. The drifted baseline resulted from the gradual depletion of AgCl on the electrodes when the current was recorded on an nA scale for several minutes. b. After 228 photopores reached a fully ON (ION=-11.4nA) or OFF (IOFF=-1.8nA) equilibrium state under continuous irradiation at 530nm (green) or 365nm (purple), no further change in conductance levels occurred even when the light intensity was reduced from the maximum LED output (Q%=100%). c. The equilibrium current level was determined by the irradiation wavelength; irradiation at 530 nm (green) resulted in a completely OFF state, irradiation at 365 nm (purple) resulted in a completely ON state, and intermediate current levels were achieved at 405 nm (purple) and 455 nm (blue). d. By irradiating with mixed wavelengths of 365 nm and 455 nm light at various ratios at 3-minute intervals, the PSS of the photopore group could be fine-tuned, and an intermediate equilibrium current state could be accessed. Current traces were recorded at -100 mV and filtered using a 20 Hz Bessel filter. Recording conditions: 2 MKCl, 10 mM Tris-HCl, 0.1 mM MEDTA, pH 8.5, 24 ± 1 °C.

[0275] Figure 5 | Remote signal transmission using photopores. a. An image or text message was coded as an optical sequence, and an ion current output was generated through a (E111C-pzMe)7 photopore. Using a provided key, the ion current data was deconvoluted and the original message was reconstructed. b. Transmission of a binary pixel pattern. A representative segment of the output trace is shown along with the binary current levels. c. Transmission of a 3-state pixel art converted to a 1D optical sequence. The pixel diagram was generated from PDB:7AHL. d. Transmission of a 3-state Morse code. The blue boxes are the Morse code decoded from the current trace, and the purple boxes are the translated text. The optical sequence was input at a rate of 20s per bit at wavelengths of 365nm, 455nm, or a mixed wavelength of 365 / 455nm, and a 3-level current pattern was generated in the photopore group.

[0276] Example 2 - ON-OFF nanopore for optical control of transmembrane ion communications overview Nanoscale photoswitchable proteins could facilitate precise spatiotemporal control of transmembrane communications and support research in synthetic biology, neuroscience, and bioelectronics. By covalently modifying α-hemolysin protein pores with arylazopyrazole photoswitches, we fabricated "photopores" that transition between iontronic resistance mode and diode mode in response to orthogonal wavelength irradiation. In diode mode, low-leakage OFF-state nanopores exhibit a reversible increase of more than 20-fold in unit conductance upon 365 nm irradiation. In the diode-state photopores, a rectification ratio > 5 was achieved with either DC or AC voltage input. Unlike conventional electronic phototransistors with intensity-dependent photoelectric responses, the photopores regulated current output based solely on the wavelength of monochromatic or bi-wavelength irradiation. Bi-wavelength irradiation at various relative intensities allowed for gradual tuning of the photopore's conductance. By utilizing these properties, photon signals can be converted into ion signals, highlighting the potential applications of photopores as components of smart devices in synthetic biology.

[0277] Introduction Remote control of transmembrane ion communications can be facilitated by various molecular tools that respond to external stimuli (e.g., light [1-5], temperature [6], magnetic field [7] or electric field [8]). Among these, photoactivated devices with photoresponsive functional groups are attracting attention, as they utilize optical input to enable spatiotemporal regulation of ion signaling across membranes [9-11].

[0278] In top-down synthetic biology, photoswitchable chromophore-containing nanodevices can be used to regulate ion flow across cell membranes. For example, native light-driven ion transporter proteins such as microbial rhodopsin[12,13] generate or dissipate electrochemical gradients via chromophores such as all-trans retinal. This makes it possible to regulate the electrical activity of cells into which rhodopsin expression has been introduced, enabling manipulation of fundamental biological activities such as calcium signaling[14,15] and neuronal firing[16,17]. These optogenetic tools can also function as integral parts of miniaturized bioelectronic systems[18,19]. Light control is also used for ionotropic glutamate receptors

[20] , transient receptor potential channels

[21] , and voltage-dependent K + Channel or Ca 2+ By using synthetic photoisomerized allosteric ligands that regulate the opening (ON) and closing (OFF) of ligand-dependent receptors such as channel[5,22], these ligands can also be introduced into natural ion channels.

[0279] In bottom-up synthetic biology, there is a strong demand for photoresponsive devices that offer modular design, ease of assembly, immediate and reversible ON-OFF responses, sustained ON and complete OFF states, and the potential to respond to additional input stimuli, in order to modulate transmembrane ion signaling. While fully synthetic systems offer a wide range of designs and applications, they face the challenge of achieving efficient, real-time, and photo-driven ion transport across membranes. For example, DNA nanopores with photoisomerizable cap strands were optically activated or deactivated before insertion into the lipid bilayer

[23] . Synthetic ion transporters with built-in photoswitches transported ions one at a time along transmembrane gradients [24,25]. Semi-synthetic approaches are also being pursued to leverage the innate ON-OFF mechanisms of protein channels. Chemical engineering of proteins with photoswitches has enabled transient photoactivated openings of mechanosensitive channels and time-controlled, irreversible formation of protein nanopores [26,27]. Novel designs are needed to achieve sustained activation and reversible ion responses to light.

[0280] This specification describes a reversible ON-OFF photopore that exhibits a long aperture lifetime in the ON state and a low background current in the OFF state. The ON-OFF mechanism is incorporated into a protein structure that has not evolved to open and close, and can be activated in real time when embedded in a membrane. This was achieved by covalently modifying an α-hemolysin (αHL) monomer with an arylazopyrazole that exhibits nearly quantitative photoswitching between E and Z isomers and high thermal stability in both states. The photopore was characterized at both the single-molecule and ensemble levels, and its ability to tolerate or limit transmembrane ionic current in response to light was demonstrated. Depending on the irradiation wavelength, the pore exhibits behavior similar to both an electron diode (i.e., unidirectional current flow) and a phototunable resistor (i.e., current regulation). The properties of the photopore have been utilized to generate the conversion of light to ionic signals.

[0281] Results and Discussion Construction of photoreversible nanopores To construct the photopore, we functionalized the heptameric protein nanopore α-hemolysin (αHL) with a chemical group that can be photoisomerized within the channel (Figure 9 / Example 2 Figure 1). αHL monomers containing a single cysteine ​​residue at positions 111, 115, 125, or 129 were individually modified using thiol-specific chemistry with one of three photoswitches (o-fluoroazobenzene (fAzo)

[28] , arylazopyrazole (pzH)

[29] , or methylarylazopyrazole (pzMe)

[30] ) (Figures 9a, b, 14 and 15 / Example 2 Figure 1a, b, Supplementary Figures 1 and 2). When assembled into a heptamer, the side chains of these residues protrude into the lumen of a transmembrane β-barrel

[31] . Modification of the αHL monomer (Figure 16 / Example 2 Supplementary Figure 3) with one molecule of either fAzo, pzH, or pzMe was confirmed by liquid chromatography-mass spectrometry (LC-MS) (Figures 9c, 17, and 18 / Example 2 Figure 1c, Supplementary Figures 4 and 5). These photoswitch-modified monomers were introduced into a synthetic lipid bilayer consisting of 1,2-difitanoyl-sn-glycero-3-phosphocholine (DPhPC) or self-assembled in the presence of sodium deoxycholate to form a homoheptamaer pore (Figure 9a / Example 2 Figure 1a).

[0282] fAzo, pzH, and pzMe photoswitches have been reported to exhibit high E / Z or Z / E isomer ratios in the photosteady state (PSS) and thermal stability in the Z state. [28,29] Bromoacetamide derivatives of pzMe (Br-pzMe), determined by ultraviolet / visible (UV / Vis) and proton nuclear magnetic resonance (1H NMR) spectroscopy, demonstrated the highest isomer ratios in PSSs generated by "orthogonal" wavelengths (Figure 9d, e / Example 2 Figure 1d, e). Upon 365 nm LED irradiation, Br-pzMe was mainly converted to the Z isomer (PSS365 = 96% Z), while upon 530 nm irradiation, Br-pzMe existed mainly as the E isomer (PSS530 = 93% E) (Figure 9e and 19 / Example 2 Figure 1e and Supplementary Figure 6). In contrast, Br-fAzo was 85% E-type in PSS405 and 91% Z-type in PSS530, while Br-pzH was 65% E-type in PSS365 and 85% Z-type in PSS530 (Figure 19 / Example 2 Supplementary Figure 6). It was expected that a higher isomer ratio would lead to more precise control of ion communication through the modified nanopores.

[0283] Reversible ON-OFF Photopore The photoreversible control of ionic currents passing through αHL nanopores containing fAzo, pzH, or pzMe photoswitches was first characterized by ensemble electrical recording in planar lipid bilayers (PLBs), i.e., bilayers containing hundreds or thousands of pores. For electrical recording under a wide range of illumination intensities from the ground side (transformer) of the bilayer, a custom poly(methyl methacrylate) (PMMA) chamber with collimated fiber-coupled LEDs was fabricated (Figures 9f and 20 / Example 2, Figure 1f and Supplementary Figure 7). To conform to the convention of reporting currents passing through αHL pores, a positive current was defined as the current in which positive ions move through the bilayer from the transformer side to the cis side of the pore (i.e., from the bottom of the barrel towards the antler). The transmembrane potential (Vm) is expressed as the transformer side potential relative to the cis side.

[0284] Interconversion between high current levels (ION) and low current levels (IOFF) was recorded at +100mV in pores with fAzo, pzH, or pzMe under alternating wavelengths (365nm / 530nm for pzH and pzMe, and 405nm / 530nm for fAzo) (Figures 10a, 14, and 15 / Example 2 Figure 2a and Supplementary Figures 1, 2). The low current state is referred to as the OFF state, where the ion flow is partially or almost completely restricted compared to the ON state, which is the highest current level obtainable. Generally, photoreversible nanopores switch to the ON state after irradiation at shorter wavelengths (Z-state photoswitch) and switch to the OFF state after irradiation at longer wavelengths (E-state photoswitch).

