METHOD FOR ELECTROSTATIC TRAPPING OF MOLECULES IN NANOPORES

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of U.S. Provisional Patent Application Serial No. 60/905,997, filed March 9, 2007, entitled "Electrostatic Trapping of Adapter Molecules in Ion Channels and Nanopores for Improved Ion Channel Sensing, Increased Temporal Translocation Control, and Detection Sensitivity in Nucleic Acid Detection and Sequencing", U.S. Provisional Patent Application Serial No. 60/906,993, filed March 13, 2007, entitled "Electrostatic Trapping of Adapter Molecules in Ion Channels and Nanopores for Improved Ion Channel Sensing, Increased Temporal Translocation Control, and Detection Sensitivity in Nucleic Acid Detection and Sequencing", U.S. Provisional Patent Application Serial No. 60/919,694, filed March 23, 2007 entitled "Nanopore Platforms for Ion Channel Recordings and

Single Molecule Detection and Analysis" and 60/926,122, filed April 24, 2007 entitled "Electrostatic Trapping of Adapter Molecules in Ion Channels and Nanopores for Improved Ion Channel Sensing, Increased Temporal Translocation Control, and Detection Sensitivity in Nucleic Acid Detection and Sequencing".

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under FA9550- 06-C-0006 awarded by the Department of Defense/Defense Advanced Research Projects Agency and under CHE-0616505 awarded by the National Science Foundation. The Government has certain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention pertains to the art of biotechnology and nanotechnology. In particular, the invention relates to a method for altering the constriction zone within protein channel size barrel(s) or lumen(s). Such methods have use in various areas, such as chemical sensors and nucleic acid sequencing.

2. Discussion of the Prior Art

Pore-forming proteins are presently being engineered for applications in biotechnology. For instance, work has been conducted on alpha-hemolysin (alpha-HL), a bacterial toxin that forms a heptameric trans-membrane pore of known three-dimensional structure. In general, it is known to use genetic engineering and targeted chemical modification to create pores having diverse functional properties. In addition, transmembrane beta-barrels are being engineered by combining fragments from various bacterial toxins and porins, as well as polypeptide segments

designed de novo. Such molecules find applications in several areas including drug delivery and the construction of biosensors.

One type of useful biosensor is the ion channel sensor. One type of ion channel sensing constitutes passing a molecule through an ion channel or protein pore present in a lipid bilayer membrane, such that each nucleotide in the molecule obstructs the pore to a different degree, causing current passing through the pore at any given moment to vary depending on the nucleotide present. One application of ion channel sensors is in nucleic acid sequencing (e.g., DNA or RNA sequencing). In such an application, DNA is driven through a biological or synthetic pore by an electrical voltage. However, key roadblocks to such sequencing methods include: (1) a fast DNA translocation rate (~2 microseconds/base (μs/base)) prevents simple electrical data acquisition, and (2) the diameter of the constriction zone in a pore may be too large to generate an electrical signature unique to a base as the base passes through the pore. For example, alpha-HL has a constriction zone of ~1.4 nanometers (nm).

Adapter molecules, such as beta-cyclodextrin, have been utilized in ion channel recordings to increase detection sensitivity. However, adapter molecules reside only briefly within the barrel or lumen of a pore. For example, the adaptor molecule heptakis (6-O-sulfo)-β-cyclodextrin (s7-βCD), resides for ~1 second within a pore when a voltage of -0.04 volts (V) is applied across the lipid bilayer membrane (trans relative to cis). The molecule then diffuses out of the barrel of the pore. This transient binding and unbinding greatly reduces the potential utility of adapter molecules in sensor applications.

With the above in mind, there exists a need for an improved ion channel sensing method, wherein control over translocation of a molecule through a pore is increased along with system sensitivity.

SUMMARY OF THE INVENTION

The present invention is directed to a method for electrostatic trapping of molecules in nanopores. More specifically, the method of the present invention provides a means for altering the constriction zone within the size barrel or lumen of a protein channel or channels within an electrostatic sensing system. The electrostatic sensing system includes a glass membrane with a lipid bilayer extending across a nanopore opening in the membrane. An ion channel or protein channel, such as alpha-HL, is inserted into the lipid bilayer and an adaptor molecule is introduced to the system. Preferably, the adaptor molecule constitutes a cyclodextrin molecule.

A trans-membrane voltage of between about -40 and -800 millivolts (mV) is applied via system electrodes and current through the ion channel is measured by a sensor. When an adaptor molecule is sensed within the barrel of the ion channel, the voltage is increased to above -100 mV, and the adaptor molecule is trapped within the barrel for an indefinite period of time. The ion channel/adaptor molecule complex may then be utilized in the analyses of other molecules, such as in nucleic acid sequencing of DNA. The adaptor molecule may then be released from the ion channel by lowering the voltage to below -100 mV.

