Patent Application
20130165348
The present invention relates to a method for synthesizing proteins from DNA molecules captured in microchambers, and the device used therefor.
In all cells, proteins are synthesized by processive enzymes called ribosomes. The dynamics of the translation process of genetic information carried by messenger RNA involving ribosomes remain to be completely elucidated (refer to Non-Patent Documents 1,2). Micrometer-size droplets and chambers are becoming extremely highly valuable tools in single-molecule enzymatic analysis procedures. Water-in-oil emulsions are used for the purpose of fractionating enzymes and gene libraries for protein synthesis (refer to Non-Patent Documents 3,4). Moreover, microchambers are also used in single-molecule enzymatic analysis (refer to Patent Document 1 and Non-Patent Document 5) and protein synthesis (refer to Non-Patent Document 6). Aqueous droplets of water-in-oil droplet emulsion are successfully applied to synthesize proteins from monomolecular DNA. Here, water-in-oil droplet emulsions are prepared in such a way that each droplet contains only DNA of no more than one molecule. In contrast, protein synthesis using microchambers lags far behind (refer to Non-Patent Document 6). Cases have been so far reported where extracellular proteins having a volume of about 100 mL were synthesized using arrays of polydimethylsiloxane (PDMS) microreactors and nano-well chips (refer to Non-Patent Documents 7 to 9). In addition, a technique has been developed with respect to low-capacity devices, wherein a green fluorescent protein (GFP) is synthesized by introducing in 1 pL PDMS chambers a gene DNA immobilized onto polymer beads (refer to Non-Patent Document 6), but which at least requires ten molecules of DNA per chamber to detect protein synthesis. Miniaturization of such microchamber arrays for protein synthesis would enable to construct protein chips at a very high density. Actually, methods to generate such protein chips by coupling extracellular protein synthesis and a DNA chip technology have been recently reported (refer to Non-Patent Documents 10 to 14). Protein chips are conventionally prepared by synthesizing proteins within viable cells and patterning the purified proteins in array format, but this methodology proved to be tedious and highly expensive.
By contrast with aforementioned methodologies to generate protein arrays by synthesizing proteins in viable cells and printing them in array format, technologies consisting in synthesizing in situ proteins on arrays such as NAPPA (Nucleic Acid Programmable Protein Array) (refer to Non-Patent Document 12) and DAPA (DNA Array to Protein Array) (refer to Non-Patent Document 13) have been recently documented. Proteins are synthesized from genetic DNA spotted onto a first slide surface, while a tag is preliminarly arranged to bind onto a second slide surface. According to these methods which allow several thousands of proteins to be synthesized within an unpartitioned area, the synthesized proteins easily get spread and there is a potential risk for protein reciprocal contamination between spots. In order to solve this problem, with respect to the NAPPA method, attempts are carried out by affixing to a slide surface a tagged genetic DNA and introducing the tag in the C-terminus of a protein to localize in the vicinity of a corresponding gene the protein synthesized by means of an antibody capable to recognize and capture it. However, this method proved to be tedious and remains problematically impractical.
As regards the DAPA method on the other hand, measures are taken in an attempt to avoid such contamination by enlarging the distance between spots of genetic DNA (0.5 mm) and using a filter membrane to prevent proteins from spreading. In either approach however, usage of microfluid devices consisting of single compartments makes it very difficult to obtain spots of highly concentrated proteins from genetic DNA since spreading and dilution of synthesized proteins would occur, whilst the array density could hardly be increased due to a large inter-spot distance.
In view of such circumstances, the present invention has as its object to provide a method and a device for synthesizing proteins from DNA molecules captured in microchambers, whereby shortening of the distance between microchambers allows for increasing array density.
