Patent Application
20190010544
This patent document relates to systems, devices, and processes that use nanoscale molecular sensor and actuator technologies.
Nucleic acids, e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), can be used to construct various structures for a wide range of applications.
Techniques, systems, devices and materials are disclosed for implementing a molecular-based nanoscale sensors and actuators including nucleic acid-based zipper tweezers and springs.
In one aspect of the disclosed technology, a molecular zipper device includes a double-stranded molecule including a first strand of nucleotide units coupled to a second strand of nucleotide units, the nucleotide units of the first strand configured in a sequence and including nucleobases, the nucleotide units of the second strand configured in a complement sequence corresponding to the sequence of the nucleotide units of the first strand, in which at least one nucleotide unit of the second strand includes a synthetic nucleobase that forms a bond with a corresponding complement nucleobase of the first strand, in which the double-stranded molecule is structured to interact with an opening molecule which includes a third strand of nucleotide units in a complementary sequence corresponding to the sequence of the nucleotide units of the first strand, and in which the opening molecule couples to the first strand by unbinding the nucleotide units of the second strand from the nucleotide units of the first strand, the nucleotide units of the third strand having nucleobases that form a substantially equal or stronger bond with the corresponding complement nucleobases on the first strand than the bond formed by the synthetic nucleobase on the second strand.
In another aspect, a molecular sensor device includes a double-stranded molecule including a binding strand and a passive strand, the binding strand including a binding zipper member in connection with a binding hinge member, the passive strand including a passive zipper member in connection with a passive hinge member, in which the passive hinge member is coupled to the binding hinge member, and in which the passive zipper member is coupled to the binding zipper member by a coupling of complementary nucleotide units of the passive zipper member and the binding zipper member, in which the double-stranded molecule is operable to interact with a target molecule initially uncoupled to the double-stranded molecule, the target molecule including an opening strand having nucleotide units in a complement sequence corresponding to a sequence of nucleotide units of the binding zipper member, and in which the opening strand couples to the binding zipper member by uncoupling the complementary nucleotide units of the passive zipper member from the binding zipper member, the nucleotide units of the opening strand bonding to the nucleotide units of the binding zipper member.
Implementations can optionally include one or more of the following features. The molecular sensor device can further include a reset molecule initially uncoupled to the target molecule and the double-stranded molecule, the reset molecule including a closing strand of nucleotide units in a complementary sequence corresponding to the sequence of nucleotide units of the opening strand. The binding strand of the molecular sensor device can further include a binding loop member that connects the binding zipper member to the binding hinge member, and the passive strand of the molecular sensor device can further include a passive loop member that connects the passive zipper member to the passive hinge member, in which the binding loop member and the passive loop member are uncoupled with one another.
In another aspect, a method of capturing a target molecule includes deploying a double-stranded molecule into a fluid environment, the double-stranded molecule including a binding strand having a sequence of nucleotides that is coupled to a passive strand having a complementary sequence of nucleotides, and attaching a target molecule in the fluid environment to the binding strand, the target molecule including an opening strand having a complement sequence of nucleotides corresponding to the binding strand, in which the attaching uncouples the passive strand as the nucleotides of the opening strand bond to the corresponding complement nucleotides of the binding strand.
Implementations can optionally include one or more of the following features. The method can further include removing the target molecule from the double-stranded molecule by coupling the opening strand to a complement closing strand of a reset molecule. The method can further include recoupling the complementary sequence of nucleotides of the passive strand to the sequence of nucleotides of the binding strand, thereby regenerating the double-stranded molecule.
In another aspect, a molecular device includes molecular components including at least a passive side molecular component, a binding side molecular component and a target molecular component, in which the passive side molecular component and the binding side molecular component are bound together by molecular interaction forces to form a molecular zipper structure, in which the target molecular component is initially unbound to the molecular zipper structure and adapted to separate the passive side molecular component and the binding side molecular component.
In another aspect, a molecular actuator device includes a double-stranded molecule including a hinge member attached at one end to a zipper member, the zipper member including a binding strand coupled to a passive strand, in which the binding strand includes a sequence of nucleotide units hybridized a corresponding complement sequence of nucleotide units of the passive strand, a first arm member connected to the binding strand of the zipper member by a first linker strand that attaches the first arm member to the binding strand, and a second arm member connected to the passive strand of the zipper member by a second linker strand that attaches the second arm member to the passive strand.
Implementations can optionally include one or more of the following features. The first arm member can include a double-stranded molecular structure, and the second arm member can include a double-stranded molecular structure. The double-stranded molecule can be structured to interact with a target molecule initially uncoupled to the molecular actuator device, the target molecule including an opening strand having nucleotide units in a complementary sequence corresponding to the sequence of nucleotide units of the binding strand, in which the opening strand couples to the binding strand by uncoupling the complement sequence of nucleotide units of the passive strand from the binding strand and binding the nucleotide units of the opening strand to the nucleotide units of the binding strand. The molecular actuator device can further include a reset molecule initially uncoupled to molecular actuator device, the reset molecule including a closing strand of nucleotide units in a complementary sequence corresponding to the sequence of nucleotide units of the opening strand, in which the closing strand couples to the opening strand by uncoupling the opening strand from the binding strand. The double-stranded molecular structure of the arm member can be structured to interact with another target molecule initially uncoupled to the molecular actuator device, the other target molecule. The molecular actuator device can operate as a spring. The molecular actuator device can be a first molecular actuator device connected to a second molecular actuator device, in which the first arm member and the second arm member of the first molecular actuator device connect with the first arm member and the second arm member of the second molecular actuator device, forming a joined molecular actuator device. The joined molecular actuator device can further include at least one other molecular actuator device, in which the hinge member of the at least one other molecular actuator device connects to a joined arm member of the first and second molecular actuator devices, thereby forming a multiple molecular actuator device. The multiple molecular actuator device can operate as at least one of a motor or a gate element. The molecular actuator device can be incorporated in a capsule, the capsule further including a container unit including a wall that forms an enclosure around an interior region, the container unit structured to include an opening, and a lid unit including a surface structured to cover the opening, in which the molecular actuator device joins the container unit to the lid by a distal end of the first arm member coupled to the surface of the lid and another distal end of the second arm member coupled to an interior surface of the interior region of the container unit. The molecular actuator device of the capsule can include a self-splicing DNA sequence as part of the first arm member that includes a DNAzyme that cleaves a single strand of the double-stranded molecular structure of the first arm member, thereby detaching the lid unit from the capsule. The capsule further can include a material initially enclosed within the capsule, the material released outside the capsule upon detaching the lid unit from the capsule, in which the material can include a drug, imaging agent, enzyme, nucleic acid, viral vector, or other molecular substance.
In another aspect, a DNA based molecular device includes a nanoscale molecular sensor, and a molecular actuator, in which, upon sensing a specific DNA sequence, the nanoscale molecular sensor detects and holds the DNA sequence and the molecular actuator contracts and imparts force to open and close the nanoscale molecular sensor.
Implementations can optionally include one or more of the following features. The nanoscale molecular sensor can operate as tweezers, and the molecular actuator can operate as a spring. The nanoscale molecular sensor and the actuator can be activated under specific environmental conditions including temperature and pH.
