The present invention relates to a method of synthesizing a microtubule in general, and particularly, to a method of synthesizing a microtubule labeled with nucleotides (DNAs or RNAs) without using a biotin-avidin binding and also to methods of preserving and resynthesizing the produced microtubule.
In Eukaryotic cells, microtubules and actin filaments which are cytoskeleton are developed and used as the rails on which cargo molecules such as intracellular organelles or transport vesicles are transported by motor proteins such as kinesins or dyneins. A mechanism of a molecular transport which is represented by an interaction between kinesins and the microtubules can be reconstituted in vitro. A gliding assay system is also widely known, where the microtubules are caused to glide on kinesins immobilized on a substrate.
As an attempt to engineer this superior biological function to create an artificial molecular transport system, the surface of the gliding microtubules is biochemically modified for functional utilization (see, for example, Non-Patent Document 1 listed below). In this attempt, by biotinylating a portion of the surface of the gliding microtubules and allowing the biotinylated surface to be bound to the cargo molecules such as microbeads, which are covered with streptavidin, via a biotin-avidin binding, the cargo molecules have been successfully loaded and transported on the microtubules. However, this biotin-avidin binding is widely known to be one of the bindings having the highest affinity in interaction of biological systems, so there is a problem that once the cargo molecules are loaded on the microtubules, it is essentially impossible to unload them from the microtubules.
In contrast, a system is disclosed in which a microtubule labeled with a single stranded DNA is produced by biotinylating a portion of the surface of the gliding microtubule and causing the biotinylated surface to be bound to the single stranded DNA, of which the end is modified with streptavidin, via the biotin-avidin binding. Loading is performed by hybridization of a DNA having base sequences complementary to the single stranded DNA bound to the microtubule, and unloading is performed by a DNA cleavage caused by an addition of restriction enzymes (see, for example, Patent Documents 1 and 2 listed below).
However, since the single stranded DNA can be bound only to the biotinylated site and avidins and streptavidins are molecules whose molecular weights are large (60-67 kDa), it leads to a problem that a modification ratio (labeling stoichiometry) of the single stranded DNAs to the microtubules cannot be set arbitrarily. Therefore, if the labeling stoichiometry is higher than a certain amount, there is a concern that the gliding movement of the microtubules will be interfered with.
Prior to the filing of this patent application, the inventors proposed DNA-labeled microtubules considering the use of the biotin-avidin binding as well as a chemical crosslinking agent, and they have proposed a system, where a mechanism of selective and autonomous loading and unloading of cargo molecules is accomplished only by hybridization (Japanese Patent Application No. 2005-307880 yet to be published). However, this proposal does not refer to a specific method of synthesizing the DNA-labeled microtubules.
Japanese Patent Application Publication No. 2006-204241.
Japanese Patent Application Publication No. 2006-271323.
Accordingly, it is an object of the present invention to provide a method of synthesizing a microtubule labeled with nucleotides without using a generally utilized biotin-avidin binding and also to provide methods for preserving and resynthesizing the produced microtubule.
In the present invention, to achieve the above object, (a) a microtubule stabilized after the polymerization is reacted with (b) a chemical crosslinking agent having a succinimide and maleimide and (c) nucleotides having a thiolated 3′ or 5′ end to synthesize a microtubule-chemical crosslinking agent-nucleotide complex.
As an example of the reaction, first, an amino group of the microtubule is reacted with succinimide of said chemical crosslinking agent to synthesize the microtubule-chemical crosslinking agent complex. Then, maleimide of the complex is reacted with said nucleotides having thiolated end to synthesize the microtubule-chemical crosslinking agent-nucleotide complex (nucleotide-labeled microtubule).
For said chemical crosslinking agent, it is preferable to use one of MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester), Sulfo-MBS, SMPB (Succinimidyl 4-[p-maleimidophenyl]butyrate), Sulfo-SMPB, GMBS (N-[γ-maleimidobutyryloxy]succinimide ester), Sulfo-GMBS, EMCS (N-[ε-maleimidocaproyloxy]succinimide ester), and Sulfo-EMCS. Molecular weights of these chemical crosslinking agents are about 280˜460 Da, two orders of magnitude smaller than the molecular weights of avidins and streptavidins so that a range can be extended in which a modification ratio (labeling stoichiometry) of the single stranded DNAs to the microtubules is set.
