Device for processing a molecule of interest with translocation control

Abstract

A device for processing a molecule of interest, comprising an aperture having an entry and an exit, the aperture being suitable for permitting the translocation of the molecule from the entry to the exit, means for causing the translocation of the molecule of interest from the entry to the exit, and a channel fluidically coupled with the aperture and suitable for guiding the molecule of interest towards the aperture, wherein the channel comprises a track adapted for binding a motor region of a walking molecule coupled to the molecule of interest and adapted for the procession of said walking molecule towards the entry of the aperture.

Description

[0001] Device with translocation control

[0002] Field of the Invention

[0003] The present invention relates to the field of aperture-based devices for processing and analyzing molecules of interest.

[0004] Background of the Invention

[0005] Efficiently guiding molecules of interest toward an aperture has multiple potential applications such as nucleotide sequencing, polypeptide analysis, biosensing, molecular sorting and separation, single molecule detection and analysis, nanotechnology, and nanofluidics, among others.

[0006] Amongst these fields, aperture-based sensing and sequencing has seen significant advancements in recent years, with the potential to revolutionize various areas such as genomics, proteomics, and molecular diagnostics. Apertures, which are nanoscale holes in a membrane or other material, allow the passage of individual molecules of interest, enabling their detection and analysis at the single-molecule level.

[0007] One of the key challenges in aperture-based sensing is controlling the translocation speed of the molecules of interest through the aperture. In many applications, such as DNA sequencing, it is crucial to slow down the translocation speed to achieve high-resolution measurements and accurate base calling. However, the rapid translocation of molecules of interest through solid-state apertures, driven by electrophoretic forces, often results in insufficient temporal resolution and signal-to-noise ratio.

[0008] Another challenge is the variation in translocation speed among identical molecules of interest under the same conditions. This speed variation can lead to inconsistencies in the measured signals and compromise the accuracy of the analysis. Addressing this issue would improve reliability and reproducibility of aperture-based sensing and sequencing.

[0009] Furthermore, the ability to fine-tune the translocation speed for specific types of molecules of interest is highly desirable. Different molecules of interest may have different optimal translocation speeds for accurate characterization, and the ability to adjust the speed accordingly can greatly enhance the versatility and performance of aperture-based systems.

[0010] Despite the progress made in aperture technology, there is still a need for further advancements to overcome one or more of the above challenges. Summary of the Invention

[0011] It is an object of embodiments of the present invention to provide a device, a method, and a system for efficiently guiding molecules of interest toward an aperture. This objective is accomplished by a device, a method, and a system for processing a molecule according to the invention.

[0012] In the first aspect, the present invention relates to a device for processing a molecule of interest, comprising: a. An aperture having an entry and an exit, the aperture being suitable for permitting the translocation of the molecule of interest from the entry to the exit, b. Means for causing the translocation of the molecule of interest from the entry to the exit, and c. A channel fluidically coupled with the aperture and suitable for guiding the molecule of interest towards the aperture, wherein the channel comprises a track adapted for binding a motor region of a walking molecule and adapted for the procession of said walking molecule towards the entry of the aperture.

[0013] In embodiments, the molecule of interest may be selected from the list consisting of lipids, sugars, and copolymers. The device allows processing of a wide range of molecules of interest.

[0014] In embodiments, the molecule of interest may be a copolymer, preferably a biopolymer. Biopolymers are of particular interest because at least some of the challenges of their analysis are well met by the device of the first aspect.

[0015] In embodiments, the copolymer may be a biopolymer selected from polypeptides with or without post-translational modifications, and nucleic acids. These are key biological molecule of interests to analyze.

[0016] In embodiments, the device may further comprise a sensor for analyzing the molecule of interest during its translocation through the aperture. Although the device of the first aspect is advantageous in all applications where a molecule of interest should be guided toward an aperture, it is especially useful for enabling real-time analysis, an application where a sensor is particularly advantageous.

[0017] In embodiments, only the channel, and not the aperture, may comprise the track. This prevents the track from interfering with translocation through the aperture. In embodiments, the track may be aligned with the entry of the aperture so as to guide the processing motor protein towards the entry of the aperture. Proper alignment is advantageous for efficient translocation.

[0018] In embodiments, a side of the track closest to the entry of the aperture may have a width at most equal to 10 times the width of the aperture. This ensures the motor protein is guided close enough to the aperture entry to be within the capture radius of the aperture.

[0019] In embodiments, a side of the track farthest to the aperture may have a width larger than the width of the aperture and the width of the track may decrease, preferably gradually decrease, from the farthest side to the closest side. The funnel shape helps capture the motor protein and guide it to the aperture.

[0020] In embodiments, the width of the track at the side farthest to the aperture may be at least 1.5 times, preferably at least 2 times larger than the width of the entry of the aperture. This provides a large capture area for the motor protein.

[0021] In embodiments, the track may comprise a plurality of primary functional groups aligned along a substrate, each being adapted for binding with a secondary functional group of the walking molecule, such that when the walking molecule is bound to the track, application of a driving force causes the walking molecule to be directionally transferred between primary functional groups on the track, thereby causing the walking molecule to move along the track. This enables controlled processive movement of the motor protein.

[0022] In embodiments, the track may comprise microtubules with their plus side oriented towards the entry of the aperture. Microtubules are well-suited for capturing and allowing the procession of kinesin motor proteins. Kinesin motor proteins move toward the plus side.

[0023] In embodiments, the track may comprise microtubules with their minus side oriented towards the entry of the aperture. Microtubules are well-suited for capturing and allowing the procession of dynein motor proteins. Dynein motor proteins move toward the minus side.

[0024] In embodiments, at least a first portion of the channel may be directly fluidically connected to the entry of the aperture, wherein said first portion has a first longitudinal axis, wherein the aperture has at least a second portion comprising the entry, said second portion having a second longitudinal axis, and wherein the first longitudinal axis makes an angle of from 20° to 160°, preferably from 40° to 140°, more preferably from 60° to 120°, yet more preferably from 80° to 100°, and most preferably 90° with the second longitudinal axis. The closer the angle between the channel and aperture axes is to 90°, the better the translocation dynamics and the easier the device is to manufacture. In embodiments, the channel may have at least one transversal cross-section having a width larger than the width of the entry of the aperture.

[0025] In embodiments, the channel may have at least one transversal cross-section having a width larger than 100 nm. In embodiments, all transversal cross-sections of a first portion of the channel may have a width larger than the width of the entry of the aperture, said first portion of the channel being directly fluidically connected to the entry of the aperture.

[0026] This allows, for instance, the side of the track closest to the aperture to be larger than the width of the aperture, which is advantageous since a capture radius typically develops around the entry of the aperture and this capture radius is larger than the radius of the aperture itself. It, therefore, allows to capture more molecules of interests. For instance, all transversal cross-section of a first portion of the channel may have a width larger than 100 nm and the width of the entry of the aperture may be at most 100 nm.