[0285] Of the homoheptamers modified at position 111 with fAzo, pzH, or pzMe, (E111C-pzMe)7 exhibited the highest gate current rate [Igate%=(1-IOFF / ION)×100%] of 85% at the ensemble level, while the other two photoswitches produced lower Igate% values ​​of approximately 20% (~20%) (Figure 15 / Example 2 Supplementary Figure 2). A decrease in Igate% was recorded when the pzMe modification was moved to positions 115, 125, or 129. Without further investigation of the mechanisms underlying the efficiency of position and photoswitch-dependent current gating, we proceeded to characterizing the (E111C-pzMe)7 photopore and considering its applications.

[0286] We investigated the photochemistry underlying the ON-OFF behavior of (E111C-pzMe)7 photopores at the single-channel level (Figures 2b-f). Sequential insertion of pre-formed heptamers pre-irradiated at 365 nm revealed that the ON-state conductance of individual photopores ranged from 0.12 to 1.27 nS. The average conductance of the pores (0.62 ± 0.30, N=42 pores) was one-third that of wild-type αHL (1.81 ± 0.17 nS, N=17 pores) (Figures 21-22 / Example 2 Supplementary Figures 8-9). Nevertheless, a consistent 95 ± 2.5% (N=21) of I was observed across all observed (E111C-pzMe)7 photopores at the single-channel level.gate% This was recorded (Figures 23-24 / Example 2 Supplementary Figures 10-11). By voltage clamping each individual photopore and repeating UV (365nm) → dark → green (530nm) → dark 10 times, reversible photogating was observed (Figures 10b and 25 / Example 2 Figure 2b and Supplementary Figure 12). Under UV irradiation, the photopore switched from ON to OFF with an average response time of 1.8 ± 0.8 s (N=9 transitions), and under green light, the transition from OFF to ON occurred within an average of 31.4 ± 7.7 s (N=9 transitions). LED irradiance value (365nm: 36.2 mW / cm 2 530nm: 30.3mW / cm 2 The result was obtained by measuring the light intensity in microwatts through a 300 μm diameter pinhole instead of a bilayer (see Figure 20 / Example 2, Supplementary Figure 7).

[0287] I ON and I OFFThe intermediate current levels between the two states are thought to have been generated by the stepwise isomerization of individual pzMe molecules (Figure 10c / Example 2 Figure 2c). Based on the number of pzMe switches in either the E or Z state within the photopore, the ON-state photopores were arranged as (0E, 7Z), the fully OFF-state photopores as (7E, 0Z), and the partially OFF pores as one of the ranges from (1E, 6Z) to (6E, 1Z) (Figure 10b / Example 2 Figure 2b). Notably, the intermediate current levels recorded during ON-to-OFF or OFF-to-ON transitions within a single photopore differed for each transition (Figure 26 / Example 2 Supplementary Figure 13). For example, during the stepwise decrease of current recorded through (E111C-pzMe)7 photopores under 530 nm irradiation, more than 10 different current levels were recorded across multiple transitions (Figure 26a / Example 2 Supplementary Figure 13a). The complexity stemmed from both the possible combinations of E / Z configurations of the seven photoswitches

[32] and the structure of the photoswitches. For example, in the (5E,2Z) state, there are three possible configurations of the two pzMe photoswitches in Z configuration around the central axis of the pore (Figure 26b / Example 2 Supplementary Figure 13b). Once a single photopore reached its final ON or OFF state, the current level fluctuated under continuous irradiation but remained relatively stable in the dark (Figures 10d-f / Example 2 Figures 2d-f). This was expected, as continuous irradiation drives the reversible isomerization of the photoswitches within the (E111C-pzMe)7 photopore: for example, switching between the (0E,7Z) and (1E,6Z) states at 365 nm and between the (7E,0Z) and (6E,1Z) states at 530 nm. In the dark, the (E111C-pzMe)7 photopore was fixed in a single configuration, and no thermal relaxation from Z to E was observed for at least one hour after irradiation was stopped (Figure 27 / Example 2 Supplementary Figure 14).

[0288] The behavior of (E111C-pzMe)7 photopores at the ensemble level was compared with the behavior at the single-channel level (Figure 10g / Example 2, Figure 2g). In the case of multiple pores, I gate%The value was found to be ~10% lower compared to the case of a single pore. This is thought to be due to the presence of a photopore in the partially ON configuration, as predicted in the single-channel observation (Figure 28 / Example 2 Supplementary Figure 15). Even when the LED bandwidth was reduced to 10 nm by incorporating an inline bandpass filter, I gate% The values ​​did not improve. By creating an asymmetric KCl concentration across the membrane and establishing a physiologically relevant membrane potential (Figure 29 / Example 2 Supplementary Figure 16), the photoswitching behavior was reproduced even without applying an external transmembrane potential (Figure 10h / Example 2 Figure 2h).

[0289] (E111C-pzMe)7 photopore switchable iontronic diode characteristics The ON-OFF behavior of the (E111C-pzMe)7 photopore was further demonstrated by widening the applied potential range and increasing the voltage in 10mV increments from -200mV to +200mV. After reaching the ON state at 365nm or the OFF state at 530nm, we characterized the current-voltage (IV) behavior of a single (E111C-pzMe)7 photopore in the dark (Figure 11a / Example 2 Figure 3a). After irradiation at 530nm, the photopore exhibited a diode-like nonlinear IV response; at positive potentials (0 to +200mV, Vm applied to the transformer side, Figure 9f / Example 2 Figure 1f), there was almost no current passing through the pore, but at negative potentials, the current behaved like a resistor (G=I / V=0.32±0.11nS at -100mV, N=3 pores) (Figure 11a / Example 2 Figure 3a). After irradiation at 365 nm, a less pronounced rectification was observed (G=I / V=0.46±0.06 nS at +100 mV and 0.58±0.12 nS at -100 mV, N=3 pores) (Figure 11a / Example 2 Figure 3a). In other words, the photopore functioned as a photoresistor after irradiation at 365 nm and as an iontronic diode after irradiation at 530 nm. We hypothesize that the voltage-dependent behavior of the (E111C-pzMe)7 photopore is due to the biased orientation of the photoswitches confined within the asymmetric nanopore. The orientation of these charge-neutral photoswitches may be influenced by electroosmotic flow, which is dependent on the direction of the applied voltage. Depending on their orientation, the seven photoswitches may adopt various configurations facilitated by the surrounding protein environment, thereby altering the ion flow.

[0290] The dual-mode functionality (i.e., diode and photoresistor) demonstrated in a single pore was reproduced in ensemble experiments (N=8 experiments, 100 to 600 pores per experiment) using several hundred pores under irradiation at 365 nm and 530 nm (Figure 11b / Example 2, Figure 3b). Irradiation at wavelengths of 405 nm and 455 nm was also tested. At these wavelengths, current levels between those generated at 365 nm and 530 nm were produced at all applied potentials (Figure 11b / Example 2, Figure 3b). To illustrate the voltage-dependent behavior in the ensemble, I gate% is, I gate% =(1-I OFF / I ON It was calculated according to ) × 100%. Here, the current level after 365nm irradiation is I ON The current level after 405nm, 455nm, or 530nm is I OFF (That is, I gate%(405nm) =(1-I 405 / I 365 ) × 100%) (Figure 11c / Example 2 Figure 3c). Between -150mV and +100mV, at all three wavelengths, I increases with decreasing negative potential and increasing positive potential. gate% It increased. From +100mV to +200mV, I gate% It reached a plateau at ~85% at 530nm, ~80% at 455nm, and ~60% at 405nm (Figure 11c / Example 2 Figure 3c). Under negative potential, as it approached -200mV, I gate% The PSS ratio plateaued at ~20% at 405nm and 455nm, and at ~35% at 530nm (Figure 11c / Example 2 Figure 3c). The difference in PSS ratio related to the irradiation wavelength was I gate% This was the main cause of the wavelength dependence. Furthermore, the voltage dependence is thought to be due to the relative occupancy of the strongly rectified (7E, 0Z) state. The rectification ratio (RR=I) of (E111C-pzMe)7 - / I +The ratio of currents recorded under reverse polarity at the same potential changed almost linearly with respect to the amplitude of the applied potential up to ±150mV (Figure 11d / Figure 3d of Example 2), and in the diode mode after irradiating at 530nm, it was 5.5±0.7 at ±100mV (Figure 11d / Figure 3d of Example 2).

[0291] Nanopore diodes can be used in soft devices as half-wave rectifiers

[33] to convert AC to DC. We tested photopores as switchable iontronic diodes and generated a unidirectional current across a composite bilayer under an AC voltage of 0.03 Hz (Figure 11e / Example 2 Figure 3e). After irradiation at 530 nm, an ensemble of ~900 (E111C-pzMe)7 photopores functioned as a half-wave rectifier, conducting current only under negative potential. After irradiation at 365 nm, the current flow reproduced the sinusoidal shape of the AC voltage input, but the rectification was weaker. At higher input frequencies, the capacitive current generated by the bilayer exceeds the resistive current through the pore, reducing the rectification. Thus, the ion current output through the (E111C-pzMe)7 photopore is determined by both wavelength and applied potential and resembles a NAND Boolean function. This NAND logic gate utilizes the switchable diode characteristics of an (E111C-pzMe)7 photopore with an irradiation wavelength of input 1 (0=365nm, 1=530nm) and an applied potential of input 2 (0=-100mV, 1=+100mV). After irradiation at 530nm, a low current output is generated only when +100mV is applied (Figure 11f / Example 2, Figure 3f).

[0292] (E111C-pzMe)7 photopore for light-to-ion signal conversion The photoisomerization rate of a photoswitch is typically proportional to the light intensity [5,34,35]. Therefore, light at 365 nm is proportional to 36.2, 28.2, and 13.4 mW / cm². 2When more than 600 (E111C-pzMe) 7-pores were irradiated (Figure 20 / Example 2 Supplementary Figure 7), the duration of the OFF-ON transition increased from 5.8±0.5s to 7.1±0.6s and then to 11.4±2.6s (N=4 ensemble, Figure 12a / Example 2 Figure 4a). After reaching an equilibrium current level under irradiation at a specific wavelength, reducing the light intensity did not result in any change to the ensemble's current level (Figure 12b / Example 2 Figure 4b). In contrast, common electronic components and optogenetic tools often exhibit transient current responses that change with light intensity.