The subject matter described in this patent document can be implemented in specific ways that provide one or more of the following features. For example, the disclosed technology can include molecular devices that can sense, hold, and release a target (e.g., DNA) upon specific interaction. For example, the disclosed molecular devices can include exemplary zipper-based tweezers to sense a target (e.g., a DNA strand) and actuate a function. For example, a driving energy to capture an exemplary target DNA strand can be distributed over the entire length of the strand, which can allow more driving energy to be employed, e.g., for holding longer DNA strands and faster opening and closing kinetics. For example, the disclosed zipper-based tweezers can be opened without the use of overhang structures, and thus allow spontaneous regeneration of the exemplary tweezers at its sensing position. For example, the disclosed zipper-based tweezers can be used in the development of new therapeutics and nanoscale machines. For example, the disclosed zipper-based tweezers can include a helix setup to be invaded by natural DNA/RNA for in vitro diagnostics.
Like reference symbols and designations in the various drawings indicate like elements.
Techniques, systems, devices and materials are disclosed for implementing molecular-based nanoscale sensors and actuators including nucleic acid-based zipper tweezers and springs.
Nucleic acids, e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), can be used to create a variety of molecular machines, with properties mimicking logic-circuit operations. For example, the small size, high binding specificity, ease of chemical synthesis and availability of inexpensive DNA or RNA oligonucleotides can make DNA/RNA-based molecular devices useful in a variety of applications. For example, the specificity with which DNA hybridizes can be applied for designing a variety of DNA based diagnostic and therapeutic systems.
A naturally-occurring double-stranded DNA (dsDNA) includes a linked chain of deoxyribose sugar as a backbone for four nucleotide bases (also referred to as nucleobases), e.g., including adenine (A), cytosine (C), guanine (G), thymine (T). These four nitrogen bases can form hydrogen bonds that hold two individual strands of the DNA together. For example, in naturally-occurring dsDNA, adenine bonds to thymine (A=T) and cytosine bonds to guanine (C≡G). The A=T and C≡G bonds are two different types of hydrogen bonds formed by the base pairs. Adenine forms two hydrogen bonds with thymine (A=T) and cytosine forms three hydrogen bonds with guanine (C≡G). For example, the energy of formation of N—H . . . O bonds is approximately 8 kJ/mol, and the energy of formation of N—H . . . N bonds is approximately 13 kJ/mol (e.g., where the dotted line represents the hydrogen bond). A naturally-occurring RNA molecule includes a linked chain of ribose sugar as a base for four nucleobases, e.g., including A, C, G, and uracil (U). For example, when RNA binds to DNA, an adenine nucleobase of DNA forms two hydrogen bonds with uracil nucleobase of RNA (A=U). RNA molecules are single stranded and can form many structural configurations.
The disclosed technology can include molecular tweezers and molecular springs to sense a target and actuate a function. For example, the disclosed molecular tweezers and molecular springs can be based on nucleotide zipper mechanisms where molecular bonds can be engaged or disengaged/released as zippers. For example, an exemplary zipper can be used to create a DNA nano-gate that can be reversibly opened and closed. The disclosed molecular zipper technology can include self-sustaining, modifiable properties that can be implemented in sensing and actuating applications exhibiting sensitivity in a range of physiologically relevant temperatures. For example, the disclosed molecular zipper technology can be implemented in various nanoscale applications, e.g., including molecular motor actuation, molecular recognition tools (e.g., molecular detection assays and molecular and biological sensors, molecular building blocks, vehicles for molecular transport (e.g., colloidal drug carriers) and as molecules modifiers and medicines.
In one aspect, the disclosed technology can include devices, systems, and techniques based on nucleotide zipper mechanisms. For example, an exemplary molecular zipper can include a closed double helix molecule (e.g., DNA) formed by the hybridization of two strands of oligonucleotides that can be opened by the capture of a target molecule, e.g., such that the double-strand separation does not use external energy. For example, the exemplary double helix molecule can include a binding strand having naturally-occurring nucleotides and a passive strand including non-naturally-occurring nucleotides. For example, the molecular zipper mechanism can be implemented by the target molecule (e.g., also referred to as an opening strand, an external strand, and a fuel strand) hybridizing with the binding strand, e.g., displacing the passive strand. For example, the passive strand does not bond to the binding side of the exemplary molecular zipper as strongly as the target molecule. The disclosed technology can function like a ‘zipper’ because the closed double helix molecule can naturally separate by interacting with the target. The physical interactions that take place between the target molecule and a closed molecular zipper can open the exemplary molecular zipper.
As a specific example, an exemplary DNA double helix can include one oligonucleotide strand that can be referred to as the normal strand (N) and the other oligonucleotide strand that can be referred to as the weak strand (W). In some implementations, the exemplary N strand can be a natural DNA strand, e.g., including the four naturally-occurring DNA nucleobases: adenine (A), cytosine (C), guanine (G), and thymine (T). For example, the exemplary N strand can be a natural RNA strand, e.g., including the four naturally-occurring RNA nucleobases: A, C, G, and uracil (U). The exemplary W strand can be an engineered or synthetic strand having a sequence of bases that includes non-naturally-occurring nucleobases. For example, the non-naturally-occurring nucleobases on the exemplary W strand can be configured to provide a weaker binding affinity to their corresponding complement nucleobases compared to the binding affinity between two naturally-occurring nucleobases. For example, when the exemplary N and W strands hybridize, there is less energy holding N and W strands together than if the W strand comprised the corresponding natural complement nucleobases of the N strand. For example, the exemplary W strand (also referred to as a synthetic strand, an engineered strand, and a passive strand) can be constructed using a deoxyribose sugar backbone identical to that occurring in natural DNA, but containing only nucleotide analog bases—nucleotide analogs are bases that can be attached to the backbone (e.g., the deoxyribose sugar backbone), but do not naturally occur in organisms.
For example, an exemplary opening strand (O) can be the natural complement of the exemplary N strand and thereby displace the W strand at each nucleotide unit along the W strand. In some examples, the exemplary O strand can include the same number or a greater number of nucleotide units than the exemplary W strand, e.g., in which the O strand hybridization with the N strand can detach the W strand from the double helix molecule. In other examples, the exemplary O strand can include a smaller number of nucleotide units than the exemplary W strand, e.g., in which the W strand can remain attached to the exemplary N strand (and part of the double helix molecule) after the O strand hybridization with the N strand.
The disclosed technology can include a variety of W strands that can be configured to provide differing binding affinities of the W strand to the N strand. In some examples, the exemplary W strand can be configured to have all of its nucleotide bases to be non-naturally-occurring nucleobases. In other examples, the exemplary W strand can be configured to have some of its nucleotide bases to be non-naturally-occurring nucleobases, e.g., spatially organized in a desired sequence with naturally-occurring nucleobases. For example, non-naturally-occurring nucleobases can include inosine (I), 2-aminopyrimidine, 5-methyisocytosine, and deoxyinosine, among others. For example, an exemplary W strand can contain the inosine (I) base along with other naturally-occurring bases. The exemplary W strands can be engineered to have differing affinities to any N strand, e.g., providing flexibility in the disclosed zipper-based devices that can also self regenerate.
Exemplary DNA based zippers can also be configured using inosine. For example, inosine preferentially hybridizes to C through two hydrogen bonds. The exemplary I═C pair has a weaker energy of formation (˜21 kJ/mol) than the G≡C pair (˜29 kJ/mol). Exemplary W strand can be configured to contain the inosine base along with other naturally-occurring bases. For example, when an exemplary N strand and the inosine-containing complementary W strand hybridize, there is less energy holding them together, e.g., than if they were the exemplary N strand and its natural complement. For example, the stronger G≡C interaction between an exemplary natural complement and the exemplary N strand outcompetes the I═C bonds and displaces the exemplary W strand from the exemplary DNA zipper structure, e.g., resulting in the opening of the zipper, to form a new double stranded DNA structure having the N strand coupled to its natural complement strand.