[0027] In embodiments, the channel may have all its transversal cross-sections having a width larger than the width of the entry of the aperture. For instance, the channel may have all its transversal cross-sections having a width of more than 100 nm and the entry of the aperture may have a width of at most 100 nm. This facilitates passage of large molecular assemblies in the channel, i.e., it facilitates the presence of a walking molecule. It also allows more space to design the track, knowing that the track preferably has a width gradually decreasing toward the aperture, and knowing that the side of the track closest to the aperture does not need to be smaller than the width of the aperture since a capture radius typically develops around the entry of the aperture.

[0028] In embodiments, the device may comprise the walking molecule coupled to the molecule of interest, said walking molecule being bound by the track. This forms allows for controlled translocation.

[0029] In embodiments, two or more identical walking molecules may be coupled to the molecule of interest so that different locations along the molecule of interest are coupled to a walking molecule. Multiple motor proteins provide redundancy and a more constant translocation speed for all parts of the molecules of interest, especially the extremities of the molecule of interest that are typically dead ends that would, otherwise, be translocated at a speed different from more central parts of the molecule of interest.

[0030] In embodiments, the molecule of interest may be a copolymer comprising concatenated identical monomer sequences, said concatenated identical monomer sequences making up at least 50%, preferably at least 70%, more preferably at least 90% of the copolymer. The repeating sequences enable more reliable analysis, especially for shorter molecules of interest.

[0031] In embodiments, the walking molecules may be selected from the group consisting of dyneins, myosins, and kinesins, preferably kinesins and dyneins. These are the most common and well-characterized walking molecules.

[0032] In embodiments, the aperture may be a solid-state aperture. Solid-state apertures are robust and tunable.

[0033] In embodiments, the aperture may have all its transversal cross-sections having a width (e.g., diameter) of at most 100 nm and at least one transversal cross-section with a width (e.g., diameter) of from at most 25 nm. This size range is suitable for analyzing many biopolymers.

[0034] In embodiments, the aperture may have all its transversal cross-sections having a width (e.g., diameter) of from 0.5 to 100 nm and at least one transversal cross-section with a width (e.g., diameter) of from 0.5 to 25 nm. An example for the lower boundary is an aperture of 0.9 nm diameter in a graphene sheet.

[0035] In embodiments, said means may comprise two electrodes fluidically separated by the aperture. The electrodes create an electric field to drive translocation.

[0036] In embodiments, the sensors may be capable of detecting changes in ionic current or tunneling current as the molecule of interest translocates through the aperture. Ionic current and tunneling current are sensitive to translocation events.

[0037] In embodiments, the sensors may be capable of detecting fluorescence signals emitted by labeled sections of the molecule of interest. Fluorescent labeling provides an orthogonal sensing modality.

[0038] In embodiments, the device may be for determining a structural sequence of the copolymer or for fingerprinting the copolymer. Sequencing and fingerprinting are two key applications facilitated by the device of the first aspect.

[0039] In the second aspect, the present invention relates to a method for processing a molecule of interest, comprising: a. Providing a device as claimed in any embodiments of the first aspect, b. Providing a molecular assembly in the channel of the device, the molecular assembly comprising one or more walking molecules coupled to the molecule of interest, and binding the motor region of the one or more walking molecules of the assembly to the track of the channel, c. Causing the procession, along the channel and toward the aperture, of the one or more walking molecules of the assembly, while simultaneously causing a free end of the molecule of interest of the assembly to enter the entry of the aperture and to progress toward the exit thereof, thereby causing the translocation of the molecule of interest from the entry to the exit.

[0040] In embodiments, the molecule of interest may be a copolymer, wherein the device further comprises a sensor for analyzing the copolymer during its translocation through the aperture, and wherein the method further comprises the step d, after step c, of analyzing the copolymer of the assembly, as it translocates towards the exit of the aperture, using the sensor. This allows real-time analysis of the copolymer.

[0041] In embodiments, causing the procession may comprise contacting the one or more walking molecules with ATP. ATP fuels the movement of the motor proteins.

[0042] In embodiments, two or more walking molecules may be coupled to the molecule of interest, e.g., the copolymer so that different locations along the copolymer are coupled to a walking molecule. Multiple attachment points provide smoother translocation wherein various parts of the copolymer are translocated at a more uniform speed.

[0043] In embodiments, the copolymer may comprise concatenated identical monomer sequences, said concatenated identical monomer sequences making up at least 50%, preferably at least 70%, more preferably at least 90% of the copolymer. The repeating sequences enable more reliable analysis, especially of smaller copolymers.

[0044] In embodiments, the one or more walking molecules may all be identical and are selected from the group consisting of myosins, kinesins, and dyneins. Using identical motor proteins simplifies the system and ensure a constant speed for all parts of the molecules of interest when more than one motor protein is attached thereto.

[0045] In embodiments, causing a free end of the copolymer of the assembly to enter the entry of the aperture and to progress toward the exit thereof, thereby causing the translocation of the copolymer from the entry to the exit, may comprise creating an electrical field between the entry and the exit of the aperture. The electric field drives translocation.

[0046] In embodiments, analyzing the copolymer of the assembly, as it translocates towards the exit of the aperture, using the sensor, may comprise detecting changes in ionic current or tunneling current as the copolymer translocates through the aperture. Ionic current and tunneling current are sensitive to translocation events. In embodiments, analyzing the copolymer of the assembly, as it translocates towards the exit of the aperture, using the sensor, may comprise detecting fluorescence signals emitted by labeled sections of the copolymer. Fluorescent labeling provides an orthogonal sensing modality.

[0047] In embodiments, the method may be for determining the sequence of or for fingerprinting the copolymer. Sequencing and fingerprinting are two key applications.

[0048] Any feature of any embodiment of the second aspect may be as correspondingly described in the first or third aspect.

[0049] In the third aspect, the present invention relates to a system for analyzing a copolymer, comprising: a. A device as claimed in any embodiments of the first aspect, b. Means for providing a molecular assembly in the channel of the device, the molecular assembly comprising one or more walking molecules coupled to the molecule of interest, c. Means for causing the procession, along the channel and toward the aperture, of the one or more walking molecules of the assembly, d. A control unit operatively connected to the device and configured to cause the system to execute the steps of the method as claimed in any embodiments of the second aspect.

[0050] Any feature of any embodiment of the third aspect may be as correspondingly described in any embodiment of the first aspect or the second aspect.

[0051] In the fourth aspect, the present invention relates to a computer program comprising instructions which, when executed by the control unit of the system of the third aspect cause the system to execute the steps of the method as claimed in any embodiments of the second aspect.

[0052] Any feature of any embodiment of the fourth aspect may be as correspondingly described in the second or third aspect.

[0053] In the fifth aspect, the present invention relates to a computer-readable medium having stored thereon the computer program as claimed in the fourth aspect.

[0054] In the sixth aspect, the present invention relates to use of one or more processing motor protein for transporting a molecule of interest, e.g., a copolymer in a channel toward the entry of an aperture of a device.

[0055] Any feature of any embodiment of the sixth aspect may be as correspondingly described in the first aspect or the second aspect. It is an advantage of embodiments of the present invention that good capture of molecular assemblies can be achieved.

[0056] It is an advantage of embodiments of the present invention that a device for processing a molecule of interest, such as a copolymer, can be provided that allows controlled translocation of the molecule of interest through an aperture. In particular, the translocation speed can be reduced and made more uniform within a molecule of interest and between molecules of interest.