For said nucleotides, by way of example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is used, which have a single stranded portion therein.
For preservation of the nucleotide-labeled microtubule thus synthesized, nucleotide-labeled tubulin is produced by cooling a solution of the nucleotide-labeled microtubule or adding a depolymerizing agent to the solution, and the solution of the tubulin is kept frozen after rapid freezing.
The preserved nucleotide-labeled microtubule can be resynthesized by mixing guanosine triphosphate (GTP) and a magnesium ion into the solution of the nucleotide-labeled tubulin and warming this mixture, which again causes a polymerization of the nucleotide-labeled tubulin and synthesis of the nucleotide-labeled microtubule.
As a preferable example, the nucleotide-labeled tubulin can be polymerized again by adding wild-type tubulin which is not labeled with nucleotides or tubulin which is labeled with nucleotides having different base sequences than said nucleotide-labeled tubulin into said mixture.
Furthermore, polymerization of the nucleotide-labeled tubulin and depolymerization of the nucleotide-labeled microtubule can be repeated several times before the rapid freezing of the nucleotide-labeled tubulin.
According to the invention, microtubule labeled with nucleotides can be synthesized without using a generally utilized biotin-avidin binding, thereby being able to set a labeling stoichiometry of the nucleotides to the microtubules arbitrarily.
Moreover, since it is possible to preserve and resynthesize the produced nucleotide-labeled microtubule, microtubules labeled with different kinds of single stranded nucleotides in a high density can be resynthesized, thereby increasing an orientation of the microtubules in patterning.
Such nucleotide-labeled microtubules increase efficiency and functionality in molecular transfer/molecular transport. In addition, these microtubules enable genetic diagnosis with high efficiency in sensing a specific DNA or RNA.
In the following, preferred embodiments of the present invention will be described with reference to the accompanying drawings. For examples below, Sulfo-GMBS was used as a chemical crosslinking agent and a single stranded deoxyribonucleic acid (ssDNA) with a thiolated 5′ end as nucleotides.
A method of synthesizing a nucleotide-labeled microtubule as an embodiment of the present invention is following.
In a solution of α,β-tubulins which were extracted and purified from porcine brains (40-60 μM), 1 mM GTP and 1 mM MgSO4 were mixed. After the mixed solution was incubated at 37° C. for 30 minutes, 80 μM Taxol was added to stabilize the polymerized microtubules and the resulting solution was let stand at room temperature.
In the solution of microtubules prepared by the above procedures, 2.5 mM Sulfo-GMBS (22324, produced by PIERCE) was mixed, of which concentration was adjusted by, for example, anhydrous DMSO (dimethyl sulphoxide), and the mixed solution was further incubated under a shaded condition at room temperature for 30 minutes. During this procedure, as shown in
Then, after the incubated solution was centrifuged (at 30 krpm at 25° C. for 10 minutes) by an ultracentrifuge, the pellet recovered after centrifugation was rinsed by a buffer A (50 mM PIPES-KOH pH 7.0, 4 mM MgCl2, and 20 μM Taxol) and suspended in the buffer A, thereby resulting in a solution of the microtubule-chemical crosslinking agent complex.
The solution of the microtubule-chemical crosslinking agent complexes prepared by the above procedures was mixed with the same concentration of ssDNAs having a thiolated 5′ end, and the mixture was incubated under a shaded condition at room temperature for 90 minutes. During this incubation, as shown in
Then, 50 mM Tris-HCl pH 7.0 and 10 mM β mercaptoethanol of which concentrations were each adjusted by the buffer A were added to the incubated solution containing the complex, and the resulting solution was further incubated under a shaded condition at room temperature for 20 minutes.
After the incubated solution of the complexes was centrifuged (at 30 krpm at 25° C. for 10 minutes) by an ultracentrifuge, the pellet recovered after centrifugation was rinsed by and suspended in the buffer A, thereby resulting in a solution of the microtubule-chemical crosslinking agent-nucleotide complexes.