The described molecular zippers can be composed of three molecular components that include a passive side, a binding side and a target that are entropy driven to interact in such a way that they perform the function of separating two individual parts held together by molecular interaction forces. For example, interaction forces can include any combination of hydrogen bonds, van der Waals attraction, hydrophobic interactions or electrostatic forces existing between the interacting molecular components. The passive and binding sides can be initially bound together forming a zipped molecule. The passive side of the molecular zipper can be separated from the binding side by interaction with the target (e.g., displaced at each nucleotide unit that the target binds to the binding side) through a process called entropy driven displacement (EDD). This exemplary separation of the passive side from the binding side is a function of the exemplary molecular zipper device. For example, the exemplary molecular zipper device can be described as being opened by a molecular key that does not require the addition of any energy. For example, the exemplary molecular zipper can be opened by a chemically engineered molecular key, or the exemplary molecular zipper can be chemically engineered to be opened by a naturally-occurring molecule to act as the key.
For example, physical principles involved in the opening of the molecular zipper include thermal fluctuations between the two individual strands of the zipper and molecular forces between the components of the zipper. The disclosed molecular zipper mechanism can rely on thermal fluctuations between the base pairs as well as the bonding energies between the three components. For example, the molecular zipper can be opened by allowing the target to statistically wiggle its way into the zipper by pushing the passive side out of the zipper. For the molecular zipper mechanism to function, the average energy of interaction between the binding side of the zipper and the target is greater than the average energy of interaction between the binding side and the passive side. In addition, the increased attraction between the binding side and the target can occur with a periodicity close enough together so that the thermal fluctuations that facilitate the opening action are statistically probable. For example, provided that the periodicity of increased bonding between the target and the binding side of the zipper occurs within statistical reason and the bonding energy between the passive side and the target are negligible, the driving energy of the unzipping action can be approximated. For example, the approximate total driving energy of the unzipping action (Eu) can be represented by Eq. (1):
E
u
=E
t-Ep (1)
where Et is the total bonding energy between the target and the binding side and Ep is the total bonding energy between the passive side and the binding side. The total driving energy of the unzipping action, e.g., represented in Eq. (2), can become:
E
u=[Mt(8 kJ/mol)+Nt(13 kJ/mol)]−[Mp(8 kJ/mol)+Np(13 kJ/mol)] (2)
where M and N represent the number of hydrogen bonds of the form N—H . . . O and N—H . . . N, respectively.
For example, the average thermal kinetic energy of a molecule is given by E=nRT where n is the number of moles, R is 8.3145 and T is the temperature in Kelvin (K). Physiological temperature is approximately 300 K, and the minimum average molecular kinetic energy at this temperature is E=2.5 kJ/mol. For example, since the biding energy of the hydrogen bonds is only several times larger than their disassociation tendency due to thermal motion, the hydrogen bonds between the nucleosides in dsDNA are constantly breaking and reforming. For example, this causes the DNA to temporarily undergo localized distortions and deformations. For example, intercalating agents such as ethidium bromide can insert into dsDNA with ease, which can suggests that the double-stranded helix temporally unwinds and presents gaps for these agents to occupy. Thus, the DNA conformation can be represented by a flickering repertoire of dynamic structures. For example, this can suggest that the ends of the two strands in a double helix must continuously undergo breaking, partially unwinding and reforming due to thermal fluctuations. For example, since the bond energy between one hydrogen bond (e.g., ˜10 kJ/mol) is only approximately 5 times grater then the thermal fluctuation energy at physiological temperatures (e.g., ˜2.5 kJ/mol), a single hydrogen bond in a double-stranded helix can be expected to be bonding only ⅘ of the time and thus be temporarily broken ⅕ of the time. It then follows, for example, that for any time sufficient in length, the probability P of n consecutive hydrogen bonds being simultaneously broken at the front of the front of a dsDNA helix is P=(⅕)n.
[N:W]+O→[N:O]+W (3)
An exemplary comparison of the hydrogen bond energies of [N:W] and [N:O] suggests approximately 140 kJ/mol is driving the reaction of Eq. (3), e.g., assuming ˜21 kJ/mol for the I═C bond and ˜29 kJ/mol for the C-G bond. For example, the W strand 216 can be configured such that to distribute of the energy along the length of the strand, e.g., periodic spacing of I with a sufficient spatial frequency along the length of the W strand can be configured for the operation of the zippers. For example, the thermal stability, kinetics and specificity of the zipper are dependent on the number of I═C bonds, their order and period of placement.
Also shown in
Table 1 shows exemplary DNA oligonucleotides base pair sequences for the individual strands of the zipper system. For example, bases presented in lower case represent the sight of a base pair mismatch in the opening strand.
In another aspect, the disclosed technology can include devices, systems, and techniques that can provide a DNA based nanoscale sensor, e.g., DNA zipper tweezers. For example, upon sensing a specific DNA sequence (e.g., a target molecule), the exemplary DNA zipper tweezers can detect and hold the target and subsequently release the target, e.g., returning to the initial position.
For example, as shown in
For example, as shown in
For example, as shown in
Exemplary implementations were performed to demonstrate the described functionalities and capabilities of the disclosed molecular zipper tweezers. Chemicals used in exemplary implementations were obtained from Sigma Aldrich (Saint Louis, Mo.) unless otherwise specified. The exemplary DNA constructs were obtained from IDT (Coreville, Iowa); the exemplary DNA ladders were obtained from Promega (Madison, Wis.); and the exemplary DNA gels were obtained from Lonza (Walkersville, Md.).
Table 2 shows base pair sequences of the individual component of the exemplary zipper tweezers system, e.g., used in exemplary implementations of the disclosed technology. The exemplary ‘+’ symbol in front of a base in Table 2 indicates that base is a locked nucleic acid (LNA). Text in parentheses represents an exemplary ssDNA overhang.
Exemplary measurements of the melting temperature (Tm) were performed in the exemplary implementations. For example, the Tm of an initial zipper helix (e.g., [N:W]) and the final state helix (e.g., [N:O]) were measured to be 54° C. and 71° C., respectively, e.g., using an AVIV 202 Circular dichroism spectrometer with a Peltier temperature controller and pH meter. Exemplary measurements were conducted using a double helix concentration of 20 μM suspended in a 10 mM PBS buffer (e.g., pH 7.4, 160 mM NaCl). Exemplary Tm calculations of natural DNA pairs were performed using the IDT online calculator with 160 mM NaCl, e.g., assuming equal concentration of 0.1 μM for both strands. Exemplary DNA calculations of sequences containing deoxyinosine were performed using deoxyadenine in the place of deoxyinosine to obtain approximate values for zipper construction. Calculated values were found to be with in a few degrees of our measured values.