[0293] By switching off the irradiation immediately after reaching the desired current level during the ON-OFF transition at a specific wavelength, I ON and I OFF An ion signal can be realized by conducting through multiple (E111C-pzMe)7 photopores at an intermediate current level between these two wavelengths. However, it was difficult to reliably capture the transient current level of interest during fast ON-OFF transitions. Another strategy was devised using multicolor light to generate the equilibrium current level determined by the PSS of the photoswitch (Figure 12c, d / Example 2 Figure 4c, d). To investigate whether mixed wavelength irradiation could yield an equilibrium current level that could not be achieved with continuous irradiation at either wavelength, light from 365 nm and 455 nm LEDs was combined via a fiber coupler. Gradually adjusting the ion signal was demonstrated by changing the relative intensity of the 365 nm and 455 nm light irradiating multiple (E111C-pzMe)7 pores every 3 minutes. An ion signal level was obtained between the ion signal levels recorded by PSS365 and PSS455 (Figure 12d / Example 2 Figure 4d). This approach allows for finer control over the ion current passing through the (E111C-pzMe)7 photopore, gradually adjusting it as needed.

[0294] Inspired by the gradual thermal relaxation of arylazopyrazoles and the subsequent gradual modulation of ion signals, we proposed the conversion of light to ion signals using an ensemble of iontronic photopores. First, we encrypted binary data carrying information within a light sequence that alternately irradiated with 365 nm and 455 nm radiation (Figure 13a / Example 2 Figure 5a). The (E111C-pzMe)7 photopore, acting as a light-to-current converter, translated the light sequence into a two-level current pattern, as shown in a representative segment of the trace with an input rate of 20 s / bit (Figure 13b / Example 2 Figure 5b) (Figure 13a / Example 2 Figure 5a). With increasing light intensity, higher input rates were achieved, reaching 1.5 s / bit at 365 nm and 1.9 s / bit at 455 nm when using the maximum LED output. No photobleaching was observed in the 90-minute experiment. By associating the ON state with black pixels and the OFF state with white pixels, pixel art spelling "OXFORD" was reconstructed from the binary optical message. Furthermore, at 365nm (36.2mW / cm²), 2 ) and 455nm (56.3mW / cm²) 2 By applying mixed irradiation in both cases, the alphabet was extended to a 3-state system and applied to the pixel art representation of protein nanopores and the demodulation of text messages written in Morse code (Figure 13c, d / Example 2 Figure 5c, d). In principle, by using mixed wavelength irradiation, the (E111C-pzMe)7 photopore can output a range of current levels between the levels of PSS365 and PSS530, as the basis for a complex coding system. In our apparatus, after a 20Hz digital filter, the variation in current levels in the dark is ±0.3%, and I gate% The range was 0-85%. Therefore, the estimated upper limit of the number of assignable levels is ~300.

[0295] conclusion In this study, we constructed protein nanopores whose conductance values ​​change under remote optical control. Among the photopores tested, we achieved quantitative, sustained, and reversible control of ion current in (E111C-pzMe)7 pores after irradiation at 365 nm (ON, high conductance) and 530 nm (OFF, low conductance). In particular, switching an (E111C-pzMe)7 pore from the ON state to OFF at +100 mV resulted in a >95% decrease in single conductance. Similarly, ensemble experiments using multiple (E111C-pzMe)7 pores also showed a >85% decrease in current. This sustained ON state and near-complete OFF state offer significant advantages for real-time control of transmembrane ion communications in nanotechnology and synthetic biology applications.

[0296] The photopore described herein differs from the photo-controlled nanopores described previously in two ways. First, the photoswitch reversibly controls the conductance of the fully assembled nanopore, rather than being an irreversible nanopore assembly process

[27] . Second, the current-voltage characteristics of (E111C-pzMe)7 exhibited two switchable states: a resistor-like ON state after irradiation at 365 nm and a diode-like OFF state after irradiation at 530 nm. In diode mode, there is no delay in rectification of (E111C-pzMe)7 after reversing the polarity of the applied potential, in contrast to the previous (non-photoswitchable) arginine-rich αHL diode

[33] .

[0297] One potential impact of photopores in the field of nanoscale iontronics may lie in the signaling of light to ionic current. In the case of (E111C-pzMe)7 pores, we demonstrated the ability to reconstruct images and text encoded in light pulses by leveraging their ability to generate multiple output current levels, such as a three-state system (blue, mixed, and UV). Thus, by arranging photo-tunable nanopores in a 2D array of lipid bilayers, it may be possible to create artificial retinas that detect color images. In our previous work on droplet hydrogel bilayer biopixels, we reported a relevant approach for monochromatic 530 nm light

[36] .

[0298] Photopores can also be incorporated into 3D-printed droplet-based synthetic tissues where spatiotemporal control of intercompartmental communication is key to controlling tissue-like behavior [37,38]. There, they can be used, among other things, to induce the release of ions or small molecules

[38] , mediate shape changes

[37] , or generate motion

[39] . Furthermore, we anticipate developing additional photopores with the conjugation protocols reported herein to gain control over additional properties such as selectivity in small molecule transport and the ability to mediate biopolymer translocations.

[0299] method General information Unless otherwise specified, all chemicals were purchased from Merck and used as is. Br-pzMe, Br-fAzo, and Br-pzH were prepared (see "Synthesis and Characterization of Photoswitches") and used as 3 mM DMSO stock solutions for bioconjugation experiments. See the Synthesis section for details of the synthesis. Fiber-optic coupled LEDs were purchased from Thorlabs (M405FP1, M455F3, and M530F2). Fiber optics (1000 μm diameter, 0.39 NA) were supplied by Mightex. LC-MS of αHL monomers was recorded on a benchtop QTOF mass spectrometer (Waters Xevo G2-XS) equipped with a ProSwift RP-2H column (ThermoFisher, linear gradient acetonitrile / water, 0.1% (v / v) TFA, 10 min), and the results were deconvoluted using MassLynx software (Waters). The Arduino UNO microcontroller was programmed to trigger and record the timing of irradiation. Molecular modeling was performed using the open-source PyMol from GitHub (Schrodinger).

[0300] Preparation of photopore monomers Site-directed mutagenesis of αHL cysteine ​​mutant Genes encoding αHL mutants with a single cysteine ​​residue at positions E111, T115, T125, or T129 were prepared from the pT7-αHL-D8H6 plasmid by site-directed mutagenesis (QuikChange II XL, Agilent) using primers listed in Table S1. The template plasmid had been previously reported

[40] , and in this plasmid, octaaspartate and hexahistidine tags (-D8H6) were designed for anion exchange (IEX) and Ni-NTA purification. Polymerase chain reaction was initiated by mixing 5 μL of 10× reaction buffer, 10 ng of DNA template, 125 ng of forward and reverse primers, 1 μL of dNTP mix, 3 μL of QuikSolution, 2.5 U of PfuUltra HF DNA polymerase, and nuclease-free water to a final volume of 50 μL. After thermal cycling (1×95°C:1 min → 18×95°C:50 s, 60°C:50 s, 68°C:5 min → 1×68°C:7 min), 1 μL of DpnI (10 U) was added, and the mixture was incubated at 37°C for 1 hour to digest the template DNA. E. coli transformation was performed by incubating 2 μL of the digested PCR product with 45 μL of XL-10 Gold competent cells (Agilent) on ice, followed by a heat shock at 42°C for 30 s. Subsequently, carbenicillin (50 μg mL) was added. -1 Transformed cells were inoculated into Luria-Bertani (LB) agar plates containing Merck. All mutants were identified by DNA sequencing.

[0301] Expression and purification of αHL cysteine ​​mutant BL21(DE3)pLysS E. coli cells (Agilent) were transformed with a mutant plasmid, and antibiotics (carbenicillin 50 μg mL) were administered. -1 Chloramphenicol 34 μg mL -1The cells were inoculated onto LB agar plates containing the antibiotic. Single colonies were taken from the plates and inoculated into 10 mL of LB for pre-culture. 4 mL of the pre-culture, which had been cultured overnight, was inoculated into 400 mL of LB medium containing the same antibiotic. This expression culture was shaken at 250 rpm at 37°C for approximately 3 hours, and when the OD reached 0.6, it was cooled to 18°C ​​and 2 mL of 0.1 M IPTG (Fluorochem) was added to induce protein expression. The culture was further shaken at 200 rpm at 18°C ​​overnight. The cells were collected by centrifugation at 5000 rpm at 4°C for 20 minutes using a Beckman J25 centrifuge and resuspended in 10 mL of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, 5% glycerol, 2 mM TCEP containing EDTA-free protease inhibitor tablets (ThermoFisher)). Subsequently, 250 μL of 40 mg / mL lysozyme (ThermoFisher), 2 μL of 250 U / mL universal nuclease (ThermoFisher), and 25 μL of 2M MgCl2 were added, and the lysate was dissolved by incubation on ice for 1 hour. The lysate was sonicated on ice for 3 minutes at 40% amplitude with a 30-second ON-30-second OFF pulse train (VCX 500, Sonics). The supernatant was clarified by centrifugation at 29000 × g for 45 minutes at 4°C and transferred to a gravity column containing 1 mL (bed volume) of Ni-NTA resin (ThermoFisher). The lysate supernatant and resin mixture were mixed on a platform rotator at 4°C for 1 hour. The column was washed twice with 15 mL of washing buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 20 mM imidazole, 2 mM TCEP, 0.1% Triton X-100, and 5% glycerol), and then eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole, 2 mM TCEP, 0.1% Triton X-100, and 5% glycerol).The fraction containing αHL (~10 mL) was mixed and loaded onto a HiLoad 26 / 600 Superdex 200 pg (Cytiva) SEC column equilibrated at 4°C with SEC buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM TCEP, and 5% glycerol). The fraction containing the αHL cysteine ​​mutant monomer was concentrated to 1 mg / mL, aliquoted, and stored at -80°C. The monomer mass was confirmed by LC-MS. On average, 9 mg of pure αHL-D8H6 monomer was obtained from 400 mL of culture medium after SEC purification.

