Patent Grant
12085534
The present disclosure relates to the fields of multiplexable super-resolution force spectroscopy that may in some embodiments resolve intermolecular bonds, reveal molecular motion, and distinguish multiple-step protein binding.
Many force spectroscopy techniques have been developed and widely used in fundamental biological research. Examples are atomic force microscopy, AFM (Florin, et al., Science 264, 415-417 (1994); Wong, et al., Nature 394, 52-55 (1998)); Bai et al., Biochem. Biophys. Res. Commun. 448, 372-378 (2014)) optical tweezers OT (Bustamante, et al., Nature 421, 423-427 (2003); Ota, et al., Appl. Phys. Lett. 87, 043901 (2005)); acoustic force spectroscopy, AFS (Sitters, et al., Nat. Method 12, 47-50 (2015)); magnetic tweezers (Strick, et al., Science 271, 1835-1837 (1996)); and force-induced remnant magnetization spectroscopy, FIRMS (Yao, et al., Angew. Chem. Int. Ed. 50, 4407-4409 (2011); Yao, et al., J. Phys. Chem. B 116, 9944-9948 (2012); De Silva, et al., J. Phys. Chem. B 117, 7554-7558 (2013)).
The majority of these techniques are single-molecule based and use optical detection. Consequently, they have several drawbacks.
The first is poor force resolution. For example, a typical force histogram by AFM is 20-40 pN wide. Although different force histograms have been obtained to indicate different binding sites or protein interactions, they have substantial overlap and are difficult to quantify (Pfreundschuh, et al., Nat. Commun. 6:8857/doi: 10.1038/ncomms9857 (2015)). Better force resolution is thus highly desired.
The second is the poor efficiency. The force traces are usually obtained one by one for the single-molecule techniques. This approach makes it very difficult to find specific ligand-receptor bonds because these bonds only represent in a small percentage (˜8-10%) among all specific and non-specific interactions on the surface. It is therefore not surprising that some studies contained a significant part of weaker, nonspecific interactions (Hu, et al., Chem. Commun. 52, 3705-3708 (2016); Dobrowsky, et. al., J. Virol. 82, 7022-7033 (2008).
In addition, for AFM, the stiffness of the cantilevers and force history often lead to inconsistent results.
While FIRMS achieves a 1.8 pN force resolution, enabling differentiation of DNA duplexes with one nucleotide difference, and recently ultrasound has been used to achieve similar force resolution. Such experimental state of the art applications are capable of distinguishing for example: the binding and non-binding of drugs with DNA based on differential binding forces, and the ribosome motion on the mRNA during translocation and frameshifting (De Silva, et al., L., Chem. Comm. 50, 10786-10789 (2014); Hu, et al., Angew. Chem. Int. Ed. 53, 14135-14138 (2014); Tsai, et al., ACS Chem. Biol. 12, 1629-1635 (2017).
However, such force resolution is not sufficient to quantify drug-DNA binding to sub-pN precision, or reveal molecular motion beyond one nucleotide length, and the approach of using a commercial ultrasound probe makes it difficult to automate the overall FIRMS device. Therefore, there is an unmet need in the field, and it is necessary to develop new experimental techniques to study such systems (Walton, et al., Biophys. J. 94, 2621-2630 (2008); Pincet, et al., Biophys. J. 89, 4374-4381 (2005)).
Multiplexable super-resolution force spectroscopy (SURFS) as disclosed herein aims to address an unmet need in the field, wherein the methods disclosed provide for resolution of intermolecular bonds, revealing molecular motion, and distinguishing multiple-step protein binding, by providing a method of highly refined force resolution.