[0057] It is a further advantage of embodiments of the present invention that the method can allow analyzing the molecule of interest as it translocates through the aperture using a sensor.

[0058] Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims and with features of other dependent claims as appropriate and not merely as explicitly set out in the claims.

[0059] The above and other characteristics, features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention. This description is given for the sake of example only, without limiting the scope of the invention. The reference figures quoted below refer to the attached drawings.

[0060] Brief description of the drawings

[0061] Fig.l is a schematic representation of a top view of a device according to embodiments of the present invention.

[0062] Fig.2 is a schematic representation of a vertical cross-section along the length of the channel of a device according to embodiments of the present invention.

[0063] Fig.3 is a flow chart of a method according to embodiments of the present invention.

[0064] Fig.4 is a schematic representation of a system according to embodiments of the present invention.

[0065] Fig.5 is a schematic representation exemplifying an embodiment of the method of the present invention.

[0066] Fig.6 is a schematic representation illustrating a problem solved by the embodiment of Fig.7 according to embodiments of the prior art. Fig.7 is a schematic representation illustrating an embodiment solving the problem illustrated in Fig.6 according to embodiments of the present invention.

[0067] Fig.8 is a schematic representation illustrating the notion of capture radius and depicting a track width which gradually decreases towards the aperture according to embodiments of the present invention.

[0068] In the different figures, the same reference signs refer to the same or analogous elements.

[0069] Detailed description of Illustrative Embodiments

[0070] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention.

[0071] The following terms are provided solely to aid in the understanding of the invention.

[0072] As used herein, and unless otherwise specified, the term "device" may include, but is not limited to, solid-state devices, biological devices, and hybrid devices combining solid- state and biological components. Solid-state and hybrid devices are preferred. Solid-state devices are particularly preferred.

[0073] As used herein, and unless otherwise specified, the term "for processing a molecule of interest" refers to the suitability of the device for performing at least one operation on a molecule of interest, typically a biopolymer such as DNA, RNA, or a protein, which may include but are not limited to:

[0074] (a) guiding the molecule of interest towards an aperture using a channel;

[0075] (b) controlling the movement of the molecule of interest using one or more walking molecules bound to a track within the channel;

[0076] (c) translocating the molecule of interest through the aperture in a controlled manner; and

[0077] (d) analyzing the molecule of interest as it passes through the aperture.

[0078] This processing may involve one or more of manipulating the molecule of interest's speed and orientation during translocation, detecting changes in ionic current or other measurable properties as the molecule of interest passes through the aperture, and interpreting these changes to derive information about the molecule of interest's structure, sequence, or other characteristics. The term encompasses various analytical techniques that leverage the controlled translocation of molecules of interest through apertures for purposes such as localizing, guiding, sequencing, structural analysis, or detection of specific molecular features.

[0079] As used herein, and unless otherwise specified, the term "molecule of interest" refers to any molecule or molecular complex that can be translocated through an aperture. Examples of molecules of interest include, but are not limited to, lipids, sugars, copolymers, biopolymers such as polypeptides (with or without post-translational modifications) and nucleic acids (DNA, RNA, etc.), and other natural or synthetic polymers.

[0080] As used herein, and unless otherwise specified, the term "aperture" refers to a nanoscale aperture in a substrate, typically having a diameter in the range of 1 to 100 nanometers. An aperture has an entry and an exit and is suitable for enabling the translocation of the molecule of interest from the entry to the exit. This suitability may include one or more of the following without being limited thereto:

[0081] (a) Having appropriate dimensions to allow the passage of the molecule of interest; (b) Possessing surface properties that allow for the molecule of interest to enter and pass through without adhering or becoming stuck; and (c) Having a structure that maintains its integrity and functionality under the conditions required for molecular translocation. This adaptation ensures that the aperture can effectively guide the molecule of interest through its constrained space. The specific adaptations may vary depending on the type of molecule of interest being processed, e.g., analyzed and the intended purpose of the device. In the context of this invention, an aperture is a key component of a device for processing molecules of interest, where it serves as a confined space through which individual molecules of interest pass, e.g., for analysis. Apertures can be:

[0082] (a) biological, formed by pore-forming proteins embedded in a lipid bilayer; (b) solid-state, fabricated in synthetic materials such as silicon, silicon nitride or graphene; or (c) hybrid, combining biological and synthetic components. Amongst others, the aperture enables the sequential passage of segments of a molecule of interest (such as individual nucleotides of a DNA strand) through its constrained space, for instance, driven by an electric field. This passage typically causes detectable changes in ionic current or other measurable properties, which can be used to analyze the molecule of interest's characteristics, such as its sequence or structure. The dimensions and surface properties of the aperture can be tailored to suit specific types of molecules of interest or analytical purposes. As used herein, and unless otherwise specified, the term "entry" refers to the opening or region of the aperture through which a molecule of interest first enters during the translocation process. Specifically:

[0083] (a) It is typically located on one side of the substrate containing the aperture and (b) It typically serves as the initial point of contact between the molecule of interest and the aperture.

[0084] As used herein, and unless otherwise specified, the term "exit" refers to the opening or region of the aperture through which a molecule of interest emerges after passing through the aperture. Specifically:

[0085] (a) It is typically located on the opposite side of the substrate containing the aperture, relative to the entry; and (b) It typically represents the final point of contact between the molecule of interest and the aperture during translocation.

[0086] In both cases, the terms "entry" and "exit" imply a directional flow of molecules of interest through the aperture, which is advantageous for the ordered analysis of molecular properties during translocation.

[0087] As used herein, and unless otherwise specified, the phrase "Means for causing the translocation of the molecule of interest from the entry to the exit" refers to any mechanism or system employed to drive the movement of a molecule of interest through the aperture in a controlled manner. This may include, but is not limited to:

[0088] (a) Electrical means: means for the application of an electric field across the aperture, creating an electrophoretic force that drives charged molecule of interests (such as DNA) through the pore;

[0089] (b) Electrochemical means: means for creating chemical gradients in conjunction with electrical fields to influence molecular movement;

[0090] (c) Mechanical means: means for creating pressure differentials or mechanical actuators to push molecules of interest through the aperture;

[0091] (d) Magnetic means: The use of magnetic fields to control the movement of magnetically labeled molecules of interest;

[0092] (e) Thermal means: The use of temperature gradients or localized heating to induce molecular movement;

[0093] (f) Combinations of the above: Systems that integrate two or more of these approaches for enhanced control over molecular translocation.

[0094] The specific means employed may vary depending on the type of molecule of interest being analyzed, the desired translocation characteristics, and the overall goals of the device. As used herein, and unless otherwise specified, the term "channel" refers to a microfluidic structure within the device that is fluidically coupled with the aperture and is suitable for guiding molecules of interest towards the aperture. Specifically:

[0095] (a) It has a width larger than the width of the entry of the aperture; (b) It can be fabricated from various materials compatible with the device's purpose, such as silicon, glass, or polymers; (c) It may incorporate surface modifications or patterns to control fluid flow or molecular interactions; (d) It may serve as a controlled environment for the initial positioning and movement of molecular assemblies before they enter the aperture. It is typically straight and uniform in cross-section, but it may also have bends and / or see its cross-section vary along its length.