[0302] Photoswitch conjugation Br-pzMe, Br-pzH, or Br-fAzo (photoswitch) was dissolved in 3 mM dimethyl sulfoxide (DMSO). The purified αHL cysteine ​​mutant monomer was buffer-exchanged and diluted to 0.5 mg / mL with low TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). The photoswitch was added stepwise to the monomer solution over 18 hours to a final concentration of 750 μM. The mixture was incubated at 20°C and cooled to 4°C before removing excess photoswitch. The reaction was evaluated by LC-MS. The conjugate protein solution was passed through a desalted spin column (ThermoFisher) equilibrated with low TE buffer to remove excess photoswitch, and the eluate was stored at -80°C.

[0303] Formation and purification of photopore homoheptamers Photopore monomers were concentrated to ~8 mg / mL using a PES protein concentrator (ThermoFisher) and then replaced with low TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) using a desalting spin column. Heptamerization was performed by adding 10% (w / v) sodium deoxycholate solution to the concentrated protein solution to a final concentration of 6.25 mM. The mixture was incubated at 22°C for 1 hour. To separate the oligomerized photopore from the remaining monomers, the mixture was loaded onto a membrane-based strong cation exchange spin column (ThermoFisher) and equilibrated at 20 mM MOPS, pH 7.5. The samples were eluted with the same buffers containing NaCl (0.2, 0.3, 0.4, 0.6, 2 M) (Figure 21b / Example 2 Supplementary Figure 8b). Homoheptamers were eluted with ~0.3 M NaCl, and the fractions were combined and frozen at -80°C. Before electrical recording, the MOPS buffer was replaced with a low-TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA).

[0304] Electrical recording in planar lipid bilayers Custom Recording Chamber The recording experiments on planar lipid bilayers were conducted in a custom-made poly(methyl methacrylate) (PMMA) recording chamber. The chamber was equipped with an LED light source via an adjustable FiberPort collimator (Thorlabs), enabling precise alignment and direct illumination of the Teflon film (100 μm diameter) aperture. Before recording began, the collimator was adjusted to ensure uniform illumination of maximum light intensity through a 3 mm thick sapphire window (Thorlabs) to the aperture. Fiber-coupled LEDs of various wavelengths (365, 405, 455, 530 nm, Thorlabs) were connected to the collimator via UV-compatible multimode fibers (0.39 NA, 1000 μm diameter, Mightex). In experiments using mixed wavelengths, a branched fiber bundle (0.39 NA, 1000 μm diameter, Thorlabs) was connected to two LEDs. In the data shown in Figure 10g / Example 2, bandpass filters (Thorlabs) were used to achieve narrow bandwidths of 10nm at 355nm and 4nm at 532nm (Figure 23c / Example 2 Supplementary Figure 10c). These filters were mounted on an integrated in-line reflective fiber optic filter (Thorlabs).

[0305] Single-channel and ensemble experiments The Teflon opening of the recording chamber was pre-treated with hexadecane (1% in pentane, ~5 μL). After 10 minutes, recording buffer (1 mL, 10 mM Tris-HCl pH 8.5, 2 M KCl, 0.1 mM EDTA) was added to both the cis-compartment and trans-compartment. A lipid bilayer was formed on the opening by adding 1,2-difitanoyl-3-sn-phosphatidylcholine (DPhPC, Avanti Polar Lipids) to each compartment. Ag / AgCl electrode pairs were placed in each compartment via a salt bridge (3 M KCl in 2% (w / v) agarose). The electrodes were covered with black tape to prevent exposure to light from the LED.

[0306] To insert a single pore into the bilayer (Figure 23a / Supplementary Figure 10a of Example 2), homoheptamer (0.1 μL, 0.2 mg / mL) was added to the cis-compartment. Once the pore was inserted and showed a photoresponse, the buffer was perfused five times to prevent further insertion. To achieve stepwise insertion of multiple pores, an additional homoheptamer (10 μL, 0.2 mg / mL) was used (Figure 22a / Supplementary Figure 9a of Example 2).

[0307] The characterization of the photopore ensemble was recorded using photopore monomers that had been pre-irradiated at 530 nm for 30 minutes before use to ensure the reproducibility of pore insertion (Figure 23c / Example 2 Supplementary Figure 10c). Monomers (~5 μL, 0.2~0.5 mg / mL) were added to the cis compartment while a 530 nm LED was lit. Pore insertion occurred under an applied AC potential of ±20 mV, and the insertion rate was slowly reduced over 15 minutes. Excess monomer was removed by perfusing the cis compartment.

[0308] Ionic currents were recorded via Ag / AgCl electrodes connected to a patch-clamp amplifier (Axopatch 200B, Axon Instruments). The signals were filtered using an in-line 4-pole low-pass Bessel filter (80 dB / decade, 5 kHz). A Digidata 1322A digitizer (Molecular Devices) was used to convert the analog signals to digital format. The data were analyzed using the pCLAMP 10.3 software suite (Molecular Devices). Electrical traces were plotted using Python (3.8.8), Pyabf (2.3.5), Matplotlib (3.3.4), and Seaborn (0.11.1).

[0309] (E111C-pzMe)7 transmittance ratio (P K+ / P Cl- ) and electroosmotic flow The current-voltage response of (E111C-pzMe)7 was measured by varying the applied potential from -200mV to +200mV in 10mV increments using an asymmetric KCl gradient (2M KCl(cis) / 0.2M KCl(trans)) (Figure 29 / Example 2 Supplementary Figure 16). Permeability ratio (P K+ / P Cl- The potential at which the current becomes zero (reversal potential, Vr) was calculated using the Goldman-Hodgkin-Katz (GHK) equation

[41] :

[0310]

number

[0311] Here, F is the Faraday constant; R is the ideal gas constant; and T is 298K.

[0312] Electroosmotic flow (J) under symmetric KCl conditions EOF The size of ) is then determined from

[42] :

[0313]

number

[0314] Here P K+ / P Cl- The values ​​were 0.15±0.01 (530nm, N=4 repeats) and 0.20±0.01 (365nm, N=4 repeats).

[0315] Transfacing signal transmission by converting light to electric current. Three steps are required to achieve the signal conversion from light to electric current. First, the encrypted digital data, either as pixel art or text, was converted into a monochromatic or multichromatic 1D light sequence. For the 4x25 pixel pattern of "OXFORD," the pixel art plotted with Matplotlib was expanded into a 100x1 string of two states (black / white), which was then assigned to "365nm" / "455nm" to obtain the light sequence. For example, a list of colors ["black," "black," "white," ..., "black"] was converted into a light sequence ["365nm," "365nm," "455nm," ..., "365nm"].

[0316] In the αHL pixel art, a snapshot of the PDB:7AHL nanopore model was first converted into a 3-color 16x16 pixel image in Adobe Illustrator. The Python Imaging Library was used to extract the value of each pixel and generate a 16x16x3 array (where 3 represents the RGB color code). This array was expanded into a 256x3 array, and the RGB color codes were further replaced with wavelengths. For example, the RGB color code for white (0,0,0) was assigned to "mixed (365+455)nm". Assigning gray and dark gray to "455nm" and "365nm" generated a 256x1 optical sequence. Similarly, an 18-word Morse code sequence (dot / space / dash) was converted to the same three wavelengths to generate an optical sequence.

[0317] In the second step, a 1D optical sequence was sent to an Arduino UNO microcontroller, illuminating 365nm and 455nm LEDs accordingly. Due to the photopore switching speed, each illumination unit (bit) was optimized to last 20 seconds to observe the rectangular signal. Higher light intensity may result in higher data transmission rates. During the bit's lifetime, the LEDs were coupled via a branched fiber bundle and switched on or off according to the optical sequence, generating high (365nm), medium (mixed), or low (455nm) ion current responses via the photopore.

[0318] The final step was to process the electrical recordings for visualization and analysis of the output signals. The electrical recording traces were processed in reverse, from a 1×N current signal to a pixel image or text. Continuous recording traces sampled at 25kHz were divided into 20-second bits (1 bit = 0.04ms, 500,000 data points), and each bit was indicated as a "high," "medium," or "low" state based on its average value compared to a manually selected threshold. The state values ​​were further replaced with ("black" / "white" for the "OXFORD" pattern), ("dark gray" / "white" / "gray" for the αHL pixel art), or ("dot" / "blank" / "dash" for Morse code). Finally, the new arrays were converted to the appropriate dimensions and displayed using Matplotlib to visualize the image or text.