The present disclosure relates in some embodiments to a method of measuring the dissociation of protein bonds using super-resolution force spectroscopy (SURFS). SURFS utilizes precise ultrasound radiation to exert an acoustic radiation force on protein bonds labelled with magnetic particles; the force-induced dissociation of the protein bonds labelled with magnetic particles may be measured as a reduced magnetic signal by a magnetic sensor, such as an atomic magnetometer, giant magnetoresistive sensor, hall probe, or superconducting quantum interference
One embodiment disclosed herein is drawn to a super resolution force spectroscopic (SURFS) apparatus configured to measure the dissociation of molecular bonds, said apparatus comprising at least one piezo plate; a compartment comprising liquid, wherein the compartment is positioned above the piezo plate; a sample-plate, placed above the compartment; a magnetic sensor; and transport apparatus configured to move the sample-plate between a first location, and a second location, wherein the sensor measures the changes in magnetism proportional to bond dissociation. In some embodiments the apparatus further comprises a function generator, in some embodiments the apparatus also comprises an amplifier, wherein in some embodiments, the amplifier is radiofrequency amplifier, in other embodiments the amplifier is an ac (alternating current) amplifier. In some other embodiments of the apparatus the first location is at the piezo plate, in other embodiments the second location is at the magnetic sensor. In some embodiments of the apparatus the transport apparatus is a motor or multiple motors. In some embodiments of the apparatus described herein the piezo plate is about 1 mm-100 mm in width, in some embodiment the piezo plate is about 1 mm-100 mm in length, and in other embodiments the piezo plate is about 0.3-10 mm in thickness. In some embodiments the first location and the second location overlap.
In some embodiments the apparatus herein described, further comprises a function generator, wherein the functional generator and an amplifier generate an amplified alternating current, in another embodiment the alternating current has a frequency of between 20 kHz and 5 MHz, and in a further embodiment the alternating current has an amplitude of between about 0.01 V and about 20 V. In some embodiments of the apparatus of the maximum power of the radiofrequency amplifier is 10-1000 watts. In another embodiment of the apparatus, the magnetic sensor is an atomic magnetometer, a superconducting quantum interference device, a Hall probe, or a magnetoresistive-based sensor. In a further embodiment of the apparatus, the sample plate comprises 1 to 10,000 sample wells. In another embodiment of the apparatus the piezo plates are in the range of 1-1000, and in a further embodiment the piezo plates are in the range of 1-10.
Another embodiment herein discloses a super resolution force spectroscopic (SURFS) apparatus configured for generating an acoustic force and measuring bond dissociation in multiple samples simultaneously, wherein the apparatus comprising: at least one piezo plate, wherein the plate is driven by an amplified ultrasound radiation; a layer of grease positioned on a first surface of the piezo plate; a sample plate comprising at least one sample well, wherein the piezo plate generates an acoustic force onto each said sample well; a magnetic sensor; a transport apparatus configured to move the sample plate between a first location, and a second location; wherein said sensor measures the changes in magnetism proportional to bond dissociation for the multiple samples. In one embodiment of the apparatus, the at least one piezo plate is in the range of about 1-1000. In another embodiment of the apparatus, the layer of grease is positioned between the sample and piezo plate, in a further embodiment the grease is a commercial vacuum grease, wherein said grease in one embodiment is a lubricant with low volatility, and in a still further embodiment the thickness of the layer of grease is 0.001-0.5 mm. in some embodiments the grease is a lubricant, and in some further embodiments a layer of lubricant is in liquid form positioned between the sample and the piezo plate, with thickness of about 0.001-5 mm.
In another embodiment an apparatus configured for producing multiple acoustic forces within a single application of ultrasound is disclosed, wherein the apparatus comprises an array of piezo plates, wherein each plate is coupled in series with a capacitor, wherein said capacitor adjusts voltage; a sample, positioned within a sample-plate at each said piezo plate; a motor; and a single magnetic sensor or an array of magnetic sensors for detection. In another embodiment of the apparatus, the array of piezo elements is configured as a one dimensional array, in another embodiment the one dimensional array is a straight line or curve. In a further embodiment the array of piezo elements is configured as a two dimensional array, and in a still further embodiment the two dimensional array are multiple lines and patterns. In some embodiments of the apparatus the capacitors comprise a range of capacitances, wherein said values of capacitances are obtained by calculation and adjusted experimentally, and in another embodiment the motor is one of a linear motor for a one-dimensional scan, or two orthogonal motors for two-dimensional scan.