[0096] The presence of the channel is advantageous in pre-positioning and orienting molecules of interest for optimal interaction with the aperture, thereby enhancing the efficiency and control of the molecular translocation process.

[0097] As used herein, and unless otherwise specified, the term "width" as applied to the channel and the entry of the aperture refers to the dimension perpendicular to the direction of molecular travel in both the channel and the aperture entry; in other words, for the channel, it is the smallest dimension of at least one cross-section perpendicular to its longitudinal axis, and for the aperture entry, it is the diameter or smallest dimension of the opening.

[0098] As used herein, and unless otherwise specified, the term "copolymer (200)" refers to a polymer composed of two or more different types of monomers. Examples of copolymers include, but are not limited to, block copolymers, alternating copolymers, and random copolymers. Biopolymers such as proteins and nucleic acids are also considered copolymers for the purposes of this disclosure.

[0099] As used herein, and unless otherwise specified, the term "walking molecule (50)" refers to a biomolecule (e.g., a protein or protein complex) or a synthetic molecule that can bind to a substrate and move along it in a directed manner. This movement is typically driven by the conversion of chemical energy into mechanical work such as by the hydrolysis of ATP or other energy sources. Examples of walking biomolecules include, but are not limited to, kinesins, dyneins, and myosins. Examples of walking synthetic molecules are DNA walkers.

[0100] As used herein, and unless otherwise specified, the term "motor region" of a walking molecule, also known as the motor domain or head domain, is the part of the walking molecule protein responsible for generating movement along a track. This region binds to the track (e.g., filament or DNA strand) and undergoes conformational changes powered by chemical energy (e.g., ATP hydrolysis), allowing the walking molecule to walk along the track. The motor region of a processive motor protein, also known as the motor domain or head domain, is the part of the protein responsible for generating movement along a filament, such as microtubules or actin filaments. This region binds to the track (filament) and undergoes conformational changes powered by ATP hydrolysis, allowing the protein to walk along the filament.

[0101] As used herein, and unless otherwise specified, the term "track" refers to a structure or surface that can bind and guide the movement of a walking molecule. The track may comprise a plurality of primary functional groups aligned along a substrate (46), each being adapted for binding with a secondary functional group of the walking molecule. Examples of tracks include, but are not limited to, microtubules, actin filaments, sequence of DNA strands (for DNA walkers), and engineered surfaces with specific binding sites for motor proteins.

[0102] As used herein, and unless otherwise specified, the term "sensor" refers to any device or system that can detect and / or analyze the molecule of interest as it translocates through the aperture. The sensor may operate independently of the aperture, may utilize effects arising from the interaction of the molecule of interest with the aperture, or may be a sensor system combining a sensor that operates independently of the aperture and a sensor that utilize effects arising from the interaction of the molecule of interest with the aperture. The sensor may may be located in the aperture, may be located outside of the aperture, or may be a sensor system combining a sensor that is located in the aperture and a sensor that is located outside of the aperture.

[0103] Examples of sensors include, but are not limited to: optical sensors, such as those detecting fluorescence signals from labeled molecules of interest or surface-enhanced Raman spectroscopy signals; electrical sensors, such as those detecting changes in ionic current or tunneling current; and force sensors for detecting changes in mechanical properties of the molecule of interest during translocation. The sensor may also utilize combinations of these sensing methods.

[0104] As used herein, and unless otherwise specified, the term "molecular assembly" refers to a complex comprising one or more walking molecules coupled to the molecule of interest. The molecular assembly is introduced into the channel of the device, and the motor proteins bind to the track, enabling the directed movement of the molecule of interest towards the aperture. As used herein, and unless otherwise specified, the phrase "aligned with the entry of the aperture" refers to a spatial relationship between the track in the channel and the entry of the aperture so that the longitudinal axis of a portion of the track closest to the aperture is oriented towards the aperture entry, preferably toward the center of the aperture entry. In other words, the track is arranged respective to the aperture so that it ensures that molecules of interest guided by the track are directed towards the aperture entry. The alignment facilitates the efficient transfer of molecules of interest from the track to the aperture. Alignment increases the probability of successful molecule of interest capture by the aperture.

[0105] As used herein, and unless otherwise specified, the phrase "the width of the track gradually decreases from the farthest side to the closest side" refers to the tapered or funnel-like shape of the track within the channel as it approaches the aperture entry. The "farthest side" refers to the edge of the track that is most distant from the aperture entry, while the "closest side" refers to the edge nearest to the aperture entry. "Gradually decreases" indicates a progressive reduction in width, rather than an abrupt change. This tapering configuration helps capture the walking molecules at proximity to the farthest side and guide and concentrate these walking molecules towards the aperture entry. The gradual narrowing may be linear (see Fig. 1) or non-linear (e.g. curved or comprise multiple steps as in Fig. 8), as long as there is an overall reduction in width from the farthest to the closest side and that this reduction is not a one-step abrupt reduction. This design feature enhances the efficiency of molecule of interest capture by the aperture by funneling the molecular assemblies towards the aperture entry. This tapering track design is advantageous for improving the probability of successful molecular translocation through the aperture by guiding the walking molecules and their attached molecules of interest towards the aperture entry in a controlled manner.

[0106] As used herein, and unless otherwise specified, the phrase "microtubules with their plus side oriented towards the entry of the aperture" refers to a specific arrangement of microtubule structures within the track of the channel. "Microtubules" are cylindrical polymers of tubulin protein, typically found in the cytoskeleton of eukaryotic cells. The "plus side" (or plus end) of a microtubule refers to the end where tubulin subunits are predominantly added during polymerization, characterized by faster growth and greater dynamic instability. "Oriented towards" indicates that the plus sides of the microtubules are consistently arranged to face the direction of the aperture entry. This orientation creates a directional path for certain types of walking molecules (particularly kinesins) that preferentially move towards the plus end of microtubules. The arrangement facilitates the guided movement of motor protein-molecule complexes along the track towards the aperture entry. This consistent orientation helps ensure that the processive motion of the motor proteins transports the attached molecules of interest in the desired direction - towards the aperture entry. This specific microtubule orientation is advantageous as it harnesses the natural directionality of certain motor proteins to guide molecules of interest efficiently and consistently towards the aperture entry, thereby enhancing the overall efficiency and control of the molecule of interest translocation process.

[0107] As used herein, and unless otherwise specified, the phrase "microtubules with their minus side oriented towards the entry of the aperture" refers to the opposite arrangement of that described for the plus side orientation. The "minus side" (or minus end) of a microtubule is characterized by slower growth and greater stability compared to the plus end. This orientation is particularly suitable for dynein motor proteins, which move towards the minus end of microtubules. As with the plus side orientation, this arrangement facilitates the guided movement of motor protein-molecule complexes along the track, leveraging the natural directionality of motor proteins to control the translocation process. The choice between plus and minus side orientation depends on the specific motor proteins and experimental design, offering flexibility in the system's configuration.