The present method provides significant advantages in application of single ion channels for chemical sensing and nucleic acid (e.g. DNA or RNA) sequencing. Additional objects, features and advantages of the present invention will become more readily apparent from the following detailed description of a preferred embodiment when taken in conjunction with the drawings wherein like reference numerals refer to corresponding parts in the several views.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic depiction of a sensing system utilized in the present invention, including a glass nanopore membrane;

Figure 2A depicts a glass nanopore membrane manufacturing process;

Figure 2B depicts a glass nanopore membrane produced by the manufacturing process of Figure 2A;

Figure 3 is an enlarged view of the nanopore of the system of

Figure 1;

Figure 4 is a simplified cross-sectional view of a protein channel/adaptor molecule complex;

Figure 5 is a graph of illustrating current versus time for trapping of a charged adapter molecule as a function of pressure; and

Figure 6 is a chart depicting current sensed over time at -40, -100, -120 and -200 mV utilizing the system of Figure 1.

DETAILED DESCRIPTION OF THE PREFERRED

EMBODIMENT

With initial reference to Figure 1 , an electrostatic sensing system utilized in accordance with the present invention is indicated at 10. In general, the system comprises a membrane 14 including a nanopore 16 located therein, electrodes 18 and 19, an internal solution indicated at 22, an external solution indicated at 26, a sensor 28, a voltage source 30 and a controller 31. In one embodiment of the present invention, system 10 also includes a pressure inducing apparatus 32 including a seal 34 for sealing an open end 35 of glass nanopore membrane 14. Electrodes 18 and 19, which are preferably silver/silver chloride (Ag/AgCl) electrodes, are utilized to bias a potential across nanopore membrane 14, with the potential being referenced to the internal solution 22. As shown, each of sensor 28, voltage source 30 and pressure inducing apparatus 32 are linked to controller (CPU) 36 for regulating the system as set forth below.

In a preferred embodiment, membrane 14 is a glass nanopore membrane constituted by a conical shaped nanopore 16 located in a thin glass membrane 17. In general, such a membrane 14 is manufactured utilizing a platinum (Pt) disk electrode 36 embedded at the bottom of a conical shaped pore 37 as shown in Figure 2 A. Pore 37 is then polished, and electrode 36 is etched out and removed, leaving a nanometer-sized opening or nanopore 16. As depicted in Figure 2B, the circular orifice of

nanopore 16 has a diameter of between approximately 5 and 5000 nm, and a glass membrane portion 17 has a thickness of approximately 20-75 μm. Advantageously, the steady-state flux of ions and molecules across glass nanopore membrane 14 is limited by transport through nanopore 16, aiding in the study of transport in nanometer-diameter pores.

Furthermore, the use of a glass nanopore membrane allow for higher trans-membrane voltages (-800 mV) to be applied than in prior systems (-30OmV).

As depicted in Figure 3, the exterior and interior surfaces of glass nanopore membrane 14 are chemically modified with 3- cyanopropyldimethylchlorosilane (represented by CN). A lipid bilayer indicated at 40 is deposited across nanopore 16 by painting techniques or other suitable methods, and a trans-membrane ion channel 44 is inserted through lipid bilayer 40. In the preferred embodiment, ion channel 44 constitutes alpha-hemolysin (alpha-HL), and lipid bilayer 40 is composed of l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Dilute solutions of monomer alpha-HL or heptamer alpha-HL may be utilized to avoid multiple insertions of protein ion channels in lipid bilayer 40.

A method for altering the constriction zone within a barrel or lumen 46 of ion channel 44 in accordance with the present invention will now be discussed with reference to Figures 1 and 3-5. Initially, system 10 is set up such that ion channel 44 extends through lipid bilayer 40 as depicted in Figure 3. Preferably, seal 34 is applied over open end 35 of glass nanopore membrane 14 and a positive trans-membrane pressure is applied through pressure inducing apparatus 32 to ensure stable ion channel insertion. Stable ion channel insertion occurs over a wide pressure range. In the

preferred method of the present invention, a pressure of between 40-300 mm Hg is applied as illustrated in Figure 5.

An adapter molecule(s), indicated at 50, is introduced into external solution 26. Adapter molecule 50 can be synthesized by standard chemical methods to produce a structure with desirable functional groups, electrical charge, and electrical dipoles. For example, with particular reference to Figure 4, adapter molecule 50 may have a cyclic structure with an inner diameter d that better matches the diameter of a nucleic acid to be sequenced, than the diameter D of an ion channel barrel 46. An adapter molecule 50 having the precise ideal structure for electrostatic trapping can be designed and synthesized, for the above applications. For example, commercially available adapters include alpha-cyclodextrin (4.7-5.3 A), β-cyclodextrin (6.0-6.5 A), and γ -cyclodextrin (7.5-8.3 A), each having a well defined pore diameter. These molecules can be modified to include charge groups and other functionality as desired. More specifically, the structure, size and charge of adapter molecule 50 may be optimized for different applications, e.g., DNA sequencing. Furthermore, adapter molecule 50 may be modified or chosen for optimal temporal translocation control of a molecule through barrel 46. In the preferred method of the present invention, adapter molecule 50 is a cyclodextrin. Most preferably, adapter molecule 50 is heptakis(6-O- sulfo)-β-cyclodextrin (s7-βCD). The preferred internal solution 22 is 1 μM alpha-HL, 1 M potassium chloride (KCl) and 10 mM phosphate buffer (PBS - pH 7.4), while the external solution 26 is 1 M KCl and 10 mM PBS (pH 7.4) containing 50 μM s7-βCD.