[0319] [Table S1]

[0320] References 1. Bozovic, O., Jankovic, B. & Hamm, P. Using azobenzene photocontrol to set proteins in motion. Nat. Rev. Chem. 6, 112-124 (2022). 2. Emiliani, V. et al. Optogenetics for light control of biological systems. Nat. Rev. Methods Primers 2, (2022). 3. Kramer, RH, Chambers, JJ & Trauner, D. Photochemical tools for remote control of ion channels in excitable cells. Nat. Chem. Biol. 1, 360-365 (2005). 4. Kocer, A., Walko, M., Meijberg, W. & Feringa, B. L. Chemistry: a light-actuated nanovalve derived from a channel protein. Science. 309, 755-758 (2005). 5. Banghart, M., Borges, K., Isacoff, E., Trauner, D. & Kramer, R. H. Light-activated ion channels for remote control of neuronal firing. Nat. Neurosci. 7, 1381-1386 (2004). 6. Vriens, J., Nilius, B. & Voets, T. Peripheral thermosensation in mammals. Nat. Rev. Neurosci. 15, 573-589 (2014). 7. Huang, H., Delikanli, S., Zeng, H., Ferkey, D. M. & Pralle, A. Remote control of ion channels and neurons through magnetic-field heating of nanoparticles. Nat. Nanotechnol. 5, 602-606 (2010). 8. Catterall, W. A. Ion channel voltage sensors: Structure, function, and pathophysiology. Neuron 67, 915-928 (2010). 9. Hirschi, S., Ward, T. R., Meier, W. P., Muller, D. J. & Fotiadis, D. Synthetic biology: bottom-up assembly of molecular systems. Chem. Rev. 122, 16294-16328 (2022). 10. Ankenbruck, N., Courtney, T., Naro, Y. & Deiters, A. Optochemical control of biological processes in cells and animals. Angew. Chem. Int. Ed. 57, 2768-2798 (2018). 11. SzymaNski, W., Yilmaz, D., Kocer, A. & Feringa, B. L. Bright ion channels and lipid bilayers. Acc. Chem. Res. 46, 2910-2923 (2013). 12. Govorunova, E. G., Sineshchekov, O. A., Li, H. & Spudich, J. L. Microbial rhodopsins: diversity, mechanisms, and optogenetic applications. Annu. Rev. Biochem. 86, 845-872 (2017). 13. Zhang, F. et al. The microbial opsin family of optogenetic tools. Cell 147, 1446-1457 (2011). 14. Maltan, L., Najjar, H., Tiffner, A. & Derler, I. Deciphering molecular mechanisms and intervening in physiological and pathophysiological processes of Ca 2+ signaling mechanisms using optogenetic tools. Cells vol. 10 (2021). 15. Nguyen, N. T. et al. CRAC channel-based optogenetics. Cell Calcium 75, 79-88 (2018). 16. Jiang, S., Wu, X., Rommelfanger, N. J., Ou, Z. & Hong, G. Shedding light on neurons: optical approaches for neuromodulation. Natl. Sci. Rev. 9, (2022). 17. Xu, X., Mee, T. & Jia, X. New era of optogenetics: from the central to peripheral nervous system. Crit. Rev. Biochem. Mol. Biol. 55, 1-16 (2020). 18. Nyns, E. C. A. et al. An automated hybrid bioelectronic system for autogenous restoration of sinus rhythm in atrial fibrillation. Sci. Transl. Med. 11, 1-12 (2019). 19. Tochitsky, I. et al. How azobenzene photoswitches restore visual responses to the blind retina. Neuron 92, 100-113 (2016). 20. Volgraf, M. et al. Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat. Chem. Biol. 2, 47-52 (2006). 21. Lam, P. Y. et al. TRPswitch - a step-function chemo-optogenetic ligand for the vertebrate trpa1 channel. J. Am. Chem. Soc. 142, 17457-17468 (2020). 22. Fehrentz, T. et al. Optical control of L-type Ca 2+ channels using a diltiazem photoswitch. Nat. Chem. Biol. 14, 764-767 (2018). 23. Offenbartl‐Stiegert, D., Rottensteiner, A., Dorey, A. & Howorka, S. A light‐triggered synthetic nanopore for controlling molecular transport across biological membranes. Angew. Chem. Int. Ed. 61, (2022). 24. Kerckhoffs, A., Bo, Z., Penty, S. E., Duarte, F. & Langton, M. J. Red-shifted tetra-ortho-halo-azobenzenes for photo-regulated transmembrane anion transport. Org. Biomol. Chem. 19, 9058-9067 (2021). 25. Johnson, T. G., Sadeghi-Kelishadi, A. & Langton, M. J. A Photo-responsive transmembrane anion transporter relay. J. Am. Chem. Soc. 144, 10455-10461 (2022). 26. Mutter, N. L., Volaric, J., Szymanski, W., Feringa, B. L. & Maglia, G. Reversible photocontrolled nanopore assembly. J. Am. Chem. Soc. 141, 14356-14363 (2019). 27. Chang, C., Niblack, B., Walker, B. & Bayley, H. A photogenerated pore-forming protein. Chem. Biol. 2, 391-400 (1995). 28. Bleger, D., Schwarz, J., Brouwer, A. M. & Hecht, S. O-fluoroazobenzenes as readily synthesized photoswitches offering nearly quantitative two-way isomerization with visible light. J. Am. Chem. Soc. 134, 20597-20600 (2012). 29. Calbo, J. et al. Tuning azoheteroarene photoswitch performance through heteroaryl design. J. Am. Chem. Soc. 139, 1261-1274 (2017). 30. Weston, C. E., Richardson, R. D., Haycock, P. R., White, A. J. P. & Fuchter, M. J. Arylazopyrazoles: azoheteroarene photoswitches offering quantitative isomerization and long thermal half-lives. J. Am. Chem. Soc. 136, 11878-11881 (2014). 31. Walker, B. & Bayley, H. Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal α-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J. Biol. Chem. 270, 23065-23071 (1995). 32. Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497-505 (1997). 33. Maglia, G. et al. Droplet networks with incorporated protein diodes show collective properties. Nat. Nanotechnol. 4, 437-440 (2009). 34. Loudwig, S. & Bayley, H. Photoisomerization of an individual azobenzene molecule in water: an on-off switch triggered by light at a fixed wavelength. J. Am. Chem. Soc. 128, 12404-12405 (2006). 35. Frank, J. A. et al. Photoswitchable fatty acids enable optical control of TRPV1. Nat. Commun. 6, (2015). 36. Schild, V. R. et al. Light-patterned current generation in a droplet bilayer array. Sci. Rep. 7, 1-9 (2017). 37. Villar, G., Graham, A. D. & Bayley, H. A tissue-like printed material. Science. 340, 48-52 (2013). 38. Booth, M. J., Restrepo Schild, V., Box, S. J. & Bayley, H. Light-patterning of synthetic tissues with single droplet resolution. Sci. Rep. 7, 1-10 (2017). 39. Li, J. et al. Metal-organic frameworks as micromotors with tunable engines and brakes. J. Am. Chem. Soc. 139, 611-614 (2017). 40. 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). 41. Lewis, C. A. Ion‐concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction. J. Physiol. 286, 417-445 (1979). 42. Gu, LQ, Cheley, S. & Bayley, H. Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore. Proc. Natl. Acad. Sci. USA 100, 15498-15503 (2003). 43. Pravda, L. et al. ChannelsDB: database of biomacromolecular tunnels and pores. Nucleic Acids Res. 46, D399-D405 (2018).

[0321] Example 3 - Synthesis and Characterization of Photoswitches material and method All reagents and solvents were purchased commercially and used without further purification. Where necessary, solvents were passed through an MBraun MPSP-800 column, dried, and degassed with nitrogen. Triethylamine was distilled with potassium hydroxide and stored. Column chromatography was performed under positive nitrogen pressure using Merck® silica gel 60. When solvent mixtures were used, the volume ratio was reported. NMR spectra were measured using Bruker AVIII 400, Bruker AVII 500 (with cryoprobe), Bruker NEO 600 with broadband helium cryoprobe, and Bruker AVIII 500 spectrometers. Chemical shifts were reported as δ values ​​in ppm. Mass spectra were performed using Waters Micromass LCT and Bruker microTOF spectrometers. UV-Vis spectra were measured using a V-770 UV-Vis / NIR spectrophotometer with a Peltier temperature controller and stirrer, using a 1 cm path length quartz cuvette. Unless otherwise specified, experiments were conducted at 25°C.

[0322] PSS determination and UV / Vis spectrum The UV / Vis spectra were determined in DMSO-d6 solution. The extinction coefficient was determined by recording the UV / Vis spectra of the E isomer at 10, 20, 30, and 40 μM in DMSO. * The absorbance at the transition maxima was plotted against concentration (Beer-Lambert plot) to determine the molar extinction coefficient ε. For each compound, a 40 μM E isomer sample was irradiated with light of the desired wavelength to generate a photosteady state, and another spectrum was processed. This spectrum was normalized to units of ε and superimposed with the dark (100% E isomer) spectrum. Light irradiation of the liquid samples was performed using Thorlabs high-power LEDs (model M530L4 (green, 530 nm), M365L2 (UV, 365 nm)) and a custom-made apparatus using optical components supplied by Thorlabs. The photosteady state (PSS) was: 1 The determination was made using 1H NMR spectroscopy.

[0323] Abbreviation AcOH: Acetic acid; Boc: tert-butyloxycarbonyl; DCM: Dichloromethane; DIPA: N,N-diisopropylamine; DIPEA: N,N-diisopropylethylamine; DMAP: 4-dimethylaminopyridine; HRMS: High-resolution mass spectrometer; MeCN: Acetonitrile; MeOH: Methanol; PhMe: Toluene; Phth: Phthaloyl; PSS: Photostatic state; rt: Room temperature; TFA: Trifluoroacetic acid; THF: Tetrahydrofuran; TMS: Trimethylsilyl.

[0324] Synthesis and Characterization Synthesis of bromoacetylazopyrazole (Br-pzMe)

[0325] [ka]

[0326] Scheme S1. Preparation of Br-pzMe. Synthesis began with commercially available S1, which was nitrated to S2, and then hydrogenated to S3. The azobenzene skeleton was generated by treating S4 with potassium phthalimide to obtain S5, which was deprotected to S6, reprotected to S7, and finally hydrogenated to S8. This was subjected to a Mills coupling reaction via S9, and S9 was reacted with S3 to produce azobenzene S10 in moderate yield. Boc deprotection was performed to obtain S11, and subsequent treatment with bromoacetyl bromide yielded Br-pzMe in good yield.

[0327] [ka]

[0328] Nitro compound S2. References [1] The solution was prepared by modifying the procedure described above. Pyrazole S1 (1.5 g, 13.6 mmol) was dissolved in sulfuric acid (7.5 mL). The solution was cooled to 0°C and nitric acid (6 mL, 150 mmol, 11 eq) was added. The solution was heated to 100°C for 2 hours. The mixture was cooled to room temperature and poured into ice water. The mixture was slowly made basic under ice using a NaOH pellet. The precipitated solid was recovered by vacuum filtration, washed with water, and dried to obtain the title compound (1.8 g, 11.6 mmol, 85%) as an off-white solid. 1 ¹H NMR (400 MHz, CDCl3) δ 3.77 (s, 3H), 2.61 (s, 3H), 2.50 (s, 3H). Data from the literature. [1] It matches what is listed.