[0108] As used herein, and unless otherwise specified, the phrase " Means for providing a molecular assembly in the channel of the device" refers to any mechanism or system that introduces and positions the molecular assembly within the channel of the device. The "molecular assembly" typically consists of one or more walking molecules coupled to the molecule of interest (such as DNA, RNA, or proteins). These means may include, but are not limited to, microfluidic injection systems, pipetting techniques, or controlled diffusion methods. The provision of the molecular assembly may involve steps such as sample preparation, dilution, and introduction. The coupling between the molecule of interest (200) and the walking molecules (50) may be achieved through biochemical or chemical conjugation techniques adapted to the functional groups present on both molecules. One common method involves covalent bond formation, such as amide bond formation between amine groups on the walking molecules and carboxyl groups on the molecule of interest, typically facilitated by carbodiimide-mediated reactions (e.g., using EDC or DCC), resulting in a stable amide linkage. Another approach is thiol-maleimide chemistry, where thiol groups on the walking molecules react with maleimide groups on the molecule of interest, forming a highly stable thioether bond. Additionally, click chemistry, particularly copper(l)-catalyzed alkyne-azide cycloaddition (CuAAC), can be employed if the walking molecules and the molecule of interest are functionalized with azide and alkyne groups, respectively, allowing for the formation of a stable triazole linkage. Non-covalent strategies may also be used, such as biotin-streptavidin interaction, where a biotinylated molecule of interest is linked to a walking molecule containing streptavidin, forming a highly specific and stable non-covalent bond. DNA hybridization is another option, particularly when the walking molecules and the molecule of interest are DNA-based, allowing them to be linked via complementary singlestranded sequences. Enzyme-mediated coupling can also be utilized, such as sortase- mediated ligation, where the sortase enzyme catalyzes the formation of a covalent bond between a specific protein or peptide sequence on the molecule of interest and the walking molecule. Once the appropriate conjugation technique is applied, the molecule of interest (200) and the walking molecules (50) are securely linked, forming the molecular assembly (300) that is ready to interact with the device's track (40) for procession and translocation.

[0109] As used herein, and unless otherwise specified, the phrase "Means for causing the procession, along the channel and toward the aperture" refers to any mechanism or system that initiates and maintains the movement of the walking molecules along the track in the channel towards the aperture entry. This may include, but is not limited to, means for the provision of ATP or other energy sources to fuel the motor proteins, means for the creation of chemical gradients, or means for the application of external forces such as electric fields.

[0110] As used herein, and unless otherwise specified, the term "control unit" refers to a computer, microprocessor, or any other device capable of executing instructions to control the operation of the system and perform the steps of the claimed methods. The control unit may be integrated into the device or may be a separate component connected to the device.

[0111] The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the technical teaching of the invention, the invention being limited only by the terms of the appended claims.

[0112] In the first aspect, the present invention relates to a device for processing a molecule of interest, comprising: an aperture having an entry and an exit, the aperture being suitable for permitting the translocation of the molecule of interest from the entry to the exit; means for causing the translocation of the molecule of interest from the entry to the exit; and a channel fluidically coupled with the aperture and suitable for guiding the molecule of interest towards the aperture. The channel comprises a track adapted for binding a motor region of a walking molecule and adapted for the procession of said walking molecule towards the entry of the aperture.

[0113] As illustrated in the particular embodiment of Fig. 1, the device (100) is designed for processing a molecule of interest (200) using an aperture (10). The device comprises a channel (30) fluidically coupled with the aperture (10), which has an entry (11) and an exit (12). In this particular embodiment, the channel (30) has a transversal cross-section which has a constant width (31) and is wider than the width (13) at the entry (11) of the aperture (10). It includes a track (40) adapted for binding a motor region (51, not depicted in this Fig.) of a walking molecule (50). The walking molecule is depicted as being bound to a molecule of interest (200), thereby forming a molecular assembly (300). The track (40) facilitates the procession of the motor protein (50) towards the aperture (10). A side of the track farthest from the aperture is depicted a having a width larger than the width of the aperture, and the width of the track is shown to decrease gradually from the farthest side to the closest side, creating a funnel shape that helps capture the motor protein and guide it to the aperture. Hence, in embodiments, e.g., in the present embodiment, the width (42) may be smaller than the width (13). In the depicted embodiment, the track (40) has a side (41), closest to the entry (11) of the aperture (10), which has a width (42) at most equal to the 1.5 times the width (13) of the aperture (10). This side (41) may be wider than the aperture because the capture radius can be wider than the radius of the aperture. For instance, the capture radius can be up to 1.5 times larger than the radius of the aperture. This is visible in Fig. 8 which is another top view of a device according to embodiments of the present invention. Fig. 8 illustrates a schematic representation of a device (100) for analyzing a molecule according to embodiments of the present invention. The device includes an aperture (10) with an entry (11) and an exit (not depicted), adapted for translocating a molecule of interest (200) from the entry (11) to the exit. The aperture (10) is depicted as a circular opening, indicating its function as a passage for the molecule of interest (200). The aperture (10) is surrounded by a capture radius (16), which is visually represented by a circular boundary around the aperture, indicating the effective area within which the molecule of interest (200) can be captured and guided into the aperture. The drawing also shows arrows pointing towards the aperture, representing the directional flow of the molecule of interest (200) captured by the aperture, driven by an external force, such as an electrical field. The aperture (10) is within a channel (30) fl uidically coupled with the aperture, suitable for guiding the molecule of interest (200) towards the aperture.

[0114] Within the channel (30), the track (40) is adapted for binding a motor region (51) of a walking molecule (50) and facilitating the procession of the motor protein towards the entry (11) of the aperture. The track (40) is represented by a series of horizontal lines within the channel, indicating the path along which the motor protein (50) moves. The molecular assembly (300) is depicted as a complex structure with a polymer (200) attached to a motor protein (50), visually represented by a chain-like structure extending from the motor protein. The long horizontal arrows represent the channel flow. The bottom panel of Fig. 8 represents a state where the molecular assemblies have progressed along the track with respect to the top panel.

[0115] In embodiments, the track may comprise tubulin bands (or other functionalized surfaces that mimic different parts of the cytoskeleton and its microfibers), allowing attachment of the motor protein motor region. In embodiments, the molecule may be selected from the list consisting of lipids, sugars, and copolymers, allowing processing of a wide range of molecules. The molecule may be a copolymer, preferably a biopolymer, which is of particular interest for analysis. The copolymer may be a biopolymer selected from polypeptides with or without post-translational modifications, and nucleic acids, which are key biological molecules to analyze.

[0116] Preferably, only the channel, and not the aperture, comprises the track, preventing the track from interfering with translocation through the aperture. The track may be aligned with the entry of the aperture to guide the processing motor protein towards the entry of the aperture, as also shown in Fig. 2, which illustrates a vertical cross-section through the length of the channel (30) of the device (100). In the depicted embodiment, the device further comprises a sensor (60) for analyzing the molecule during its translocation through the aperture, enabling real-time analysis, and means (20) for causing the translocation of the molecule of interest (200) from the entry (11) to the exit (12). These means can, e.g., be electrodes.