A DC voltage ranging between approximately -40 to -800 mV (trans relative to cis) is then applied via electrodes 18 and 19. An electrical parameter, such as current through ion channel 44, is measured by sensor 28. When a single adapter molecule 50 enters barrel 46 of ion channel 44, a characteristic drop in current is sensed by sensor 28 as a consequence of the increased resistance through ion channel 44. Once the presence of adapter molecule 50 within barrel 46 is sensed, a potential, such as between -120 and -800 mV, is applied and electrostatic trapping of adapter molecule 50 within barrel 46 occurs. Preferably, a potential in the order of -150 mV is utilized to insert adapter molecule 50 into barrel 46, and a disassociation voltage of less than -150 mV is utilized to remove adapter molecule 50 from barrel 46. Adaptor molecule 50 can be trapped for an indefinite period of time (e.g., minutes, hours, days, etc.) inside ion channel 44. Thus, trapping creates a long-lived adapter molecule/ion channel structure, which can be utilized in nucleic acid sequencing. At this point, molecules of interest 60, such as nucleic acids, may be introduced to system 10 such that translocation of molecule 60 can occur through the reduced lumen diameter d of adapter molecule 50, thus increasing sensitivity of sensing system 10. More specifically, electrostatic trapping of s7-βCD and other adapter molecules 50 in the lumen or barrel 46 of ion channel 44 reduces the size of the channel through which molecule 60 passes, thereby reducing the translocation rate and reducing the size of the constriction zone in which a base signature is generated.

After electrostatic trapping in the order of -120 mV for an extended period of time, the voltage may be reduced to release adapter molecule 50 from barrel 46 of ion channel 44. It should be noted that the conductivity

of ion channel 44 is the same before and after electrostatic trapping of adapter molecule 50, demonstrating that the method of the present invention does not damage or alter ion channel 44.

EXAMPLE 1

The following experimental methods and materials where used to demonstrate electrostatic trapping of a -7 charged molecule s7-βCD within the internal cavity of an alpha-HL trans-membrane protein ion channel. The experiment was performed using a glass nanopore membrane modified with 3-cyanopropyldimethylchlorosilane. The internal nanopore solution was 1 μM alpha-HL, 1 M KCl and 10 mM phosphate buffer (PBS - pH 7.4). The external solution was 1 M KCl 10 mM PBS (pH 7.4) containing 50 μM s7-βCD. Two Ag/AgCl electrodes were used to bias a potential across the nanopore membrane, with potential referenced to the internal solution. A bilayer composed of DPhPC was painted across the nanopore (450nm) and a single alpha-HL was inserted into the bilayer membrane. A DC voltage ranging between -40 to -200 mV (trans relative to cis) was applied to monitor the effects of s7-βCD binding events inside the alpha-HL channel as a function of the applied voltage. A positive trans-membrane pressure from 40-300 mm Hg was applied to ensure stable protein insertion. Important to the present invention is the use of high voltages (>-0.1V) to extend the lifetime of s7-βCD within the lumen of the alpha-HL channel. After electrostatic trapping at about -120 mV or less for an extended period of time (e.g., 10 minute test), the voltage is reduced to release s7-βCD from the alpha-HL channel. The conductivity of the alpha-HL channel is the same before and after electrostatic trapping of s7-βCD.

Figure 6A-D illustrates the current sensed over time respectively utilizing -40, -100, -120 and -200 mV of applied potential. When a potential of -40 mV is applied, reversible stochastic s7-PCD binding events are readily seen with relatively short lifetimes as expected. When a single s7-βCD molecule enters the alpha-HL channel a characteristic drop in current is seen as a consequence of the increased resistance through the channel. An increase of potential to -100 mV causes the stochastic binding events to occur more frequently, but still reversibly. Once an applied potential >-120 mV is used, electrostatic trapping of the s7-βCD can be seen. This is shown by the onset of the characteristic current drop associated with s7-βCD entering the channel, without the return to the normal open state of the channel or initial current level. The electrostatic trapping is clearly labeled in the two current-time plots titled -120 mV and -200 mV in Figure 6.

Based on the above, it should be readily apparent that, in accordance with the invention, a charged adapter molecule, such as s7- βCD, can be electrostatically trapped for indefinite periods of time inside a protein ion channel, e.g., alpha-HL, by applying an electrostatic voltage across the lipid bilayer membrane in which the ion channel is inserted. The foregoing experiment was performed with various other cyclodextrins (e.g., both positively and negatively charged different functional groups, and alpha and gamma cyclodextrin), proteins and nucleic acids (e.g., RNA), with similar results being achieved. Although described with reference to a preferred embodiment of the invention, it should be readily understood that various changes and/or modifications can be made to the invention without departing from the spirit thereof. For instance, although a protein pore is shown, it should be understood

that electrostatic trapping of adapter molecules in synthetic or other pores is possible utilizing the system and method of the present invention. In general, the invention is only intended to be limited by the scope of the following claims.