Exemplary measurements of the zipper mechanism activity were performed in the following manner. For example, zipper action was visualized by tagging N and W strands with fluorescent probes and observing the change in fluorescence with time. For example, fluorescent quenchers were placed at both ends of the N strands (e.g., 3′-IBFQ and 5′-IBRQ); and 6-carboxyfluorescein (FAM) and Cy5 were placed on W strands at 5′ and 3′ ends, respectively; while O was left unlabeled, e.g., as shown in Table 1. Exemplary fluorescence measurements were conducted using a Jobin Yvon FluoroMax-3 luminescence spectrometer. For example, fluorescent observations (Excitation/Emission) of FAM were performed at 495/520 nm, of Cy5 were performed at 648/688 nm, and of Cy5.5 were performed at 668/706 nm. Exemplary measurements were performed using quartz cuvettes with 40 μL sampling volume (e.g., Sterna Cell 16.40F-Q-10/Z15) filled with 100 μL of sample at the start of each experiment. Exemplary experimental implementations were carried out on samples dissolved in nuclease free reaction buffer (e.g., 30 mM Tris-HCl, 160 mM NaCl, and pH 8.0). Basal fluorescence of the quenched zipper was measured on each sample prior to data collection. For example, basal fluorescence in the exemplary implementations is a measure of the degree of colocalization of the quencher and Cy5.5, e.g., in a closed zipper tweezers. Basal fluorescence can represent the minimum fluorescence of the system prior to any dilution effects. The data was collected typically at every second for ˜90 min and at every 5 s for experiments involving more than 90 min. Exemplary zipper-opening implementations were conducted by adding 10 times more opening strands than zippers, unless stated otherwise. Exemplary initial tweezers-opening implementations were performed by adding 10 times more opening strand, and successive opening and closing experiments were performed by consecutively adding 2 times more of each strand, unless stated otherwise (as shown in Tables 3 and 4). For example, after the initial opening of the zipper tweezers, successive opening and closing cycles were conducted by adding 30 and 50 times Oi opening constructs and 20 and 40 times Ci closing strands, respectively. For example, excessive concentrations can ensure that the reactions can be stabilized with a terminating value and drive the reactions to completion significantly faster than equal concentrations.
Table 3 shows the kinetics of the opening reaction with different constructs at 37° C.
Exemplary gel electrophoresis analyses of the exemplary DNA zipper tweezers were performed in the following manner. For example, the initial and final states of the zipper system were confirmed by DNA gel electrophoresis. For example, the final double helix conformation [N:O] was created by thermally annealing [N:W]+10 O the oligonucleotides (e.g., to ensure the reaction was driven to completion) and used as a control sample. Thermal annealing was accomplished using a custom program in a PCR thermocycler (e.g., Mastercycler personal, Eppendorf) to quickly raise the solution temperature to 94° C. beyond the double strand melting temperature (e.g., N:W 54° C.; N:O 71° C.), followed by a slow, controlled, cooling at a rate of 1° C./2 min to a final temperature of 4° C. DNA gel electrophoresis was performed with 4% agarose gel at 5 V/cm in 1× Tris/Borate/EDTA (TBE) buffer while monitoring the solution temperature not to exceed 20° C. For example, in order to resolve single and double-stranded DNA, the positions of the strands within the gel were determined using fluorescent gel imaging and Ethidium Bromide (EtBr) staining. Exemplary gels were imaged with a Bio-Rad FX-Imager Pro Plus and analyzed with the Quantity One software package (Bio-Rad).
Exemplary implementations of the exemplary DNA zipper tweezers included performing fluorescence observation of the zipper tweezers activity. For example,
For example, when O was added to the [N:W] helix, a continuous increase in fluorescence was determined, e.g., that stabilized to a final steady state as shown by the Opening plot 401 in the spectra plot 400. An increase in the fluorescence can be considered to be due to delocalization of the fluorophores and quenchers (e.g., separation of W from N). For example, completion of the reaction was confirmed by comparing the peak signal produced by the thermal annealing of [N:W] with O, e.g., producing the highest fluorescence and lowest energy configuration of the system, as shown by the Max plot 404 in the spectra plot 400. The exemplary results indicated that the zipper reaction was driven to its completion in about ˜42 min at 37° C. Table 3 presents the time required for 50% completion of zipper opening reactions (t1/2) at 37° C.
For example, in these exemplary implementations, the increase in fluorescence observed in the zipper reaction could also result from spontaneous strand dissociations, random base pair mismatches (e.g., resulting in the formation of overhangs), and slipping between the strands (e.g., resulting in delocalization of fluorescent probes, due to weaker interactions in [N:W] helix). For example, to rule out these possibilities, the [N:W] helix was probed by observing the change in basal fluorescence after adding a ten-fold higher concentration of No (e.g., 10×NO, the N sequence without any quenchers). If any of the above possibilities should take place, then the formation of [NO:W] would result in an increase in the fluorescence. Absence of any such increase can suggest that such possibilities are either absent or insignificant, e.g., as seen in
Exemplary implementations were also performed to probe the specificity and efficiency of zipper action for seven different opening strands with significant (e.g., 16-24%) sequence mismatches OM1-OM7, shown in Table 1, measured at 37° C. Exemplary results are shown in
Exemplary implementations of the exemplary DNA zipper tweezers included performing DNA gel electrophoresis of the zipper tweezers action. For example, the zipper action was validated using fluorescent gel imaging, and the products and reactants of the zipper reaction along with thermally annealed sample [N:O] as a control were analyzed. For example, since the mass-charge ratio of double- and single-stranded DNA is the same in the exemplary implementations, the exemplary products and reactants ran collinear on the gel electrophoresis. For example, the double strands were identified with Ethidium Bromide (EtBr), and the single strands were identified with fluorophores.
Exemplary implementations of the exemplary DNA zipper tweezers included characterizing the zipper tweezers activity. For example, the activity of the exemplary DNA zipper tweezers was examined by tagging the W strands with Cy5.5; the N strands with Iowa Black RQ; and both opening and closing strands without fluorophores. Exemplary time lapse fluorescence measurements and fluorescence images from DNA gel electrophoresis from three successive opening and closing cycles of the disclosed DNA zipper tweezers using the O1, C1-LNA pair are shown in
[WZ:NZ]+O1→WZ+[NZ:O1] (4)
WZ+[NZ:O1]+C1→[O1:C1]+[WZ:NZ] (5)
For example, the exemplary O1 strand contained 28 nt and was configured to be complementary to NZ (21 nt), e.g., the additional 7 nt formed a DNA overhang, which enabled the exemplary O1 strand to be removed by the exemplary C1-LNA strand. The exemplary C1-LNA strand had 21 nt and contained six LNA base modifications (as shown in Table 2). For example, the exemplary C1-LNA strand was configured to be complementary to the entire 7 nt overhang of the exemplary O1 strand and its remaining 14 nt. For example, since the exemplary C1-LNA strand and the exemplary WZ strand are complements (as shown in Table 2), the exemplary C1-LNA strand was made shorter than the exemplary O1 strand to reduce the affinity between them. For example, this can necessitate the condition that the Tm of [WZ:C1-LNA] be sufficiently less than the operating temperature of the exemplary zipper tweezers. Otherwise, the exemplary WZ strand can hybridize with the C1-LNA strand, e.g., preventing the exemplary zipper tweezers from closing [WZ:C1-LNA]. The six exemplary LNA bases were positioned near the overhang binding end of the C1-LNA strand in order to preferentially increase the binding affinity between the C1-LNA strand and the O1 strand.