[0117] We now refer to Fig. 2 which shows a vertical cross-section through the length of the channel (30) of a device (100) according to embodiments of the present invention. The drawing depicts the channel (30) as a rectangular structure extending horizontally, with an aperture (10) located at one end. The aperture (10) is depicted as a rectangular structure extending vertically. The aperture (10) has at least a second portion (14) comprising the entry (11), said second portion (14) having a second longitudinal axis (15), and the first longitudinal axis (33) makes an angle (a) of from 20° to 160° (here 90°) with the second longitudinal axis (15).

[0118] In embodiments, the device may utilize a channel flow to guide the motor protein complexes near the track for attachment, and then one or more of channel flow, a translocation flow, and an electrical field to pull the biopolymer through the aperture.

[0119] In the second aspect, the present invention relates to a method for processing a molecule, comprising: providing a device as claimed in any embodiments of the first aspect; providing a molecular assembly in the channel of the device, the molecular assembly comprising one or more walking molecules coupled to the molecule, and binding the motor region of the one or more walking molecules of the assembly to the track of the channel; causing the procession, along the channel and toward the aperture, of the one or more walking molecules of the assembly, while simultaneously causing a free end of the molecule of the assembly to enter the entry of the aperture and to progress toward the exit thereof, thereby causing the translocation of the molecule from the entry to the exit. This is illustrated in Fig. 3 which is a flow chart illustrating a method according to embodiments of the second aspect of the present invention. The flow chart outlines the sequential steps involved in the method, starting with the provision of the device (100) and the molecular assembly (300), followed by binding the motor region (51) to the track (40), causing procession along the channel (30), and ultimately along the aperture. An optional step of analyzing the molecule of interest (200) using a sensor (60) may be performed during the translocation of the molecule through the aperture (10). In such embodiments, the molecule may preferably be a copolymer, wherein the device further comprises a sensor for analyzing the copolymer during its translocation through the aperture. The method further comprises the step of analyzing the copolymer of the assembly as it translocates towards the exit of the aperture using the sensor, allowing real-time analysis of the copolymer.

[0120] As illustrated in Fig. 5, which depicts successive steps of the method in a vertical cross-sectional view of the device (100) along its longitudinal axis. The device (100) is designed for processing a molecule of interest (200) through an aperture (10). The aperture (10) is shown with an entry (11) and an exit (12), facilitating the translocation of the molecule of interest (200) from the entry (11) to the exit (12). The channel (30) is depicted as having a width (31) larger than the width (13) of the aperture's entry (11), and the track (40) is designed to facilitate the procession of the motor protein (50) towards the aperture's entry (11). Within the channel (30), a track (40) is visible, which is adapted for binding a motor region (51) of a walking molecule (50) and facilitating the procession of the motor protein (50) towards the entry (11) of the aperture (10).

[0121] The track (40) is depicted as a series of aligned primary functional groups (45) along a substrate (46), which interact with secondary functional groups (52) of the walking molecule (50). The motor protein (50) is shown bound to the track (40), moving directionally along it due to the application of a driving force.

[0122] The molecule of interest (200) is depicted as a biopolymer, such as a polypeptide or nucleic acid, coupled to the walking molecule (50). In the lower panels, the drawing shows the molecule of interest (200) being guided towards the aperture (10) by the motor protein (50), which moves along the track (40). The aperture (10) is a solid-state aperture, with its transversal cross-sections having a width (13) of at most 100 nm and at least one transversal cross-section with a width (13) of from 2 to 20 nm.

[0123] The device (100) includes means (20) for causing the translocation of the molecule of interest (200) from the entry (11) to the exit (12) of the aperture (10), which may involve creating an electrical field between the entry (11) and the exit (12). The sensors (60) are capable of detecting changes in ionic current or tunneling current as the molecule of interest (200) translocates through the aperture (10), providing analysis of the molecule of interest (200) during its translocation.

[0124] In embodiments, causing a free end of the copolymer of the assembly to enter the entry of the aperture and to progress toward the exit thereof may comprise creating an electrical field between the entry and the exit of the aperture, which drives translocation. Analyzing the copolymer of the assembly, as it translocates towards the exit of the aperture, using the sensor may comprise detecting changes in ionic current or tunneling current as the copolymer translocates through the aperture.

[0125] In a third aspect, the present invention relates to a system (400) for analyzing a copolymer, comprising: a. A device (100) according to any embodiment of the first aspect, b. Means (80) for providing a molecular assembly (300) in the channel (30) of the device (100), the molecular assembly (300) comprising one or more walking molecules coupled to the molecule, c. Means (90) for controlling the speed of (or for causing) the procession, along the channel (30) and toward the aperture (10), of the one or more walking molecules (50) of the assembly (300), and d. A control unit (500) operatively connected to the device (100) and configured to cause the system (400) to execute the steps of the method as claimed in any embodiment of the second aspect.

[0126] Fig. 4 illustrates a system (400) for analyzing a copolymer, comprising a device (100), means (80) for providing a molecular assembly (300) in the channel (30) of the device (100), means (90) for controlling the speed of the processing (or for causing the procession), along the channel (30) and toward the aperture (10), of the one or more walking molecules (50) of the assembly (300), and a control unit (500) operatively connected to the device (100) and configured to cause the system (400) to execute the steps of the method according to any embodiment of the second aspect.

[0127] The device (100) includes a channel (30) fl uidically coupled with an aperture (10). The aperture (10) has an entry (11) and an exit (not depicted), and is adapted for translocating the molecule of interest (200) from the entry (11) to the exit (12). The channel (30) is suitable for guiding the molecule of interest (200) towards the aperture (10). The width (31) of the channel (30) is larger than the width (13) of the entry (11) of the aperture (10). The channel (30) comprises a track (not shown) adapted for binding a motor region of a walking molecule (not shown) and adapted for the procession of said walking molecule towards the entry (11) of the aperture (10).

[0128] The means (80) for providing a molecular assembly (300) in the channel (30) of the device (100) are depicted as a separate block connected to the device (100). Similarly, the means (90) for controlling the speed of the procession (or for causing the procession) of the walking molecules (50) along the channel (30) and toward the aperture (10) are shown as another separate block connected to the device (100). In embodiments, said means (90) may be a system for providing in the channel (30) an activator like ATP or inhibitors. Activator or inhibitors may be used to adjust the resistance provided by the motor protein, enabling fine-tuning of the translocation speed for specific polymer types to optimize the speed-accuracy ratio. In embodiments, causing the procession may comprise contacting the one or more walking molecules with ATP, which fuels the movement of the motor proteins.

[0129] The control unit (500) is operatively connected to the device (100) and is configured to cause the system (400) to execute the steps of the method as claimed in any embodiment of the second aspect. The control unit (500) is depicted as a large block, indicating its operational connection to the other components of the system (400). The system (400) is designed to facilitate the analysis of copolymers by controlling the procession of walking molecules (50) along the track (40) in the channel (30) and guiding the molecule of interest (200) through the aperture (10) for analysis.