In order to achieve this purpose, the invention characteristically relates to:
[1] a method for synthesizing proteins from DNA molecules captured in microchambers, wherein microchambers are arranged at a high density, a DNA solution diluted to capture one DNA molecule on average is enclosed into microchambers, mRNA is synthesized using as a template said one DNA molecule on average, and synthesis of proteins is carried out extracellularly on the basis of this mRNA;
[2] the method for synthesizing proteins from DNA molecules captured in microchambers as mentioned in [1] above, wherein the microchambers refer to tiny chambers having a volume not exceeding 200 femtoliters;
[3] the method for synthesizing proteins from DNA molecules captured in microchambers as mentioned in [2] above, wherein the microchambers having a diameter of about 7 μm are arranged at distances corresponding to a center-to-center spacing of about 10 μm;
[4] the method for synthesizing proteins from DNA molecules captured in microchambers as mentioned in either [1] to [3] above, wherein the DNA solution is diluted 500-fold to give a bulk concentration of 8.0 pM versus a bulk concentration of 4.2 nM for usual reaction solutions;
[5] the method for synthesizing proteins from DNA molecules captured in microchambers as mentioned in either [1] to [3] above, wherein the DNA solution is diluted 400-fold to give a bulk concentration of 10.5 pM versus a bulk concentration of 4.2 nM for usual reaction solutions;
[6] the method for synthesizing proteins from DNA molecules captured in microchambers as mentioned in either [1] to [5] above, wherein the microchamber surface is subjected to surface modification by means of a polymer so as to restrain the adsorption of extracellular proteins thereto;
[7] the method for synthesizing proteins from DNA molecules captured in microchambers as mentioned in [6] above, wherein a MPC polymer [poly (2-methacryloyl oxyethyl phosphorylchloline-co-3-methacryloyl oxypropyl trimethoxysilane)] provides for the polymer;
[8] a device for synthesizing proteins from DNA molecules captured in microchambers, whereby protein arrays are generated at a high density and a high purity by extracellularly synthesizing proteins from one DNA molecule on average in each of the microchambers arranged at a high density;
[9] the device for synthesizing proteins from DNA molecules captured in microchambers as in [8] above, wherein each microchamber has a volume of not more than 200 femtoliters;
[10] the device for synthesizing proteins from DNA molecules captured in microchambers as in [9] above, wherein said microchambers having a diameter of about 7 μm are arranged at distances corresponding to a center-to-center spacing of about 10 μm;
[11] the device for synthesizing proteins from DNA molecules captured in microchambers as mentioned in either [8] to [10] above, wherein proteins are synthesized from one DNA molecule on average by introducing a diluted DNA solution into said microchambers.
[12] the device for synthesizing proteins from DNA molecules captured in microchambers as in [11] above, wherein said DNA solution is diluted 500-fold to give a bulk concentration of 8.0 pM versus a bulk concentration of 4.2 nM for usual reaction solutions;
[13] the device for synthesizing proteins from DNA molecules captured in microchambers as in [11] above, wherein said DNA solution is diluted 400-fold to give a bulk concentration of 10.5 pM versus a bulk concentration of 4.2 nM for usual reaction solutions;
[14] the device for synthesizing proteins from DNA molecules captured in microchambers as mentioned in either [8] to [13] above, wherein a polymer is provided to perform surface modification in said microchambers so as to restrain the adsorption of extracellular proteins thereto; and
[15] the device for synthesizing proteins from DNA molecules captured in microchambers as in [1,4] above, wherein the polymer is a MPC polymer [poly (2-methacryloyl oxyethyl phosphorylchloline-co-3-methacryloyl oxypropyl trimethoxysilane)].
According to the invention whereby proteins can be synthesized from one DNA molecule on average in micro-volume reactors (about 200 femtoliters) arranged at distances corresponding to a center-to-center spacing of about 0.01 mm, not only the purity of protein-encoding mRNA is guaranteed in each microchamber, but also protein arrays can be achieved at a high density (in principle, at least 2500 times that of the conventional devices).
The method for synthesizing proteins from DNA molecules captured in microchambers consists in setting up microchambers at a high density, enclosing thereinto a diluted DNA solution to capture one DNA molecule on average and synthesizing mRNA using this one DNA molecule on average as a template to perform extracellular protein synthesis on the basis of this mRNA.
Furthermore, the device designed for synthesizing proteins from DNA molecules captured in microchambers allows for preparing high densityhigh purity protein arrays by extracellularly synthesizing proteins from one DNA molecule on average in each of the microchambers from a plurality thereof arranged at a high density.
Synthesis of proteins is accomplished by coupling transcription and translation reactions. mRNA (2) is constructed by transcripting GFP-encoding DNA (1). The resulting mRNA (2) is translated to protein (GFP) (3) through vectors of the protein synthesis system. DNA (1) is bound to ribosome (4) to act as a template in protein synthesis. Here, the substance which is individualized inside the microchambers is a substrate for protein synthesis (DNA), but not an enzyme (ribosome to be specific). Each microchamber contains enough translational components (ribosomes, nucleotides, translational factors) to efficiently synthesize proteins, and these factors are not limitative.
According to the invention, the minimal gene DNA enables to easily synthesize proteins at a high purity in compartments consisting of microchambers using one DNA molecule on average. It is noteworthy that this method is kept free from any adherence of single DNA molecules onto the surface of the microchambers. Such DNA molecules remain in a state of free diffusion inside those microchambers.
This method enables to construct high-density chips having arrayed spots of highly concentrated proteins.