For example, to examine the robustness of the exemplary zipper tweezers, they were driven further for three opening/closing cycles (as shown in the plot 810 in
It is noted, for example, that as the peaks shown in the plot 810 in
For example, to confirm that the loss of functionality was due to the excess waste product and not from the destruction of the exemplary zipper tweezers, exemplary reactions from four successive opening and closing cycles were subjected to DNA gel electrophoresis. For example,
Exemplary implementations of the exemplary DNA zipper tweezers included characterizing zipper tweezers kinetics, and for example, the role of overhangs and locked nucleic acid (LNA) bases. LNA bases are known to be highly selective and capable of single nucleotide discrimination when hybridizing and have increased target specificity. The exemplary results shown in
For example, the closing rates of the zipper tweezers were examined using the exemplary O1 strand and the exemplary closing C1-LNA implemented to obtain the opening/closing cycling data of the plot 810 in
For example, zipper tweezers with overhangs can be more prone to random hybridizations. In these situations in which overhangs are undesirable, LNAs can be employed. For example, LNA/DNA helices have higher Tm than DNA/DNA helices for a given sequence, and this energy difference can be used to invade small DNA duplex. However, such reactions can be relatively slow. For example, one such system is demonstrated with the O3 opening strand and the C3-LNA closing strand, as shown in
In another example, an exemplary closing strand C4-LNA was configured to have the same base pair sequence as C3-LNA containing 8 LNA modifications evenly distributed along its length. For example, the even distribution of the LNA modifications along the C4-LNA strand resulted in a significant decrease in the opening rate of the zipper tweezers (˜3 times). This exemplary decreased opening rate may be caused by a higher affinity between C4-LNA and the WZ portion of the zipper tweezers (e.g., because the LNA bases are positioned along the section that is complementary to WZ). The disclosed DNA based nanomachines can be configured without overhangs to achieve rapid open/close cycling functionality, e.g., by using locked nucleic acids (LNAs) and peptide nucleic acids (PNAs) together with the exemplary zipper tweezers.
Exemplary examinations into different zipper tweezers states and actions were performed by fluorescent DNA gel electrophoresis.
Exemplary opening schemes (e.g., zipper alone and NL hybridizing overhang) and exemplary different closing schemes (e.g., overhang, overhang with LNAs, and LNAs only) are described for implementing the disclosed zipper tweezers of the disclosed technology. For comparing their kinetics, time required for the 50% completion of the opening and closing reaction (t1/2) with different strand configurations are shown in Tables 3 and 4, respectively.
Exemplary techniques and principles for creating the disclosed molecular zipper-based devices and systems include engineering the functional zipper with regards to the total driving energy and how this energy is distributed along the length of the strands. For example, the nucleotide units (e.g., nucleobases) providing the driving energy must occur with a sufficient frequency along the length of the weak strand in order for a favorable displacement reaction by a target strand. For example, if too many natural DNA bases occur between the driving bases (e.g., inosine), the reaction may terminate. The entropy-induced statistical fluctuations between the bases can enable the reaction to progress along sufficiently small sections of natural base pairs. For example, the length of the natural section that could be overcome by the statistical fluctuations is a temperature- and sequence-dependent property. Also, for example, the bases used to supply the driving energy need not be inosine, as other synthetic bases can be used (e.g., in an engineered strand) that hybridize with less or more than natural affinity. For example,
Exemplary techniques and principles for creating the disclosed molecular zipper-based devices and systems include engineering the functional zipper with regards to the cross-binding nature of the closing strands. For example, a difference between the energies of the hybridization of [Ci:WZ] and [Ci:Oi] can be incorporated into the configuration of the molecular zipper-based devices and systems. For example, a temperature window can be incorporated in which the zipper tweezers can function, e.g., an operating temperature of the tweezers can be significantly chosen below the Tm of the zipper portions of the tweezers (e.g., [WZ:NZ]) and significantly above the Tm of [Ci:WZ]. Exemplary implementations of the disclosed technology demonstrated the increase of the operating temperature range of the disclosed zipper tweezers, e.g., by DNA overhangs, truncating the length of Ci relative to Oi, and using LNA base modifications concentrated at sequence portions that are uncommon between Ci and WZ. For example, DNA strands naturally self-assemble into energetically stable configurations. The disclosed technology can control the interaction energies of the systems constituents to minimize unwanted self-assembly from DNA. For example, if semi-stable unwanted hybridization between the different system elements occurs, it can significantly affect the kinetics of the system, and if stable hybridizations occur (unwanted self-assembly), the function of the system can completely cease.
The disclosed molecular zipper-based tweezers include a variety of advantages, e.g., including having a driving energy that is distributed over the entire length of the fuel strands, which allows more driving energy to be employed. Exemplary molecular zipper-based tweezers devices can sense and capture longer DNA strands with additional abilities to tune the kinetics (e.g., open/close mechanisms) as compared to non-zipper-based tweezers that contain all of their driving energy at short overhangs or loops. Exemplary molecular zipper-based tweezers devices can also allow for the use of longer fuel strands, e.g., because the disclosed zipper tweezers do not have sticky ssDNA overhangs that protrude from the ends of the tweezers in the sensing (e.g., closed or zipped) position. This can enable the exemplary molecular zipper-based tweezers devices to be opened without the use of overhangs, e.g., which can allow spontaneous regeneration to its closed position.
In another aspect, the disclosed technology can include devices, systems, and techniques that can provide a nanoscale molecular-based actuator, e.g., molecular zipper based springs. For example, the exemplary molecular zipper based springs can contract and impart force. For example, the molecular zipper based springs that can be implemented in applications that require tools that are small and sensitive enough to interact with molecules of interest, e.g., including smart drug carriers, sensors and devices for nanoscale transport and manipulation of biological macromolecules. DNA can be employed in the molecular zipper based springs of the disclosed technology, e.g., which can offer innate self-assembly properties, flexibility in design of secondary structures, and desirable length scale. In some examples, a DNA zipper based spring can include an inosine-based zipper mechanism at its functional core in which an inosine-containing strand creates a weak complement to a natural DNA strand.
The exemplary schematic illustration 1120 shows the opening of the exemplary zipper spring using the disclosed zipper mechanism. An exemplary extended DNA based zipper spring 1131 is shown in an extended position, which includes the two zipper strands AN and AW separated, e.g., by uncoupling the hybridized complementary nucleobases between the AN and AW strands to an unzipped or open position. For example, the exemplary extended DNA based zipper spring 1131 can be unzipped to an extended position by a target molecule that includes an extending strand 1132 (e.g., also referred to as an SE strand) which can hybridize to the AN strand of the zipper member 1122, thereby displacing AW from AN. The extending strands 1132 (SE) can be configured as an opening fuel strand (AO) with toeholds on either end or both ends, e.g., to assist in contraction and extension of the zipper springs. For example, when the SE extending strand 1132 was introduced to the contracted spring (e.g., the contracted DNA based zipper spring 1121), the SE extending strand 1132 hybridizes to the AN portion of the zipper member 1122 by competitively displacing AW away from AN using the zipper process causing the zipper spring to extend (e.g., into the extended DNA based zipper spring 1131). For example, the displacement reaction occurs because the enthalpies of the C≡G bonds between SE and AN are stronger by ˜8 kJ/mol than those of the I═C bonds between AW and AN.