[0329] [ka]

[0330] Aniline S3. References [2]The preparation was carried out by modifying the procedure described above. Nitro compound S2 (1.8 g, 11.6 mmol) was dissolved in MeOH (25 mL) and placed under nitrogen. 10% palladium on carbon (250 mg) was added, and the reaction was stirred under a hydrogen atmosphere at room temperature for 16 hours. The reaction was filtered through Celite and washed with CH2Cl2. The filtrate was concentrated to obtain the title compound as a yellow solid (1.44 g, 11.5 mmol, 99%). 1H NMR (400 MHz, CDCl3) δ 3.66 (s, 3H), 2.46 - 2.31 (br s, 2H), 2.15 (s, 3H), 2.13 (s, 3H). The data are consistent with those described in the literature.

[0331] [ka]

[0332] Phthalimide S5. References [3] The following procedure was modified to prepare the compound: p-nitrobenzyl bromide S4 (7 g, 32.40 mmol) was dissolved in DMF (45 mL). Phthalimide potassium (6.3 g, 34 mmol, 1.05 eq) was added, and the reaction mixture was stirred under reflux temperature for 1 hour. The reaction mixture was cooled, and ice water was added. The filtrate was dissolved in hot ethyl acetate (300 mL), and recrystallized in a 500 mL flask at room temperature for 2 days. The solid was collected and washed with cold ethyl acetate to obtain the title compound as white crystals (5.9 g, 20.9 mmol, 65%). 1 H NMR (400 MHz, CDCl3) δ 8.22 - 8.15 (m, 2H), 7.88 (dd, J = 5.4, 3.1 Hz, 2H), 7.75 (dd, J = 5.5, 3.1 Hz, 2H), 7.62 - 7.56 (m, 2H), 4.93 (s, 2H). Data are from literature. [3] It matches what is listed.

[0333] [ka]

[0334] Amine S6. References[4] The preparation was carried out by modifying the procedure. Nitro compound S5 (10 g, 35.43 mmol) was dissolved in THF (100 mL). Hydrazine hydrate (8 mL) was added, and the reaction mixture was stirred at reflux temperature for 16 hours. The reaction mixture was concentrated and then added to RINKAN. The suspension was filtered through cotton, and the filtrate was washed with 1 M NaOH. The organic layer was dried and then concentrated to obtain the title compound as a yellow oil (3.7 g, 24.3 mmol, 69%). 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​- 8.16 (m, 2H), 7.50 (d, J = 8.3 Hz, 2H), 4.01 (s, 2H). Data from literature [4] It matches what is listed.

[0335] [ka]

[0336] Nitro compound S7. References [5] The reaction was prepared by modifying the procedure described above. Amine S6 (3.7 g, 24.32 mmol) was dissolved in CH2Cl2 (50 mL). NEt3 (10.2 mL, 73 mmol, 3 eq) was added. Next, Boc2O (8 g, 36.5 mmol, 1.5 eq) dissolved in CH2Cl2 (50 mL) was added dropwise. After stirring the reaction mixture for 3 hours, it was poured into ice water (50 mL). The reaction mixture was recrystallized from isopropyl ether (40 mL) to obtain the title compound as a yellow crystalline solid (5.1 g, 20.2 mmol, 83%). 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​- 8.14 (m, 2H), 7.45 (d, J = 8.6 Hz, 2H), 4.99 (s, 1H), 4.42 (d, J = 6.3 Hz, 2H), 1.47 (s, 9H). Data from literature [5] It matches what is listed.

[0337] [ka]

[0338] Phthalimide S8. References [5] The preparation was carried out by modifying the procedure. Nitro compound S7 (2.42 g, 9.59 mmol) was dissolved in EtOH (40 mL). The reaction mixture was placed under nitrogen and 10% Pd / C (200 mg) was added. The reaction mixture was stirred under an H2 atmosphere for 2 hours, then filtered through Celite and concentrated to obtain the title compound as a white solid (2.13 g, 9.59 mmol, 100%). 1 H NMR (400 MHz, CDCl3) δ 7.07 (d, J = 8.0 Hz, 2H), 6.69 - 6.60 (m, 2H), 4.73 (s, 1H), 4.18 (d, J = 5.7 Hz, 2H), 1.45 (s, 9H). [5]

[0339] [ka]

[0340] Azobenzene S10. To a solution of aniline S8 (554 mg, 2.45 mmol, 1.2 equiiv.) in CH2Cl2 (40 mL), a solution of Oxone® (4.9 g, 16 mmol, 6.5 equiiv.) in H2O (40 mL) was added. The two-phase reaction mixture was vigorously stirred at room temperature for 16 hours. The phases were separated, and the aqueous phase was extracted with CH2Cl2 (20 mL). The combined organic extracts were washed with saturated NaHCO3 (aq) solution and H2O. The washed organic layer was treated with S3 (260 mg, 2.1 mmol, 1.0 equiiv.) and AcOH (30 mL). Next, CH2Cl2 was removed under reduced pressure at 35°C, and the solution was stirred at room temperature for 16 hours. The solution was concentrated and purified by flash silica gel chromatography (0-10% Â in CH2Cl2) to obtain the title compound as a yellow solid (300 mg, 0.87 mmol, 42%). 1H NMR (600 MHz, CDCl3) δ 7.74 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.88 (s, 1H), 4.36 (d, J = 6.0 Hz, 2H),3.78 (s, 3H), 2.57 (s, 3H), 2.49 (s, 3H), 1.47 (s, 9H). 13 C NMR (151 MHz, CDCl3) δ 156.03, 153.08, 142.55, 140.31,138.89, 135.25, 128.13,122.15, 79.76, 44.58, 36.11, 28.56, 13.91, 10.11. HRMS-EI (m / z) C18H26N5O2 [M+H] + Calculated value: 344.2081; Measured value: 344.2084.

[0341] [ka]

[0342] Amine S11. S10 (100 mg, 0.29 mmol) was dissolved in CH2Cl2 (5 mL), and TFA (0.6 mL) was added to the solution. The reaction mixture was stirred at room temperature for 5 hours. After removing the TFA under a nitrogen stream, the residue was dried under reduced pressure to obtain the title compound as a yellow solid (104 mg, 0.29 mmol, 100%). 1 H NMR (600 MHz, MeOD) δ 7.85 - 7.81 (m, 2H), 7.58 - 7.55 (m, 2H), 4.18 (s, 2H), 3.79 (s, 3H), 2.61 (s, 3H), 2.45 (s, 3H). 13 C NMR (151 MHz, MeOD) δ 155.30, 143.22,141.59, 136.05, 135.46,130.82, 123.36, 44.01, 36.13, 13.87,9.76. HRMS-EI (m / z) C13H18N5 [M+H] + Calculated value: 244.1557; Measured value: 244.1557.

[0343] [ka]

[0344] Br-pzMe. TFA salt S11 (98 mg, 0.3 mmol) was suspended in CH2Cl2 (2.5 mL). NEt3 (95 μL, 0.69 mmol, 2.5 eq) was added, the solution was cooled to 0°C, bromoacetyl bromide (37 μL, 0.37 mmol, 1.5 eq) was added, and the solution was stirred at 0°C for 30 minutes. The solution was diluted with CH2Cl2, and volatile substances were removed using a rotary evaporator (water bath at 35°C). The residue was purified by flash column chromatography (15% Â in CH2Cl2), and the product was obtained as a yellow solid (64 mg, 175 μmol, 64%). 1 H NMR (600 MHz, DMSO) δ 8.84 (t, J = 6.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 4.36 (d, J = 6.0 Hz, 2H), 3.93 (s, 2H), 3.74 (s, 3H), 2.55 (s, 3H), 2.37 (s, 3H). 13 C NMR (151 MHz, DMSO) δ 166.64, 152.55, 140.77, 140.77, 139.94, 134.81, 128.49, 121.87, 42.73, 36.42, 29.91, 14.22, 9.94. HRMS-EI (m / z) C15H19N5OBr [M+H] + The calculated value is 364.0767; the measured value is 364.0763. HMBC shows two peaks at 140.77.

[0345] Synthesis of Br-pzH

[0346] [ka]

[0347] Scheme S2. The synthesis began with commercially available S4, which was nitrated to obtain S5, then decomposed on hydrazine to obtain S6, which was reprotected to obtain S7, and finally hydrogenated to obtain S8. This was subjected to a Mills coupling reaction via S9, and S9 was reacted with S12 to obtain azobenzene S14 in high yield. After deprotection of S14 with Boc, treatment with bromoacetyl bromide yielded Br-pzH in high yield.

[0348] [ka]

[0349] Azobenzene S13. A solution of aniline S8 (452 ​​mg, 2 mmol, 1.2 equiiv.) in CH2Cl2 (37 mL) was treated with a solution of Oxone® (4 g, 13 mmol, 6.5 equiiv.) in H2O (37 mL). The resulting two-phase reaction mixture was vigorously stirred at room temperature for 16 hours. The phases were then separated, and the aqueous phase was extracted with CH2Cl2 (20 mL). The combined organic extracts were sequentially washed with saturated sodium bicarbonate aqueous solution (50 mL) and H2O (50 mL). The organic layer containing S9 after washing was treated with S12 (147 μL, 1.69 mmol, 1.0 equiiv.) and AcOH (30 mL). Next, CH2Cl2 was removed under reduced pressure at 35 °C, and the solution was stirred at room temperature for 16 hours. Next, AcOH was removed under reduced pressure. The residue was purified by flash silica gel chromatography (0-5% Â in CH2Cl2) to obtain the title compound as a yellowish-brown solid (204 mg, 0.95 mmol, 56%). (E)-S13: 1 H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.3 Hz, 2H), 7.45 - 7.35 (m, 3H), 6.65 (d, J = 2.4 Hz, 1H), 4.93 (s, 1H), 4.38 (s, 2H), 4.02 (s, 3H), 1.47 (s, 9H). 13C NMR (151 MHz, CDCl3) δ 163.81, 156.06, 152.18, 142.24, 131.98, 128.13, 123.35, 96.38, 79.85, 44.54, 39.80, 28.54. HRMS-EI (m / z) C 16 H 22 N5O2[M+H] + Calculated value: 316.1768; Measured value: 316.1768.