If either end of the nucleotide is labeled with the fluorochrome, it is possible to check visually the result of labeling the nucleotides to the microtubules by observing through a fluorescent microscope. For example, the fibrous microtubules can be identified, as shown in
From an experimental result from the measurements of absorbance or concentration of the microtubules, nucleotides, and chemical cross-linking agents, it is also possible to calculate a concentration ratio of the microtubules and nucleotides and quantify the labeling stoichiometry of the nucleotides to the microtubules. In the above example, the labeling stoichiometry was 0.61 meaning that at least one nucleotide chain was attached to one of the two molecules of tubulin heterodimer and the nucleotides were labeled to the microtubules in a quite high density. The labeling stoichiometry can be set arbitrarily by adjusting a concentration of the Sulfo-GMBS or nucleotides to be reacted with the microtubules which are already polymerized and stabilized.
A gliding speed of the nucleotide-labeled microtubules can be evaluated by a gliding assay, where the microtubules labeled with the nucleotides are caused to glide on a slide glass which motor proteins such as kinesin or dynein are adsorbed to.
It is also possible for the nucleotide-labeled microtubules to glide even if the motor proteins are exchanged for dyneins (e.g. HFB380) which can cause the nucleotide-labeled microtubules to glide with a speed several times faster than that of kinesins. In this case, the gliding speed easily exceeds 1.0 μm/s even if the microtubules are labeled with the nucleotides at high density.
Now, methods of preserving and resynthesizing the synthesized nucleotide-labeled microtubule will be described.
The solution of the microtubule-chemical crosslinking agent-nucleotide complexes prepared by the above procedures was centrifuged (at 30 krpm at 25° C. for 10 minutes) by an ultracentrifuge. The pellet recovered after centrifugation was suspended in the ice-cold buffer B (80 mM PIPES-KOH pH 6.8, 1 mM MgCl2, and 1 mM EGTA), and the suspension was incubated at 4° C. for 30 minutes. The nucleotide-labeled microtubules may also be depolymerized by adding a depolymerizing agent such as colcemids.
Then, the solution of the depolymerized microtubules was centrifuged (at 80 krpm at 2° C. for 10 minutes) by an ultracentrifuge, and the supernatant fluid was recovered. The solution to be centrifuged may be dispensed, if necessary. The recovered solution (nucleotide-labeled tubulins) was frozen by rapid freezing with liquid nitrogen and then kept in storage of liquid nitrogen. Also, before the rapid freezing of the nucleotide-labeled tubulins for preservation, the purity of the active nucleotide-labeled tubulins may be increased. This increase is accomplished by repeating several times a process of polymerizing the nucleotide-labeled tubulins by the following procedures and depolymerizing the polymerized nucleotide-labeled microtubules.
The solution of the nucleotide-labeled tubulins preserved by the above procedures was thawed, in which the buffer B, 1 mM GTP, and 1 mM MgSO4 were further added. After the resultant solution was mixed, it was incubated at 37° C. for 30 minutes. Glycerol may be added to the mixed solution to increase the efficiency of polymerization, if necessary. Moreover, the labeling ratio of the nucleotides to the microtubules can be set arbitrarily by mixing a moderate amount of wild-type tubulins which are not labeled with the nucleotides. Microtubules which are labeled with nucleotides having one of several kinds of base sequences can be synthesized by mixing a moderate amount of tubulins which are labeled with nucleotides having different base sequences than the above nucleotide-labeled tubulins.
Then, 80 μM taxol was added to the incubated solution to stabilize the polymerized microtubules. After the solution of the stabilized microtubules was centrifuged (at 30 krpm at 25° C. for 10 minutes) by an ultracentrifuge, the pellet recovered after centrifugation was suspended in the buffer A, thereby resulting in a solution of the resynthesized nucleotide-labeled microtubules.
Even though the invention has been described based on specific examples thereof, the invention is not limited to these examples. By way of example, in the above examples, Sulfo-GMBS was used as the chemical crosslinking agent, but the chemical crosslinking agent may comprise MBS (22311, produced by PIERCE), Sulfo-MBS (22312, produced by PIERCE), SMPB (22416, produced by PIERCE), Sulfo-SMPB (22317, produced by PIERCE), GMBS (22309, produced by PIERCE), EMCS (22308, produced by PIERCE), Sulfo-EMCS (22307, produced by PIERCE), and the like.
In addition, deoxyribonucleic acid (DNA) was used as the nucleotides in the above examples, but also ribonucleic acid (RNA) may be used.
The present application is based on Japanese priority application No. 2007-047933 filed on Feb. 27, 2007, with the Japanese Patent Office, the entire contents of which are hereby incorporated by reference.
Number | Date | Country | Kind |
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2007-047933 | Feb 2007 | JP | national |