Once the exemplary zipper springs have been extended by the SE extending strand 1132, the exemplary extended DNA based zipper spring 1131 can once again be reset (e.g., contracted) by introducing contracting fuel stands 1333 and 1334 (e.g., also represented as an SC1 strand and an SC2 strand, respectively). For example, the SE extending strand 1132 that is bound to the AN strand of the zipper member 1122 on the extended DNA based zipper spring 1131 can be removed by the contracting strands 1333 and 1334 and the AW and AN portions can re-hybridize together, e.g., resetting the zipper spring back to the contracted state. For example, the SC1 and SC2 contracting fuel strands 1333 and 1334 can remove the SE extending strand 1132 by hybridizing to exemplary toehold nucleotide units (e.g., 12 nt toeholds) on the SE extending strand 1132 and subsequently to bases of the zipper-hybridizing portion on the SE extending strand 1132. In some examples, the three strands (e.g., SE, SC1 and SC2) form a waste product 1135, which can drift away and leave the exemplary zipper springs to re-hybridize and contract. For example, the two strands SC1 and SC2 can remove the SE strand from the AN portion of the zipper spring because there is additional energy in the exemplary toeholds (e.g., 12 nt toehold) of SC1 and SC2 driving them to hybridize with the complementary 12 nt toehold on the SE strand. For example, at 37° C. there is considerable amount of free energy (e.g., ΔG37=−91.46 kJ/mol), e.g., favoring the SE strand to extend the contracted zipper spring; and once the SE strand is removed, there is also a considerable amount of free energy favoring the zipper spring to contract (e.g., ΔG37=−87.90 kJ/mol).
Exemplary implementations were performed to demonstrate the described functionalities and capabilities of the disclosed molecular zipper tweezers. Chemicals and buffer solutions used in exemplary implementations were obtained from Sigma Aldrich (Saint Louis, Mo.) unless otherwise specified. The exemplary DNA constructs were obtained from IDT (Coreville, Iowa); the exemplary DNA ladders were obtained from Promega (Madison, Wis.); and the exemplary DNA gels were obtained from Lonza (Walkersville, Md.). Exemplary DNA constructs were suspended in DNAase-free 30 mM Tris and 0.16 M NaCl buffer solution pH 8.0.
Exemplary time-lapse fluorescence measurements of the exemplary zipper actions of exemplary zipper springs were visualized, for example, by tagging the strands with fluorescent probes (shown in Table 6) and observing the change in fluorescence with time using appropriate excitation (Ex) and emission (Em) wavelengths for the fluorophores. Exemplary Ex/Em conditions of FAM, Cy5 and Cy3 were observed at 495/520, 550/564 and 648/668 nm, respectively. Exemplary fluorescence measurements were conducted using a Perkin Elmer LS-50B luminescence spectrometer. Exemplary measurements were performed at 37° C. using quartz cuvettes with a 40 μL sampling volume (e.g., Sterna Cell 16.40F-Q-10/Z15) filled with 100 μL of sample at the start of each experimental implementation. The exemplary basal fluorescence of the quenched zipper was measured on each sample prior to data collection. For example, data was collected every 5 seconds. Each exemplary experimental implementation was repeated at least three times, e.g., to obtain an average. Exemplary error bars depict standard error of the mean, which are included in some of the exemplary data plots in the patent document. For example, the addition of exemplary fuel or anti-fuel strands included pausing measurements, e.g., for approximately 20 seconds.
Exemplary gel electrophoresis and fluorescence imaging analyses were performed in the exemplary implementations. For example, DNA gel electrophoresis was performed with 4% agarose gel at 5 V/cm in TBE buffer while monitoring the solution temperature to be less than 20° C. Exemplary reactions were incubated at 37° C. for at least 2 hours prior to gel examination. For example, each constituent of the gel was run in duplicate with a 25 base pair DNA ladder in the first and last lanes. Exemplary extension reactions were conducted, e.g., by adding ten times more extending strands than springs, and exemplary contractions reactions were conducted, e.g., by adding 20 times more contracting strands than springs to over saturate the existing extending strands. Exemplary reactants and controls were thermally annealed with equal concentrations of its components. For example, in order to observe single and double stranded DNA, positions of the strands within the gel were determined using fluorescent gel imaging and Ethidium Bromide (EtBr) staining. Exemplary gels were imaged with a Bio-Rad FX-Imager Pro Plus (Bio-Rad, Hercules, Calif.) and analyzed with the Quantity One software package (Bio-Rad). Modifications to the original gel images included brightness, contrast, cropping of the image area, over laying lines for reference and symbols for identification of the components. Exemplary Cy3 and EtBr imaging was performed with the internal 532 nm laser and 555 nm band pass filter, while exemplary Cy5 imaging uses an external 632 nm helium neon laser and a Newport 670 nm band pass fluorescence filter. Exemplary FAM imaging is performed using a 20 mW argon ion laser and a 530 nm band pass filter.
Exemplary fluorescence measurements and monitoring of the zipper springs were performed in the exemplary implementations. For example, time-lapsed fluorescence measurements of the zipper springs were performed using a temperature controlled Tecan Infinite (San Jose, Calif.) 200 M plate reading spectrometer at 37° C. For example, each experimental implementation was run with an initial 50 μL sample volume with a spring concentration of 100 nM in black 96 well plates. The exemplary plates were covered with a sticky film covers instead of the traditional clear plastic plate cover, e.g., because they reduced the error in measurements caused by evaporation. Addition of the extending or contracting strands in-between cycles may yield about 30 seconds of error in the measurements, e.g., because of the time required to add the strands and restart the machine. The successive extension and contraction cycles of the zipper springs were performed as follows. For example, the first extension and contraction cycle was performed by adding 10 times more extending strands and 20 times more contracting strands than springs. The second extension and contraction cycles were performed by adding 30 times more extending strands and 40 times more contracting strands than springs. The final extension of the zipper springs was performed by adding 50 times more extending strands than springs. For each exemplary cycle, 1 μL of the appropriate extending or contracting strand was added. Exemplary internal controls were included in each plate to monitor intensity shifts from removing and reinserting the plate, evaporation, photo bleaching and dilution from the additional volumes. For example, appropriate slight corrections to the data plots were performed to correct for variations from these effects. The exemplary values including average values and standard errors were calculated using Microsoft Excel, and the average values were plotted and a trend line was added when appropriate.
Thermally annealed zippers self-assembled into their lowest energy configuration. For example, a custom cycling program was run in a PCR thermocycler (Mastercycler Personal, Eppendorf, Westbury, N.Y.) to accomplish this. The solution temperature was quickly raised to 94° C., beyond the double strand melting temperature, followed by a slow, controlled, cooling at a rate of 1° C. every 2 min. to a final temperature of 4° C.
Exemplary implementations were performed to demonstrate tunability of the extension and contraction functionalities of the disclosed zipper springs. For example, the kinetics of extension and contraction can be tuned, e.g., using two different toehold schemes. For example, a first scheme used single stranded toeholds with 6 nt built into the SN side of the springs. These were positioned between the B and AN sections and fluorescent labels were placed on BO(IbFQ) and LO(FAM)) strands. The exemplary 6 nt extending strands (SDE+6) were created by placing a complementary 6 nt toehold into the SE sequence. The 6 nt toeholds on the extending strands hybridized to the 6 nt toehold on the exemplary zipper springs. Likewise, subsequent contraction of the spring was performed with SC1 and SC2+6 (e.g., fitted with an appropriately placed a 6 nt complementary section). Also, for example, the two arms of the zipper spring were modified to accommodate the 12 nt toehold, which included for example, 6 nt being removed from BO(IbFQ) creating Bo-6(IbFQ) and 6 nt being added to LO(FAM) creating, LO+6(FAM), respectively.
Exemplary sequences of the nucleotide units used in exemplary implementations are shown in Table 5 and Table 6. Estimated energies of interaction for exemplary extending and contracting reactions performed in exemplary implementations are presented Table 7.