[0350] [ka]

[0351] Amine S14. TFA (0.6 mL) was added to a solution of S13 (100 mg, 0.32 mmol) in CH2Cl2 (5 mL). The reaction mixture was stirred at room temperature for 5 hours. After removing the TFA under a nitrogen stream, the residue was dried under reduced pressure to obtain the title compound as a yellow solid (104 mg, 0.32 mmol, 100%). (E)-S14: 1 H NMR (400 MHz, MeOD) δ 7.99 - 7.92 (m, 2H), 7.69 (d, J = 2.5 Hz, 1H), 7.66 - 7.60 (m, 2H), 6.64 (d, J = 2.5 Hz, 1H), 4.21 (s, 2H), 4.03 (s, 3H). 13 C NMR (151 MHz, MeOD) δ 164.83, 154.36, 137.41, 134.28, 130.99, 124.30, 96.25, 43.92, 39.74. HRMS-EI (m / z) C 11 H 14 N4[M+H] + Calculated value: 216.1244; Measured value: 216.1245.

[0352] [ka]

[0353] Br-pzH. TFA salt S14 (98 mg, 0.3 mmol) was suspended in CH2Cl2 (2.5 mL). NEt3 (104 μL, 0.74 mmol, 2.5 eq) was added, the solution was cooled to 0°C, bromoacetyl bromide (40 μL, 0.45 mmol, 1.5 eq) was added, and the solution was stirred at 0°C for 30 minutes. The solution was diluted with CH2Cl2, and volatile substances were removed using a rotary evaporator (35°C water bath). The residue was purified by flash column chromatography (10% Â in CH2Cl2), and the product was obtained as a yellow solid (68.2 mg, 202 μmol, 68%). (E)-Br-pzH: 1 H NMR (400 MHz, DMSO) δ 8.87 (d, J = 6.1 Hz, 1H), 7.83 (d, J = 2.4 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 6.53 (d, J = 2.4 Hz, 1H), 4.39 (d, J = 6.0 Hz, 2H), 3.97 (s, 3H), 3.95 (s, 2H). 13 C NMR (151 MHz, DMSO) δ 166.72, 163.46, 151.56, 143.69, 133.57, 128.64, 122.67, 95.00, 41.83, 40.55, 29.90. HRMS-EI (m / z) C 13 H 15 N5OBr [M+H] + Calculated value: 336.0454; Measured value: 336.0457.

[0354] Synthesis of Br-fAzo

[0355] [ka]

[0356] Scheme S3. Preparation of fAzo derivative. S15 was reduced to the unstable S16, followed by Fmoc protection to obtain S17. This was azo-coupled with S18 to produce S20, which was deprotected with DIPA and treated with bromoacetyl bromide to obtain the Br-fAzo derivative.

[0357] [ka]

[0358] Aniline S17. S15 (1.45 g, 9.41 mmol) was dissolved in THF (20 mL). Under an N2 atmosphere, borane-THF (1 M, 38.4 mL, 38.4 mmol, 4 eq) was added at 0°C, and the reaction mixture was stirred at reflux temperature for 16 hours. At this temperature, MeOH (5 mL) was added dropwise until foaming stopped, and the reaction mixture was stirred at reflux temperature for 30 minutes. The solvent was removed under reduced pressure, redissolved in SiO2, and washed with water. The organic layer was concentrated. Off-white S16 was redissolved in CH2Cl2 (30 mL). DIPEA (1.46 mL, 9.41 mmol, 1 eq) was added, followed by Fmoc-Cl (2.43 g, 9.41 mmol, 1 eq), and the mixture was stirred at room temperature for 16 hours. The solution was concentrated and purified by silica gel chromatography (6:1 hexane:acetone) to obtain the title compound as a white solid (1.37 g, 3.60 mmol, 38%). 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 6.75 (d, J = 7.4 Hz, 2H), 5.02 (s, 1H), 4.47 (d, J = 6.8 Hz, 2H), 4.30 - 4.01 (m, 3H), 3.70 (s, 2H). 13C NMR (126 MHz, CDCl3) δ 156.49, 152.05 (dd, J = 242.30 Hz, 8.10 Hz), 143.98, 141.49, 127.86 (t), 127.20, 125.11, 123.24 (t, J = 16.2 HRMS-ESI (m / z) C 22 H 18 F2N2O2[M+H] + Calculated value: 381.1409; Measured value: 381.1408

[0359] [ka]

[0360] Aniline S18 (170 mg, 1.31 mmol, 2 eqs) was dissolved in CH2Cl2 (10 mL). Oxon (5.35 g, 13.1 mmol, 20 eqs) was added to water (20 mL), and the two-phase solution was vigorously stirred at room temperature for 16 hours, after which the organic layer turned green. The organic layer was separated and washed sequentially with 1N HCl (20 mL), saturated bicarbonate solution (20 mL), and water (20 mL). The organic layer was dried and concentrated. The residue was redissolved in 6:6:1 AcOH:toluene:TFA (20 mL), S17 (250 mg, 657 μmol, 1 eq) was added, and the solution was stirred at room temperature for 3 days. The mixture was concentrated and purified by flash column chromatography (100% CH2Cl2) to obtain the title compound (66 mg, 133 μmol, 17%) as an orange solid and a mixture of isomers. When the sample was irradiated with 405 nm, 1 ¹H NMR analysis yielded (E)-S20 and (Z)-S20 in a ratio of 17:1. (E)-S20: 1¹H NMR (600 MHz, CDCl₃) δ 7.77 (d, J = 7.1 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.37 (t, J = 8.9 Hz, 1H), 7.33 (t, J = 7.7 Hz, 2H), 7.06 (t, J = 8.9 Hz, 2H), 6.95 (d, J = 10.4 Hz, 2H), 5.16 (t, J = 6.4 Hz, 1H), 4.55 (d, J = 6.5 Hz, 2H), 4.39 (d, J = 6.4 Hz, 2H), 4.24 (t, J = 6.4 Hz, 1H). 13 ¹³C NMR (151 MHz, CDCl₃) δ 156.56, 155.89 (dd, J = 263.0, 4.4 Hz), 155.73 (dd, J = 261.4, 4.0 Hz), 143.97, 143.86, 141.55, 132.01, 131.53 (t, J = 10.3 Hz), 130.78, 127.95, 127.25, 125.04, 120.18, 112.77 (dd, J = 20.1, 3.8 Hz), 111.25 (d, J = 20.9 Hz), 66.94, 47.48, 44.34. HRMS-EI (m / z) C 28 H 20 N₃O₂F₄[M+H] + calculated value 506.1486; found 506.1488.

[0361]

Chemical Formula

[0362] S20 was dissolved in DMF (66 mg, 131 μmol, 70 mM). The reaction mixture was mixed with DIPA (73 μL, 522 μmol, 4 eq) under N2 conditions and stirred at room temperature for 16 hours. The reaction mixture was concentrated and then purified by flash column chromatography to obtain the product as an orange solid. Free amine (16 mg, 56 μmol) was dissolved in CH2Cl2 (40 mM). NEt3 (12 μL, 84 μmol, 1.5 eq) was added, the solution was cooled to 0°C, bromoacetyl bromide (7 μL, 84 μmol, 1.5 eq) was added, and the solution was stirred at 0°C for 30 minutes. The solution was diluted with CH2Cl2, and volatile substances were removed using a rotary evaporator (water bath at 35°C). The residue was purified by flash column chromatography to obtain the product (7.3 mg, 18 μmol, 32%) as an orange solid. (E)-Br-fAzo: 1 H NMR (600 MHz, CDCl3) δ 7.37 (tt, J = 8.4, 5.8 Hz, 1H), 7.09 - 7.03 (m, 2H), 7.00 (d, J = 9.8 Hz, 2H), 6.91 (s, 1H), 4.52 (d, J = 6.2 Hz, 2H), 3.98 (s, 2H). 13 C NMR (151 MHz, CDCl3) δ 165.72, 155.76 (dd, J = 262.2, 4.7 Hz), 155.63 (dd, J = 261.2, 4.0 Hz), 142.39 (t, J = 9.3 Hz), 131.80 (t, J = 10.0 HRMS-ESI (m / z)C 15 H 11 ON3BrF4[M+H] + Calculated value: 404.0016; Measured value: 404.0017

[0363] PSS determination and UV-Vis spectrum The UV-Vis spectra were determined in DMSO solution. The extinction coefficients were determined by recording the UV-Vis spectra of the E isomer in DMSO at 10, 20, 30, and 40 μM, respectively. * The absorbance at the transition maxima was plotted against concentration (Beer-Lambert plot) to determine the molar extinction coefficient ε. For each compound with a slow relaxation rate, a 40 μM E isomer sample was irradiated with light of an appropriate wavelength to generate a photosteady state, and another spectrum was processed. This spectrum was normalized to ε units and superimposed with the dark (100% E isomer) spectrum.

[0364] The deconvoluted spectrum was created using Origin Pro 2020b by fitting each λmax to the "Multiple Peak Fit" function.

[0365] The liquid samples were irradiated using Thorlabs high-power LEDs (Model M530L4 (green, 530nm), M365L2 (UV, 365nm)) and a custom-made system using optical components supplied by Thorlabs. The photosteady state (PSS) was determined as follows: 1 The determination was made using 1H NMR spectroscopy.

[0366] References [1] E. De Vita, P. Schuler, S. Lovell, J. Lohbeck, S. Kullmann, E. Rabinovich, A. Sananes, B. Hesling, V. Hamon, N. Papo, J. Hess, EW Tate, N. Gunkel and AK Miller, J Med Chem, 2018, 61, 8859-8874. [2] P.-Y. Lam, AR Thawani, E. Balderas, AJP White, D. Chaudhuri, MJ Fuchter and RT Peterson, J Am Chem Soc, 2020, 142, 17457-17468. [3] N. Tran-Hoang and T. Kodadek, ACS Comb Sci, 2018, 20, 55-60. [4] DP Nguyen, RN Sladek and LH Do, Tetrahedron Letters, 2020, 61, 152196. [5] S. Ait Amiri, C. Deboux, F. Soualmia, N. Chaaya, M. Louet, E. Duplus, S. Betuing, B. Nait Oumesmar, N. Masurier and C. El Amri, J Med Chem, 2021, 64, 5667-5688.