In another embodiment a method of measuring bond dissociation with super resolution force spectroscopy (SURFS), is disclosed, method comprising: (a) transporting a sample-plate comprising at least one sample to a first location, wherein said first location comprises a magnetic sensor, and measuring a first magnetic signal; (b) transporting said sample plate to a second location, wherein said second location comprises a piezo plate, and applying a first amplitude of ultrasound radiation, (c) transporting the sample plate to said first location and measuring a second magnetic signal; (d) transporting said sample plate to said second location, and applying a second amplitude of ultrasound radiation, wherein said second amplitude of ultrasound radiation is greater than said first amplitude of ultrasound radiation; and (e) Calculating the difference between the first magnetic signal, and said second magnetic signal, (f) repeating steps a-e until a statistically significant difference is measured, wherein the difference between the first and the second magnetic signal is proportional to the force required to dissociate molecular bonds of said sample. In some embodiments of the method transporting further comprises a motor. In other embodiments of the method herein disclosed the sample comprises biomolecules, wherein said biomolecules are labeled with magnetic particles, and the biomolecules are nucleic acids, proteins, ribosome complexes, receptors on cell surfaces, or combinations thereof.
The force resolution may be in the order of about 0.5 pN, which is approximately one order of magnitude better than the force spectroscopy techniques of the prior art, wherein for example, the associated spatial resolution for molecular motion is about 0.2 nm, which cannot be revealed by structural techniques such as X-ray crystallography and cryo-electron microscopy. In some embodiments, with a range of differential force fields, time resolution of about 1-3 min may be achieved, allowing the resolution of the multiple steps of protein binding.
The concept of multiplexed force spectroscopy with gradient forces has not been realized in force spectroscopy applications of the prior art. It is therefore disclosed herein that generating different forces within a single force application enables efficient data acquisition of force spectra. It also implements time resolution to resolve multiple steps of ligand-receptor binding, which is common in in vivo biological and biochemical processes, for example, during the fusion of a HIV virus.
Further, it is disclosed herein, that in some embodiments the force resolution can be correlated to high spatial resolution for molecular motion. By measuring the binding force between a probe DNA and a targeted molecular system the dimensional information of the system can be extracted. For example, in some embodiments it can be shown that the ribosome-mRNA complex has a very subtle motion of 0.2 nm from the post-translocation state to the subsequent pre-translocation state.
In another embodiment, the SURFS technique may be used to precisely resolve protein bonds on living cells. Compared to other force spectroscopic techniques, primarily atomic force microscopy (AFM) and optical tweezers, the force resolution method disclosed herein is about fifty times more accurate as compared to single-molecule techniques which typically provide a force distribution of 20-40 pN. Therefore, the methods disclosed herein may be applicable to revealing different protein bonds on living cell surface and identifying the influence of drug molecules.
For a detailed description of the disclosed embodiments, reference will now be made to the accompanying drawings, wherein:
The following description is directed to various exemplary embodiments of the invention. However, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be exemplary of that embodiment, and that the scope of this disclosure, including the claims, is not limited to that embodiment.
Certain terms are used throughout the following description, and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not function. The drawing figures are not necessarily to scale. Certain features and components herein may be shown exaggerated in scale or in somewhat schematic form, and some details of conventional elements may be omitted in interest of clarity and conciseness. In the following discussion and in the claims, the terms “including” and “comprising” are used in an open-ended fashion, and thus should be interpreted to mean “including, but not limited to . . . .”. As used herein, the term “about,” when used in conjunction with a percentage or other numerical amount, means plus or minus 10% of that percentage or other numerical amount. For example, the term “about 80%,” would encompass 80% plus or minus 8%. References cited herein are incorporated in their entirety. Also, the term “couple” or “coupled” is intended to mean either an indirect or direct electrical connection. Thus, if a first device is coupled to a second device, that connection may be through a direct electrical connection of the two devices, or through an indirect electrical connection, established via other intermediate devices and connections.