Table 5 shows the exemplary DNA zipper sequences for nucleotide units of strands used in exemplary implementations of the disclosed DNA based zipper springs technology. Nucleotide sequences that are included in the exemplary hinge members are represented in white text and highlighted in black. Nucleotide sequences that are included in the exemplary arm members are in black text and highlighted in gray. Nucleotide sequences that are included in the exemplary linking toehold members (e.g., toeholds used for fast extension on the zipper springs) are represented in lower case text.
Table 6 shows the exemplary DNA zipper sequences for nucleotide units of strands used in exemplary implementations of the disclosed DNA based zipper springs technology.
Table 7 shows the energy calculations of the transitions, e.g., assuming equal concentrations of all interacting strands with a 160 mM NaCl concentration. The presented ΔG37 energy values can be representative of the actual usable energy of the interaction for which they were calculated. The energy calculations also take the helix formation energy of the incoming extending and contracting strands into account.
Exemplary implementations of the disclosed molecular zipper based springs were performed to examine the functionality of the zipper spring, e.g., with several different extension and contraction strands. For example, the reversible actuation of the zipper springs was visualized through gel electrophoresis (as shown in
For example, the zipper springs were monitored by tagging the inward facing ends of an L strand and a B strand with a fluorescent reporter (FAM) and quencher (IbFQ), respectively. For example, when the two fluorophores co-localized, the zipper springs contracted and quenched the fluorescence (as seen in the plot 1310 in
For example, the extension rate for the zipper springs was sped up by extending one of the exemplary toeholds on the SE strand by an extra 6 nt or 12 nt (e.g., the SE+6 or SE+12 strands shown in illustrations 1321 and 1331, respectively). These exemplary extra sequences were complementary to the toehold built into the zipper springs between its AN and B sections (as shown in the illustration 1120 of
Exemplary implementations were performed to examine the hybridization rate of single closing strands compared to the closing rate of an exemplary zipper spring. Small exemplary DNA hairpins have been shown to re-hybridize closed in a few milliseconds once disassociated. This was investigated by placing a fluorescent reporter on SE+6(FAM) and a quencher on SC+6(IbFQ). Experimentally, this observes the hybridization rate of SC+6(IbFQ) with SE+6(FAM) which should be relatively close to the spring's contraction reaction. Their hybridization rate was found to be k=7.9±3.3×104M−1 s−1. Comparison of this rate constant with that of the contracting spring (k=1.7+0.3×104 M−1 s−1) suggests that the contracting rate of the spring is mostly dominated by the rate at which the extending strand is removed.
The specificity of the contracting strands can be further enhanced by increasing the length of the contracting strands and by incorporating a small zipper duplex into the toehold of the extending strands. For example, for the contracting strand to hybridize with the toehold on the extending strand, it can first displace the zipper and then remove the extending strand. These exemplary modifications can increase the specificity to the contracting strands, but may also slow down the kinetics.
For example, the extension rates of the zipper springs can be decreased by substituting inosine in the place of guanine in the extending strand sequence (as shown in Table 5). For example, this decreased the driving energy of the zipper mechanism by ΔH≈8 kJ/mol for each inosine included in the extending strands. In this example, the weak side of the exemplary zipper sequence built into the zipper springs contained 17 inosines. The exemplary results in
Table 8 shows exemplary data of the extending controls of the spring. Exemplary zipper springs were extended with 10 times and 110 times more SE strands and SE+6 strands than zipper springs. The similarities in the fold change of the different strands with different energies driving the extension reaction and the lack of change with increased extending strand concentrations suggests that the extension reactions using the full zipper mechanism are all relatively complete.
Exemplary implementations were performed to examine the contraction times of the exemplary zipper springs using a single contracting strand as compared to two separate contracting strands. For example, single contracting strands (SC+6) and (SC+12) closed the springs in about the same amount of time as their two-strand counterparts, but the use of a single contracting strand may increase the practicality of the exemplary zipper springs, e.g., by using a single DNA sequence to trigger the extension or contraction of the zipper springs.
The contraction rate of an individual zipper spring, after the extending strand is removed by the contracting strands, is on the order of a few milliseconds. This suggests that the contraction rate of the springs should mostly be dominated by the hybridization rate of the contracting strand with the extending strand. This was verified by placing a FAM fluorescent reporter on SE+6 and an IbFQ quencher on SC+6 shown in
The disclosed zipper mechanism can be produced to be highly sequence specific, which can allow for more than one zipper to function independently within a single device. Exemplary implementations were performed to demonstrate the independence of functionality of the disclosed technology. For example, the B arm members of the zipper springs were transformed into a zipper by changing all of the guanines in its sequence to inosines (e.g., as shown in Table 6). This demonstrated the feasibility of incorporating multiple zipper or spring systems of the disclosed into a more elaborate device or system. For example, fluorescence analysis and gel electrophoresis data shown in
The zipper spring mechanisms and the B arm members (e.g., which can also be configured to have zipper functionality) zipper actions can be configured to function independently from each other. Exemplary implementations were performed to demonstrate the functionality.
For example, opening of an exemplary B arm member zipper is visualized with the exemplary BW strand, e.g., used for time-lapse fluorescence measurements, e.g., BW strand can be tagged with two fluorescent reporters (3′Cy5 and 5′Cy3). However, the Cy3 fluorophore cannot be visualized independently in the gel because of the spectral overlap between Cy3 and EtBr. The springs' extensions are performed with SE and the contractions by SC1 and SC2. For example, BW can be removed by the opening strand BO. The exemplary data in the data panels 1810 and 1820 demonstrate the stability, specificity and independent operation of the arm member zipper actions and the zipper spring actions.
Exemplary calculations of kinetic rates of the exemplary DNA zipper springs are described. The rate constants (k) for the opening and closing of the DNA zipper springs were calculated in Matlab. The modeling was performed utilizing the function “lsqcurvefit” for least squares fitting of the parameters. For example, due to the stiff nature of the kinetics data and equations, integration of the differential equations was carried out using “ode23s”. For curve fitting, the data was scaled from 0 to 1 with 0 relating to the fully quenched state (e.g., all springs contracted) and 1 to maximum observed fluorescence when all the springs are extended.
The opening of the zipper springs from the contracted to the extended state was modeled as a second order reaction between the contracted spring (CS) and the extending strand (SE) to produce a fluorescent extended spring (F) as represented by Eq. (6):
[SE]+[CS]k→[F] (6)
The standard second order kinetics equation was utilized for least squares fitting in Eq. (7):
The concentration of extending strand ([SE]) and contracted springs ([CS]) can be approximated utilizing the fluorescence data using the following relations in Eq. (8) and Eq. (9):
[CS]=1−[F] (8)
[SE]=[SE]0−[F] (9)
where [SE]0 is the concentration of extending strand added to the reaction vessel.
When the spring extension did not run to completion (as determined by the fluorescence not reaching the maximum fluorescence observed when all strands are extended), the reaction was treated as being reversible. This was observed for the inosine substitution spring extension experiments. In this case, it was assumed that the weak portion (AW) on the spring displaced the extending strand.