Claims

1. A nanopore that can be selectively converted between an open state and a closed state using light, wherein substantially no substance can move through the nanopore when it is in the closed state.

2. The nanopore has one or more photoisomerizable groups within its lumen, and each of these one or more photoisomerizable groups independently comprises formula (I): 【Chemistry 1】 (In the formula: * indicates a connection point to a nanopore; L is a linker; and Ar １ and Ar ２ (Each of these groups is independently selected from an aryl group that may be substituted and an heteroaryl group that may be substituted.) The nanopore described in claim 1, which is the basis of the

3. - R with 1 to 4 substituted aryl groups １ A 6- to 10-membered aryl group which may be substituted with; and - The substituted heteroaryl group is a 5- to 10-membered ring heteroaryl group having 1 to 4 ring heteroatoms independently selected from the group consisting of N, O, and S; the 5- to 10-membered ring heteroaryl group has 1 to 4 R ２ It may be replaced with; and -R １ and R ２ are each independently optionally substituted by COO － , SO ３ － , or NR ３ ３ ＋ C alkyl; COO optionally substituted by １－６ alkyl; COO － , SO ３ － , or NR ３ ３ ＋ C alkenyl optionally substituted by ２－６ alkenyl; COO － , SO ３ － , or NR ３ ３ ＋ C alkynyl optionally substituted by ２－６ alkynyl; halogen; CN; NO ２ ; C １－６ haloalkyl; OH; COO － , SO ３ － , or NR ３ ３ ＋ C alkoxy optionally substituted by １－６ alkoxy; C １－６ haloalkoxy; COO － , SO ３ － , or NR ３ ３ ＋ COOC alkyl optionally substituted by １－６ alkyl; CONH ２ ; COO － , SO ３ － , or NR ３ ３ ＋ CONR optionally substituted by ４ C １－６ alkyl; COO － , SO ３ － , or NR ３ ３ ＋ NR optionally substituted by ４ COC １－６ alkyl; SO ２ NH ２ ; COO － , SO ３ － , or NR ３ ３ ＋ SO which may be substituted with ２ NR ４ C １－６ alkyl; COO － , SO ３ － , or NR ３ ３ ＋ NR which may be substituted with ４ SO ２ C １－６ alkyl; COO － , SO ３ － , or NR ３ ３ ＋ (wherein each R ３ is independently H or C １－６ alkyl, and each R ４ is independently H or C １－６ alkyl), the nanopore according to claim 2.

4. - R with 1 to 4 substituted aryl groups １ A phenyl group which may be substituted with; and - The substituted heteroaryl group is a five-membered ring heteroaryl group having 1 to 3 ring heteroatoms independently selected from the group consisting of N and O; the five-membered ring heteroaryl group has 1 to 3 R ２ It may be replaced with; and -R １ and R ２ are each independently C １－３ alkyl; halogen; C １－３ haloalkyl; COO － , SO ３ － , or NHCOC which is optionally substituted with NR ３ ３ ＋ alkyl; NHSO optionally substituted with １－３ COO － , SO ３ － , or NR ３ ３ ＋ C ２ alkyl, COO １－３ , SO － , or NR ３ － ; ３ ３ ＋ wherein each R ３ is independently C １－３ alkyl), the nanopore according to claim 3, which is selected from the group consisting of.

5. Ar １ and Ar ２ The nanopore according to any one of claims 2 to 4, wherein at least one of the groups may be substituted with a heteroaryl group.

6. Each of the one or more photoisomerizable groups independently has the chemical formula (IB): 【Chemistry 2】 (In the formula: * indicates a connection point to a nanopore; and The nanopore according to claim 5, wherein L is the base of a linker.

7. Linker, structure (L1) 【Transformation 3】 (In the formula: * indicates a connection point to a nanopore. ** is Ar １ This indicates the connection point. LL is a substituted 5- to 10-membered carbocyclic ring or a substituted 5- to 10-membered heterocyclic ring; Y is independently a halogen, OH, or C １－６ C may be substituted with alkyl. １－２０ Alkylene group; amide group; sulfonamide group; ester group; thioester group; ether group; thioether group; and amine (i.e., -NR) ５ - (wherein, R ５ Preferably H or C １－６ It is an alkyl group; q is 0 or 1; and (m, o, and r are each independent integers between 0 and 5.) A nanopore according to any one of claims 1 to 6, which is a linker.

8. Linker, structure (L4): 【Chemistry 4】 (In the formula: * indicates a connection point to a nanopore; ** is Ar １ The nanopore according to claim 7, which is a linker (indicating a connection point to the...).

9. Each of the one or more photoisomerizable groups independently has the chemical formula (IC): 【Transformation 5】 (In the formula: A nanopore according to any one of claims 1 to 8, which is the base of (where * indicates a connection point to the nanopore).

10. The nanopore is a transmembrane nanopore; preferably - The transmembrane nanopore is a transmembrane protein pore, a DNA origami pore, a solid-state pore, or a polymer-based pore; more preferably - The transmembrane protein pore is a porin, pore-forming protein, or channel protein; and / or - The nanopore according to any one of claims 1 to 9, wherein the transmembrane protein pore is an alpha-hemolysin (αHL) pore, preferably an αHL pore formed from seven αHL monomers.

11. The nanopore has one or more nucleophilic amino acids exposed in the lumen of the pore, preferably: - The nanopores are αHL pores, and the αHL pores preferably contain nucleophilic amino acids at positions corresponding to positions 111, 113 and / or 147 of SEQ ID NO: 1; and, more preferably - The nanopore according to claim 10, wherein the αHL pore includes substitutions corresponding to E111C, M113C, or K147C of SEQ ID NO: 1, most preferably E111C substitution.

12. One or more photoisomerizable groups are covalently attached to the nanopore, preferably: - One or more photoisomerizable groups are covalently attached to a nucleophilic amino acid exposed in the lumen of the nanopore, and more preferably: - The nanopore according to any one of claims 2 to 11, wherein the nucleophilic amino acid is cysteine.

13. The nanopore according to claim 12, wherein the nanopore is αHL and the nucleophilic amino acids are located at positions corresponding to positions 111, 113 and / or 147 of SEQ ID NO:

1.

14. The nanopore according to any one of claims 1 to 13, wherein the nanopore is an oligomeric nanopore, and each monomer of the nanopore contains at least one of the photoisomerizable groups.

15. A nanopore according to any one of claims 1 to 14, wherein the nanopore is located within an amphiphilic membrane.

16. A method for producing a nanopore according to any one of claims 1 to 15, comprising introducing one or more photoisomerizable groups into the nanopore.

17. One or more photoisomerizable groups form a nanopore of formula (II): 【Transformation 6】 (In the formula: Ar １ and Ar ２ The method according to claim 16, wherein Lelec is introduced into the nanopore by treatment with a compound of any one of claims 2 to 6 (corresponding to the linker described in any one of claims 2, 7, or 8, except that the atom connected to the nanopore is an electrophile capable of reacting with a nucleophile).

18. The compound is given by formula (IIC): 【Transformation 7】 The method according to claim 17, wherein X is a leaving group, preferably a compound selected from halogens (preferably F, Cl, Br, or I) and sulfonate groups (preferably tosylate or trifluoromethanesulfonate), most preferably Br.

19. The method according to any one of claims 16 to 18, wherein one or more photoisomerizable groups are introduced into the nanopore by treating the monomer of the nanopore with a compound of formula (II) as defined in claim 17 or a compound of formula (IIC) as defined in claim 18, and preferably subsequently oligomerizing the monomer to provide the nanopore.

20. A method for regulating the flow of one or more substances through a nanopore according to any one of claims 1 to 15, or a nanopore obtained by the method according to any one of claims 16 to 19, the method comprising irradiating the nanopore with light.

21. The method comprises irradiating a nanopore with a first wavelength of light and a second wavelength of light, preferably the nanopore being a nanopore according to any one of claims 2 to 14, and: - The first wavelength of light isomerizes the photoisomerizable group to its Z-type, and the second wavelength of light isomerizes the photoisomerizable group to its E-type, more preferably: - The first wavelength of light is the wavelength in the UV / Vis spectrum where the ratio of εE / εZ is maximized, and the second wavelength of light is the wavelength in the UV / Vis spectrum where the ratio of εZ / εE is maximized, where: εE is the molar extinction coefficient of the E-type of the photoisomerizable group, εZ is the molar extinction coefficient of the Z-type of the photoisomerizable group. εE and εZ are determined using a UV / Vis spectrophotometer; and / or The method according to claim 20, wherein the wavelength of the first light is 365 nm and the wavelength of the second light is 455 nm or 530 nm, preferably 530 nm.

22. The method according to claim 20 or 21, wherein one or more substances are independently selected from ions, water, sugars, inorganic salts, lipids, amino acids, peptides, nucleotides, nucleic acids, metabolites, neurotransmitters, and drugs, most preferably being ions.

23. Use of the nanopore according to any one of claims 1 to 15 in regulating the flow of ions across an amphiphilic membrane.

24. A device comprising a vesicle surrounded by an amphiphilic membrane, wherein the vesicle contains one or more drugs within its lumen, and the amphiphilic membrane comprises a nanopore as described in any one of claims 1 to 14.

25. Use of the nanopore according to any one of claims 1 to 15 as an iontronic element that can be photoswitched between a resistor and a diode.

26. Use of nanopores according to any one of claims 1 to 15 in biocomputing and / or bioiontronics.

27. A method for transmitting information, comprising the step of irradiating a nanopore according to any one of claims 1 to 15 with a plurality of light pulses, wherein each pulse includes light having one of a predetermined group of wavelengths.

28. A method for receiving information, comprising the step of measuring the ion current passing through a nanopore according to any one of claims 1 to 15 while irradiating the nanopore with a plurality of light pulses, wherein each pulse includes light having one of a predetermined group of wavelengths.

29. An iontronic element comprising a nanopore as described in any one of claims 1 to 15, wherein the element is optionally a logic gate.

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