[0130] As depicted in Fig. 6, the drawing illustrates a problem encountered in the translocation of molecules through an aperture (10) when using a walking molecule (50) to control the movement. The figure highlights the issue of uncontrolled rapid translocation of the molecule of interest (200) end segment when the motor protein (50) is in relative close proximity to the aperture (10), leading to inaccuracies in the measurement and analysis of the molecule of interest (200). The top panel shows the molecular assembly shortly after it bound to the track. The middle panel shows that the end segment (in the rectangle) of the molecule already passes though the aperture even before the motor protein started to move. The lower panel shows that, because the motor protein will not be able to reach the entry of the aperture, a segment of the molecule attached to the motor protein (in the rectangle) will pass through the aperture without being slowed down by the motor protein. These dead ends (both extremities of the molecule) will not be analyzed as well as the rest of the molecule.

[0131] To address this issue, Fig. 7 illustrates an embodiment designed to ensure a controlled and averaged translocation speed of the molecule of interest (200) through the aperture (10). In this embodiment, two or more walking molecules may be coupled to the copolymer so that different locations along the copolymer are coupled to a walking molecule, providing smoother translocation.

[0132] In embodiments, short polymer segments may be linked together into a repeating chain, transforming them into a longer repeating sequence suitable for the motor proteinaperture system. In embodiments, the copolymer may comprise concatenated identical monomer sequences, said concatenated identical monomer sequences making up at least 50%, preferably at least 70%, more preferably at least 90% of the copolymer or even 100% of the copolymer. The repeating sequences enable more reliable analysis. The one or more walking molecules may all be identical. In embodiments, they may for instance be myosins, kinesins, or dyneins.

[0133] In the third aspect, the present invention relates to a system for analyzing a copolymer, comprising: a device as claimed in any embodiments of the first aspect; means for providing a molecular assembly in the channel of the device; means for causing the procession, along the channel and toward the aperture, of the one or more walking molecules of the assembly; and a control unit operatively connected to the device and configured to cause the system to execute the steps of the method as claimed in any embodiments of the second aspect.

[0134] In the fourth aspect, the present invention relates to a computer program comprising instructions which, when executed by the control unit of the system of the third aspect, cause the system to execute the steps of the method as claimed in any embodiments of the second aspect.

[0135] In the fifth aspect, the present invention relates to a computer-readable medium having stored thereon the computer program as claimed in the fourth aspect.

[0136] In the sixth aspect, the present invention relates to the use of one or more processing motor proteins for transporting a copolymer in a channel toward the entry of an aperture of a device.

[0137] Example 1: Single Kinesin Motor Protein Attachment for DNATranslocation Control

[0138] In this example, illustrated in Fig. 5, a novel method is developed to enable higher precision readout of DNA molecules using a single kinesin motor protein attached to each DNA strand in combination with aperture-based sensing. The aim is to fine-tune the translocation speed by leveraging the walker motion of kinesin motors, addressing the challenges of excessive speed and lack of control in free translocation through solid-state apertures.

[0139] Materials used in this study include ATP (adenosine triphosphate), kinesin motor proteins, microtubules, and DNA molecules of various lengths. The experimental setup consists of a microfluidic channel with a solid-state aperture at one end. The channel surface is functionalized with microtubules arranged in a linear pattern, with their plus ends oriented towards the aperture.

[0140] DNA molecules, each attached to a single kinesin motor protein, are introduced into the channel. The kinesin-DNA complexes are captured by the microtubule tracks and begin to "walk" along them towards the aperture. The movement of the kinesin motors is powered by ATP hydrolysis, with a characteristic speed of approximately 800 nm / s.

[0141] As the kinesin-DNA complexes approach the aperture, an electrical field is applied to create a translocation force. This force, combined with the walking motion of the kinesin, results in a controlled insertion and translocation of the DNA through the aperture. Changes in the ionic current between electrodes placed near the aperture are measured, indicating the nature of the DNA bases passing through. The use of a single kinesin motor protein on each DNA strand slows down the translocation compared to free translocation, providing a "medium" alternative between the fast but inaccurate solid-state pores and the accurate but slow biological pores. The walking speed of kinesin motors (approximately 800 nm / s) is much slower than free DNA translocation, allowing for improved temporal resolution of the ionic current measurements.

[0142] The introduction of ATP allows for fine-tuning of the translocation speed by adjusting the walking rate of the kinesin motors. By modulating the ATP concentration, the speed can be optimized for different DNA lengths or aperture geometries. However, some variation in translocation speed is still observed between different molecules, as each DNA strand is controlled by only one motor protein.

[0143] Example 2: Multiple Kinesin Motor Protein Attachment for Enhanced DNA Translocation Control

[0144] This example, shown in Fig. 7, builds upon the previous method by attaching multiple kinesin motor proteins to each DNA molecule. The aim is to further improve translocation control and reduce speed variations between molecules.

[0145] The experimental setup is similar to Example 1, but now each DNA molecule is treated and attached to multiple kinesin motor proteins along its length. The channel surface is functionalized with microtubules arranged in a funnel-shaped pattern, with the narrow end of the funnel aligned with the aperture entrance. This arrangement guides the kinesin-DNA complexes towards the aperture, significantly increasing the capture rate compared to random diffusion or the linear track in Example 1.

[0146] As the DNA molecules are pulled through the aperture, the multiple attached kinesin motors work in concert to control the translocation. The pulling force from the electrical field, combined with the presence of ATP, induces the kinesin motors to "walk" step by step along the microtubules, allowing for even more precise control of the DNA's movement through the aperture.

[0147] The use of multiple kinesin motors on a single DNA strand not only slows down the translocation but also averages out the speed, significantly reducing the variation in translocation speed observed in both free translocation and the single motor protein method. This approach provides a more consistent "medium" speed alternative between fast solid-state pores and slow biological pores. Furthermore, the ability to fine-tune the translocation speed by adjusting the ATP concentration is enhanced with multiple motors. The collective behavior of the motors provides a more stable and controllable system. This method addresses the issues of excessive speed and speed variation in solid-state apertures more effectively than the single motor protein approach.

[0148] The results of this multiple kinesin motor approach demonstrate several significant advantages over conventional methods. The controlled translocation speed, achieved through the collective action of multiple motors, provides a consistent and slower passage of DNA through the aperture compared to free DNA movement. This slower pace, coupled with the reduced speed variations between different molecules, leads to more uniform analysis conditions. The averaging effect of multiple motors attached to a single DNA strand effectively smooths out speed fluctuations, resulting in more consistent translocation rates across different samples.

[0149] Moreover, the slower and more controlled translocation process yields longer dwell times for individual DNA bases within the aperture. This extended presence of each base in the sensing region translates to an improved signal-to-noise ratio, enhancing the quality of the ionic current measurements and leading to better base discrimination. The system's flexibility is further enhanced by the ability to fine-tune the collective walking speed of the kinesin motors through adjustments in ATP concentration. This precise speed control, more refined than in the single-motor approach, allows researchers to optimize the translocation rate for various DNA lengths or specific aperture geometries, tailoring the system to different experimental requirements.

[0150] These combined advantages of enhanced control, reduced variability, improved signal quality, and tunable speed make the multiple kinesin motor method a promising approach for high-precision applications in aperture-based DNA sequencing. The method offers the potential for significant improvements in base-calling accuracy and overall consistency, addressing key challenges in the field of aperture sensing and pushing the boundaries of DNA analysis capabilities.