[SE]+[CS]⇄[F]+[AW] (10)
The concentration of the weak portion (AW) was approximated by its local concentration (=160 μM=1600×). The kinetics equation then becomes:
Closing of springs from extended to the contracted state was modeled as either a reversible second order or third order reaction depending on whether 2 or 1 contracting strands (SC) were used to remove the extending strand from the spring device. The fluorescence decreases as a result of the addition of the contracting strands, however, adding excess contracting strands does not result in the contraction of all of the devices, e.g., indicating that removal of the SE is a reversible process. The contracting strand was not able to extend the spring when added by itself at 100× concentrations to the contracted spring demonstrating a weak affinity to its compliment on the spring device. Thus, the closing was modeled as reversible reaction. The resulting equation becomes:
In the models, it was assumed that free extending strands would bind quickly with free contracting strands reducing the effective concentration of the free contracting strands. The concentrations of the unbound and bound contracting strands were approximated as:
[SC]=[SC]0[SE] (13)
[SESC]=[SE] (14)
The amount of extending strand was calculated similarly when in excess of the contracting strand for the cycling implementations.
Table 9 shows the kinetics of the opening reaction with different constructs at 37° C. Reaction rate constants (k) together with their standard deviations (σk) and R2-value for the indicated zipper and spring reactions are shown.
The “local concentration” of a DNA zipper spring can be determined as the estimated bulk solution equivalent concentration of the two spring strands unhybridized. This exemplary value can describe the driving force for interaction that two co-localized strands have. In the exemplary calculations, a sequence of DNA can have a maximum interaction volume that is approximated by a sphere with the diameter equal to the length of the strand. For example, a 24 base pair (bp) DNA spring fully extended forms an isosceles right triangle with the hypotenuse that is 10.9 nm (e.g., assuming 0.32 nm/bp). A sphere with a 10.9 nm diameter has a volume of 671 nm3. For example, with one zipper spring contained within this volume, the local concentration of the zipper springs can be determined to be 2.47 mM. In other words, with all else being the same, the propensity for an assembled DNA spring to hybridize is equivalent to 2.47 mM of unhybridized DNA spring strands.
Exemplary implementations of the disclosed molecular zipper based springs can be employed to create composite devices. For example, to demonstrated this, the 26 nt BO strand on the B arm of the springs was converted to a zipper by changing the 11 guanines in its sequence to inosines. This gives the springs a removable arm and could be chemically coupled to a surface or an object using a variety of functional groups, e.g., such as thiol modification, then unzipping BW to release the objects from the springs (as exemplified in the illustration 1710 in
The force created by the zippers can also be tuned by changing the base pair sequence of the zippers. For example, a strand including only C-G bonds requires a force of ˜20 pN to be torn apart, where as a strand solely composed of A-T bonds requires ˜9 pN, and a mixture of the bases is somewhere in-between these force values. The disclosed zipper mechanism of the zipper springs can be modified to contain C bases, and thereby tuning the force created by the zipper springs.
The disclosed molecular zipper based spring technology is compact, performs a defined contractile mechanical function, and can be implemented as an actuator (e.g., a motor to actuate DNA origami structures). The disclosed molecular zipper based spring technology includes tunable reaction kinetics with repeatable extension and contraction cycles. For example, exemplary DNA zipper springs demonstrate repeatable extension and contraction cycles and generate ˜9 pN of force during contraction, e.g., which is enough force to manipulate biological macromolecules. In addition, by changing the toehold length of an exemplary DNA zipper spring, the DNA zipper spring's extension and contraction duration can be tuned. Exemplary zipper springs of the disclosed technology can be useful in a variety of applications, e.g., including biomolecular interactions. For example, by using the exemplary zipper springs in dynamic DNA origami structures, these assemblies can become useful functional components in larger microfluidic lab-on-a-chip systems or in nanomedicine as part of a drug delivery system.
The exemplary DNA zipper tweezers and springs can be implemented as separate devices or on a single device, and these devices can be activated under specific environmental conditions, e.g., including temperature, pH, etc. For example, the DNA zipper-based tweezers and springs are self-regenerating, utilize longer fuel strands, and are reliably efficient (e.g., energetically self-sufficient, requiring no external energy, and preventing nonspecific binding of non-target molecules). Also, for example, the described zipper-based technology can provide flexibility in designing robust, compact and modular devices and systems that can be incorporated into multi-component and/or more elaborate DNA based nanomachines.
In another aspect, the disclosed technology can include engineering new structures and materials with the disclosed zipper constructs and integrating the disclosed zipper constructs with other materials, devices, systems, and techniques. For example,
Various configurations of the disclosed molecular zipper can be engineered as structures that include multiple molecular zipper constructs, which can be implemented in nanoscale devices and systems. For example, the double zipper structure 2000 can be configured as a multiple zipper structure implemented in devices and systems that include array structures, position motors, gating elements, vehicles, and carriers.
The disclosed molecular zipper technology can include controlled drug delivery devices, systems, and techniques using integrated nanocapsules with kinetically tunable lids employing the disclosed zipper mechanism. For example, exemplary controlled drug delivery devices can be implemented in a variety of applications, e.g., including biomedical applications such as using controlled release of biocompatible material to treat diseases and disorders. For example, an exemplary biodegradable nano-capsule with a movable lid of the disclosed technology can be implemented for long-term delivery of age-related macular degeneration (AMD) therapeutics, e.g., by controlling the lid opening/closing over an extended time and frequency using exemplary DNA zipper springs. For example, the DNA springs can include engineered nucleic acids constructs that allows tunable and regenerative motor and spring-like action. Other exemplary materials can be included within the exemplary controlled drug delivery device, e.g., including functionalized nanoparticles, imaging agents, enzymes, nucleic acids, or viral vectors, as well as other materials.
For example, intravitreal delivery of drugs and compounds can experience rapid clearance and hence require frequent injections. Controlled drug release over an extended period can reduce the frequency of these injections and allow on-demand release, e.g., for ocular diseases and disorders such as AMD but other diseases. The disclosed controlled drug delivery vehicles can include a degradable nanoscale container (e.g., a nanobowl or nanojar), an actuating molecular zipper construct, and a nanoscale degradable lid. The exemplary drug delivery vehicles can be configured to be biocompatible and immune protected.
For example, the degradable nanoscale container can be configured as a metal capsule or a hollow colloidal capsule. For example, gold can be used as initial plating material to create the hollow colloidal capsule, e.g., by evaporating gold onto polystyrene beads. The exemplary polystyrene beads can include biocompatible and biodegradable polymer materials, e.g., poly-1-lactic acid, poly(glycolic acid), and polycaprolactone. For example, the exemplary capsule can be coated with subsequent layers, e.g., by coating silica using the evaporation techniques.
While this patent document contains many specifics, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this patent document in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.
Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Moreover, the separation of various system components in the embodiments described above should not be understood as requiring such separation in all embodiments.
Only a few implementations and examples are described and other implementations, enhancements and variations can be made based on what is described and illustrated in this patent document.
This patent document is a divisional of U.S. patent application Ser. No. 14/003,442 entitled “MOLECULAR ZIPPER TWEEZERS AND SPRING DEVICES” filed on Nov. 18, 2013, which is a 35 USC § 371 National Stage application of International Application No. PCT/US2012/028383 entitled “MOLECULAR ZIPPER TWEEZERS AND SPRING DEVICES” filed Mar. 8, 2012, which claims the priority of U.S. provisional application No. 61/450,544 entitled “MOLECULAR ZIPPER, TWEEZERS AND SPRING DEVICES” filed on Mar. 8, 2011, the entire disclosures of which are incorporated by reference as part of this document.
This invention was made with government support under grant 5R01DA025296-04 awarded by the National Institute on Drug Abuse (NIDA) of the National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61450544 | Mar 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14003442 | Nov 2013 | US |
| Child | 15952152 | US |