Embodiments disclosed herein are intended to overcome certain above mentioned limitations by using SURFS, which provides for a number of advancements over the prior art as disclosed herein and throughout the disclosure. One such advancement is to use precisely controlled ultrasound as the force source. By using a water reservoir as the medium between the piezo disk or plate and the sample, a precise force field may be obtained, and the thermal effect is significantly reduced. A second advancement is to automate the associated apparatus such that the sample can be alternately scanned for magnetic measurement and fixed for force application in many cycles (see
δF=(2δV/V)*F=(2*0.001/0.204)*45=0.5 pN (1)
Here, F is the bond dissociation force, and V is the dissociation voltage for ultrasound. δF and δV represent resolution of F and V, respectively.
Multiplexed SURFS using a single piezo plate: To accommodate multiple samples, a larger piezo plate is used. Depending on the size of each sample well and the total number of sample wells needed, the typical in-plane dimension of the piezo plate may be 5-100 mm in various shapes, and the thickness is typically 0.2-10 mm. Various large piezo plates are commercially available, for example, from American Piezo Company, and can be easily cut into customized sizes. Larger piezo plates have larger capacitances than the smaller ones, which lead to lower applied voltages. The equation governing the maximum voltage (Vmax) is:
Vmax=Imax/(2πfC) (2)
Herein, Imax is the current rating of the amplifier, f is the frequency of sine ultrasound wave, and C is the capacitance. Because the acoustic force is proportional to the square of voltage, a high-current amplifier is required to drive a large piezo plate.
In addition to the scheme shown in
The sample plate contained fifteen sample wells (
Gradient force field using piezo plates coupled with voltage dividers: Prior to the current disclosure, the time resolution of force spectroscopy has been poor because the data points were acquired one at a time. For example, to obtain a force spectrum with 15 data points, 15 different forces are needed and magnetic measurement will follow each force application. Each data point will take about 3 minutes. Hence, the time resolution is on the order of one hour. This may not be sufficient to study multiple-step protein binding events, such as the sequential binding of gp120 onto CD4 receptor and CXCR4 co-receptor for example.
The time resolution of such measurements is advanced by embodiments of SURFS as disclosed herein, and is thus achieved by generating a gradient force field. Herein, in some embodiments an array of individual piezo plates may be used instead of a single one. The plates are in parallel, but each is coupled with a different capacitor in series. The capacitances of the capacitors (C1-C4 in
Ci=(√χ+1)C (3)
The gradient force field produced by adjusting the coupling capacitors may therefore improve the time resolution of SURFS. For example, in some embodiments the number of force applications may be reduced by 4 if four identical samples are used at different forces, which improves the force resolution by a factor of 4. By using more piezo elements in parallel, the whole force spectrum can be obtained within a single ultrasound application.
Applications of SURFS
With high force resolution, spatial resolution, and time resolution, SURFS may be utilized in biological research and biotechnology industrial branches, some such embodiments are disclosed herein.
Drug Molecules
Key questions in the design and development of drug molecules include the issue of how a drug interacts with their molecular target(s), including how effective is the drug for inhibiting the targeted ligand-receptor pair. Another is how the drug functions, i.e., whether it works through allosteric effect or directly blocks the ligand's binding pocket. Typically, binding affinity in concentration is used to characterize drug's efficacy. However, it does not provide information on the binding mechanism. Specifically, considering as an example CXCR4 and CCR5, both have been viable drug targets for HIV. A number of drug candidates have been developed, with one of them “Maraviroc” having been approved. X-ray structure showed that Maraviroc functions through allosteric effect that weakens the binding between gp120 and CCR5. However, it is unknown how much weaker the binding becomes and how effective it is on the cell surface.