[0151] Example 3: Dynein Motor Protein for RNATranslocation Control

[0152] In this example, a system is developed to control the translocation of RNA molecules through an aperture using dynein motor proteins. The experimental setup consists of a microfluidic channel with a solid-state aperture at one end. The channel surface is functionalized with microtubules arranged in a funnel pattern, with their minus ends oriented towards the aperture. RNA molecules, each attached to a single dynein motor protein, are introduced into the channel. The dynein-RNA complexes are captured by the microtubule tracks and begin to move along them towards the aperture, powered by ATP hydrolysis. As the complexes approach the aperture, an electrical field is applied to create a translocation force. The combination of the dynein's movement and the electrical force results in controlled insertion and translocation of the RNA through the aperture. Changes in the ionic current are measured to determine the nature of the RNA bases passing through.

[0153] Example 4: Myosin Motor Protein for Polypeptide Analysis

[0154] This example showcases the application of the invention to polypeptide analysis using myosin motor proteins. A microfluidic device is fabricated with a channel leading to a biological aperture embedded in a lipid bilayer. The channel surface is coated with actin filaments arranged parallel to each other. Polypeptide molecules are attached to myosin V motor proteins and introduced into the channel. The myosin-polypeptide complexes bind to the actin filaments and move towards the aperture entrance. An electrochemical gradient is applied across the aperture to facilitate polypeptide translocation. The stepping motion of myosin V, combined with the electrochemical force, enables controlled passage of the polypeptide through the aperture. Ionic current measurements can be used to identify amino acid sequences, indicating the invention's capability in protein sequencing applications.

[0155] Example 5: Sugar Polymer Analysis Using Engineered Motor Proteins

[0156] This example showcases the versatility of the invention in analyzing sugar polymers such as cellulose or chitin. Engineered kinesin motor proteins with cellulose-binding domains are created to interact specifically with sugar polymers. These motor proteins are used to control the translocation of cellulose fibers through a solid-state aperture. The microfluidic channel is functionalized with microtubules arranged in a radial pattern converging at the aperture. Cellulose fibers attached to the engineered kinesin motors are introduced into the channel. The motor proteins guide the cellulose fibers along the microtubules towards the aperture, where an electrical field facilitates their translocation. Ionic current measurements during translocation provide information about the structure and composition of the sugar polymers. It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope of this invention. For example, any formulas given above are merely representative of procedures that may be used. Functionality may be added or deleted from the block diagrams and operations may be interchanged among functional blocks. Steps may be added or deleted to methods described within the scope of the present invention.

Claims

28Claims1. A device (100) for processing a molecule of interest (200), comprising: a. An aperture (10) having an entry (11) and an exit (12), the aperture (10) being suitable for enabling the translocation of the molecule of interest (200) from the entry (11) to the exit (12), b. Means (20) for causing the translocation of the molecule of interest (200) from the entry (11) to the exit (12), and c. A channel (30) fluidically coupled with the aperture (10) and suitable for guiding the molecule of interest (200) towards the aperture (10),Wherein the channel (30) comprises a track (40) adapted for binding a motor region (51) of a walking molecule (50) and adapted for the procession of said walking molecule (50) towards the entry (11) of the aperture (10).

2. The device (100) according to claim 1, wherein the molecule of interest (200) is a copolymer, preferably a biopolymer.

3. The device (100) according to claim 1 or claim 2, further comprising a sensor (60) for analyzing the molecule of interest (200) during its translocation through the aperture (10).

4. The device (100) according to any one of the preceding claims, wherein the track (40) is aligned with the entry (11) of the aperture (10) so as to guide the processing motor protein (50) towards the entry (11) of the aperture (10).

5. The device (100) according to claim 4, wherein a side (41) of the track (40) closest to the entry (11) of the aperture (10) has a width (42) at most equal to the 1.5 times the width (13) of the aperture (10).

6. The device (100) according to claim 4 or claim 5, wherein a side (43) of the track(40) farthest to the aperture (10) has a width (44) larger than the width (13) of the aperture(10) and wherein the width of the track (40) decreases, preferably gradually decreases, from the farthest side (43) to the closest side (41).

7. The device (100) according to any one of the preceding claims, wherein the track (40) comprises microtubules (47) with their plus side oriented towards the entry (11) of the aperture (10).

8. The device (100) according to any one of the preceding claims, wherein at least a first portion (32) of the channel (30) is directly fluidically connected to the entry (11) of the aperture (10), wherein said first portion (32) has a first longitudinal axis (33), wherein the aperture (10) has at least a second portion (14) comprising the entry (11), said second portion (14) having a second longitudinal axis (15), and wherein the first longitudinal axis (33) makes an angle (a) of from 20° to 160°, preferably from 40° to 140°, more preferably from 60° to 120°, yet more preferably from 80° to 100°, and most preferably 90° with the second longitudinal axis (15).

9. The device (100) according to any one of the preceding claims, comprising the walking molecule (50) coupled to the molecule of interest (200), said walking molecule (50) being bound by the track (40).

10. A method for processing a molecule of interest (200), comprising: a. Providing a device (100) as claimed in any one of claims 1 to 9, b. Providing a molecular assembly (300) in the channel (30) of the device (100), the molecular assembly (300) comprising one or more walking molecules (50) coupled to the molecule of interest (200), and binding the motor region (51) of the one or more walking molecules (50) of the assembly (300) to the track (40) of the channel (30), c. Causing the procession, along the channel (30) and toward the aperture (10), of the one or more walking molecules (50) of the assembly (300), while simultaneously causing a free end of the molecule of interest (200) of the assembly (300) to enter the entry (11) of the aperture (10) and to progress toward the exit (12) thereof, thereby causing thetranslocation of the molecule of interest (200) from the entry (11) to the exit (12).

11. The method according to claim 10, wherein the molecule of interest (200) is a copolymer, wherein the device (100) further comprises a sensor (60) for analyzing the copolymer during its translocation through the aperture (10), and wherein the method further comprises the step d, after step c, of analyzing the copolymer of the assembly (300), as it translocates towards the exit (12) of the aperture (10), using the sensor (60).

12. The method according to claim 10 or 11, wherein two or more walking molecules (50) are coupled to the molecule so that different locations along the molecule are coupled to a walking molecule (50).

13. A system (400) for analyzing a copolymer, comprising: a. A device (100) as claimed in any one of claims 1 to 9, b. Means (80) for providing a molecular assembly (300) in the channel (30) of the device (100), the molecular assembly (300) comprising one or more walking molecules coupled to the molecule, c. Means (90) for controlling the speed of the procession, along the channel (30) and toward the aperture (10), of the one or more walking molecules (50) of the assembly (300), and d. A control unit (500) operatively connected to the device (100) and configured to cause the system (400) to execute the steps of the method as claimed in any one of claims 10 to 12.

14. A computer program comprising instructions which, when executed by the control unit (500) of the system (400) of claim 32 cause the system (400) to execute the steps of the method as claimed in any one of claims 23 to 31.

15. A computer-readable medium (600) having stored thereon the computer program as claimed in claim 33.

16. Use of one or more processing motor protein (50) for transporting a molecule in a channel (30) toward the entry (11) of an aperture (10) of a device (100).

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