Firstly, a PDMS chip for protein synthesis was constructed. The PDMS chip and the template used for this purpose were generated by a commonly used replica molding technique and photolithography using a negative-type photoresist (SU-8 2005, MicroChem, Co.). Using as a template a cylindrically-patterned structure (7 μm in diameter, 5 μm in height) (refer to
The volume of each microchamber on this PDMS chip was about 190 femtoliters (fL). These SEM images indicate that a PDMS chip may be modified with a MPC polymer without breaking down the structure of any chamber. In order to prevent the reaction solution in the recipients from evaporating in the midst of the protein synthesis reaction process taking place in the microchambers, the MPC polymer-modified PDMS chip was soaked overnight in water and taken out therefrom just prior to usage.
With respect to extracellular synthesis of proteins, GFP or emerald GFP (EmGFP; Invitrogen) were synthesized using a commercially available kit (RTS 100 E. coli HY Kit, Roche Co.). This kit which contains all the factors necessary for in vitro protein synthesis was implemented according to directions for use. In bulk synthesis, the variation with time of the synthetized amount of proteins was quantified using a multilabel reader [Arvo (registered trademark)×2; PerkinElmer, Inc.; excitation filter: 485 nm; emission filter: 535 nm]. The reaction solution was introduced in microchambers by inserting a droplet of solution for protein synthesis between the PDMS chip and a microscope cover glass. The removal of the solution in excess was carried out by pressing the PDMS chip with a blunt-tipped plastic rod, etc. In this way, it was possible to hermetically-seal the solution inside each microchamber (refer to Patent Document 1 and Non-Patent Document 5). Hermetically-sealed microchambers were set onto the stage of an optical microscope and reacted at room temperature (25° C.). Observations of how protein synthesis was going on were done using a high sensitivity electron multiplying charged coupled device (EMCCD) camera (iXonEM+885 EMCCD Camera, Andor Technology Plc).
As a first step, bulk synthesis of natural type GFP and EmGFP was performed using a RTS100 Kit (reaction volume of 50 μL).
Because of a slight gap between excitation and emission wavelengths of these proteins, no accurate comparison of protein synthesis amounts could be done with current experimental setups. However, as shown in
Next, extracellular synthesis of proteins from one DNA molecule was performed using microchambers. A DNA solution with a concentration adjusted to 10.5 pM through a 400-fold dilution so that one DNA molecule on average fits into one microchamber was placed over the PDMS chip with the RTS100 Kit, and was hermetically sealed inside the microchamber according to the aforementioned method, then maintained at room temperature (25° C.) to incubate.
Also, adjustment of the DNA solution through a 500-fold dilution to a concentration of 8.0 pM versus 4.2 nM for an usual bulk reaction solution enables to capture one DNA molecule per microchamber.
As evidenced in
With reference to
The microchambers in
The viability of this concept is demonstrated with reference to two different types of DNA molecules bearing the EmGPF gene and the yellow fluorescent protein (YFP) gene. The DNA molecules are respectively added to a cell-free protein synthesis system at a concentration of 0.31 DNA molecule per microchamber. The synthesis system having DNA molecules is incubated in PDMS microchambers.
Protein synthesis was extracellularly performed from one DNA molecule on average in microchambers exhibiting a volume of 190 femtoliters. mRNA was synthesized using DNA as a template in microchambers, then proteins were synthesized on the basis of this mRNA. The present invention allows for efficiently synthesizing RNA and proteins from one DNA molecule on average as contained in a microchamber. According to the invention and as shown in
RNA molecules play an important role in signal transduction, structure, catalysis and genetic information control. Through genome analysis, it has been clarified that they code for approximately 1.5% of proteins in the human genome. 60 to 70% of RNA molecules do not code for proteins [refer to Non-Patent Document 5 above].
In
In
The device in
As aforementioned, usage of minuscule microchambers requires less expensive reagents for RNA and protein synthesis, thus enabling to curb at a large extent costs for generating RNA or protein chips. In principle, a tight arrangement of microchambers makes it possible to generate using a DNA library protein chips capable to produce at a high density proteins coded in one or several complete genomes on one chip of only a few square millimeters in size. In that way, drug screening tests and functional analysis of genes in one or several complete genomes could be easily performed. Furthermore, proteins (or RNA) associated with the method to construct protein (RNA) chips according to the invention would constitute a powerful tool for in vitro evolution systems if a mutated-gene library is employed.
The present invention is construed not to be limited to the aforementioned example and allows for various changes and modifications pursuant to its purpose, which are not excluded from the scope thereof.
The method for synthesizing proteins from DNA molecules captured in microchambers and the device aimed at this purpose can be used as tools to synthesize proteins in a manner which does not require any large distance between microchambers and therefore that enables to highly densify arrays.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009281673 | Dec 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/007144 | 12/8/2010 | WO | 00 | 3/14/2013 |