SURFS, in some embodiments may be used to provide a precise differential binding force, ΔF, to quantify the effect of drug molecules: wherein the presence of the drug altered the binding force of ligand-receptor bonds from F to F′, so ΔF=F−F′. [See U.S. Pat. No. 9,778,249 further incorporated herein in its entirety] Given that ΔF in some will be different for allosteric vs. direct blocking: ΔF will be smaller for allosteric effect than for direct blocking. Embodiments of the methods disclosed herein have showed that ΔF accurately revealed the chiral selectivity of drug molecules and sequence specificity of DNA duplexes. The values of ΔF were typically 5-16 pN. ΔF may have a greater range in such an embodiment because drug molecules usually have higher affinity for proteins than for DNAs.
Another embodiment of the SURFS method herein disclosed was used to measure a series of DNA and DNA-drug complexes, and correlated the dissociation voltages with forces obtained by FIRMS. Linear fitting produces:
F=740V2+11 (4)
This equation confirms that the dissociation force linearly depends on the ultrasound power (represented by V2).
Molecular Motion Revealed by SURFS
Ribosomal translocation is a key step in protein synthesis. For each translocation step, the ribosome moves by three nucleotides from the pre-translocation state (Pre-complex) to the post-translocation state (Post-complex). Then, the ribosome will be in the pre-translocation state for the next translocation cycle. No technique of the prior art has revealed any difference between the “Post-complex of the previous step” and the “Pre-complex of the next step”. In other words, in light of the prior art it is unclear how the ribosome moves along the mRNA to decode genetic information.
However, embodiments are herein disclosed that comprise a probing scheme based on SURFS to precisely determine the position of the ribosome on the mRNA. Shown in
The force spectra in
Resolving Multiple Binding Steps
Embodiments of the scheme of differential force field disclosed herein, enables the study of multiple-step binding, such as the infusion of the HIV infection. The most current prior art model for the infusion mechanism is that the viral envelope glycoprotein, gp120, first binds with the CD4 receptor on the cell surface and then with a co-receptor, CCR5 or CXCR4; fusion subsequently takes place between the virus and the cell. However, direct evidence for the molecular mechanism of this process remains elusive.
SURFS with time resolution as disclosed herein is well suited to address the resolution of this mechanistic question. It has been shown that gp120 binds with CD4 fairly rapidly on the substrate surface, within 2-3 min, whereas the complete fusion between the HIV virus and the cells takes approximately 60 min. Therefore, both force resolution and time resolution should be used to resolve the two steps. Shown in
SURFS may also be employed in some embodiments to investigate drug molecules targeting the second binding step. The efficacy will be shown as two parameters, one is the differential binding force and the other is the second step binding amplitude, ΔB3.
Thus, disclosed herein is a multiplexable SURFS technique that addresses the unmet need for sub-pN force resolution for the binding forces of intermolecular bonds, 0.2 nm spatial resolution on molecular motion along nucleic acids, and ˜2 min time resolution for multiple-step protein binding, capabilities of SURFS as disclosed herein may be extended to study protein binding on living cells.
The present application is a National Phase Entry of, and claims priority to PCT Application No. PCT/US2019/016822, filed Feb. 6, 2019, which claims priority to U.S. Provisional Patent Application No. 62/628,188, filed Feb. 8, 2018, entitled “Multiplexable Super-Resolution Force Spectroscopy Using Ultrasound Methods and Magnetic Detection,” the entire contents of each being hereby incorporated herein by reference in their entirety for all purposes.
This invention was made with government support under R01 GM111452 awarded by the National Institutes of Health, and 1508845 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/016822 | 2/6/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/157038 | 8/15/2019 | WO | A |
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| 4074070 | Gaus | Feb 1978 | A |
| 20130104667 | Koyano | May 2013 | A1 |
| 20130139575 | Lee | Jun 2013 | A1 |
| 20150117156 | Xu | Apr 2015 | A1 |
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| Number | Date | Country | |
|---|---|---|---|
| 20210041395 A1 | Feb 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62628188 | Feb 2018 | US |
