Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-19
2004-11-02
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091200, C435S287200, C536S022100, C536S023100, C536S024300, C521S053000, C521S059000, C521S084100, C521S143000, C521S146000, C428S453000, C428S543000, C525S054100
Reexamination Certificate
active
06811980
ABSTRACT:
DESCRIPTION
The invention relates to a process of immobilization of nucleic acid molecules on a substrate, immobilized nucleic acid obtainable therefrom and use thereof.
Immobilization (binding) of nucleic acid molecules on a substrate, such as a solid surface, is a well known problem in a large number of applications. The binding of nucleic acid molecules on substrates is of high interest for the development of nucleic acid based nanotechnology, including nucleic acid based nanoelectronics, like wires, biosensors, chips, see Storhoff, J. J., Mirkin, C. A. (1999) Chem. Rev., 99, 1849-1862 “Programmed Materials Synthesis with DNA.”
Another strong motivation for immobilizing nucleic acid molecules on substrates and membranes is the characterization and engineering of nucleic acids in the field of medicine and biology, see Allison, D. P., Bottomley, L. A., Thundat, T., Brown, G. M., Woychik, R. P., Schrick, J. J., Jacobson, K. B. and Warmack, R. J. (1992) Proc. Natl. Acad. Sci. USA, 89,21 10129-10133 “Immobilization of DNA for scanning probe microscopy”; Bezanilla, M., Manne, S., Laney, D. E., Lyubchenko, Y. L. and Hansma, H. G. (1995) Langmuir, 11, 655-659 “Adsorption of DNA to mica, silylated mica and minerals: characterization by atomic force microscopy.”
Also, purification of nucleic acid solutions by attachment of nucleic acid molecules to substrates is of interest, see U.S. Pat. Nos. 5,523,392 and 5,503,816.
The binding problem for nucleic acids to solid surfaces has so far been solved by using a number of different approaches.
The most common ones employ the modification of the substrate surface by chemical treatment. One useful approach is the silanization of surfaces leading, for example, to exposed vinyl groups which bind to nucleic acid molecules, see Bensimon, D., Simon, A. J., Croquette, V., Bensimon, A. (1995) Physical Review Letters 74, 23, 4754-4757 “Stretching DNA with a Receding Meniscus: Experiments and Models.” On mica substrates, effective binding of the nucleic acid was found by using the counterion method: this method is done by adsorbing the nucleic acid onto mica in the presence of a divalent (+2 charged) ion, like Mg
2+
. The idea is that the counterion will provide binding to the negatively charged nucleic acid backbone and at the same time also to the negatively charged mica surface, see Ye, J. Y., Umemura, K., Ishikawa, M., Kuroda, R. (2000) Analytical Biochemistry 281, 21-25 “Atomic Force Microscopy of DNA Molecules Stretched by Spin-Coating Technique.”; Dunlap, D. D., Maggi, A., Soria, M. R., Monaco, L. (1997) Nucl. Acid Res. 25, 3095 “Nanoscopic Structure of DNA Condensed for Gene Delivery.”; Lyubchenko, Y. L., Shlyakhtenko, L. S. (1997) Proc. Natl. Acad. Sci. USA 94,496 “Direct Visualization of Supercoiled DNA in situ with Atomic Force Microscopy.”; Yokota, H., Sunwoo, J., Snikaya, M., van den Engh, G., Aebersold, R. (1999) “Spin-Stretching of DNA and Protein Molecules for Detection by Fluorescence and Atomic Force Microscopy.”
Also, the adjustment of the degree of immobilization through the chemical control of the pH-value was described for a large variety of different surfaces, see Allemand, J.-F., Bensimon, D., Julien, L., Bensimon, A, Croquette, V. (1997) Biophysical Journal, 73, 2064-2070 “pH-Dependent Binding and Combing of DNA.”
Yoshida, K., Yoshimoto, M., Sasaki, K., Ohnishi, T., Ushiki, T., Hitomik, J., Yamamoto, S., Sigeno, M. (1998) Biophysical Journal, 74, 1654-1657 “Fabrication of a New Substrate for Atomic Force Microscopic Observation of DNA Molecules from an Ultrasmooth Sapphire Plate.” describes the hydrophilization of a sapphire surface treated with Na
3
PO
4
aqueous solution. It is reported that the hydrophilic surface character after the wet treatment makes it easy for nucleic acid molecules to adhere to the substrate surface. Other approaches utilize the specific binding (chemisorption) of thiol-group terminated nucleic acid to gold surfaces and electrodes. Washizu, M., Kurosawa, O., Arai, I., Suzuki, S., Shimamoto, N. (1995) IEEE Trans. Industr. Appl., 31, 3, 447-456 “Applications of Electrostatic Stretch-and-Positioning of DNA.” reported on strong, covalent-like binding of nucleic acids to fresh aluminum electrodes in an alternating electrical field.
Oxygen plasma treatment is a well-known method to clean surfaces from organic impurities by oxidation, which supports the generation of OH-groups. U.S. Pat. No. 5,055,316 teaches the oxygen plasma supported tight binding of proteins to surfaces. Molecular tailoring of surfaces using a plasma treatment is disclosed in U.S. Pat. No. 5,876,753. A method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals was described in U.S. Pat. No. 5,369,012.
As mentioned above, various nucleic acid immobilization methods have been proposed. Most of them employ wet chemical treatment to modify the substrate. Therefore, the use of expensive chemical components is necessary, often not providing reproducible and permanent immobilization of nucleic acid molecules on that substrate.
Only a small variety of substrate materials can be employed using the above mentioned wet chemical treatment.
Accordingly, it is an object of the present invention to provide a process for immobilization of nucleic acid molecules on a substrate to overcome the drawbacks of prior art, especially to provide a process not requiring a wet chemical treatment of the substrate and to provide a reproducible process for the permanent immobilization of nucleic acid molecules on a substrate.
A further object underlying the present invention is to provide an immobilized nucleic acid which may be used in nucleic acid based nanotechnology.
The first object is solved by a process for immobilization of nucleic acid molecules on a substrate wherein the substrate is treated with atomic oxygen plasma prior to immobilizing the nucleic acid molecules thereon.
In a preferred embodiment the nucleic acid is selected from the group consisting of DNA,RNA, PNA (peptidic-NA), CNA (aminocyclohexylethane acid-NA), HNA (hexitol nucleic acids), p-RNA (pyranosyl-RNA), oligonucleotides, oligonucleotides of DNA, oligonucleotides of RNA, primers, A-DNA, B-DNA, Z-DNA, polynucleotides of DNA, polynucleotides of RNA, T-junctions of nucleic acids, domains of non-nucleic acid polymer-nucleic acid blockpolymers and combinations thereof. Suited non-nucleic acid polymers for blockcopolymers can be polypeptides, polysaccharides such as cellulose, or artificial polymers, such as polyethylene glycol, and are generally known to the person skilled in the art.
In another embodiment the nucleic acid is double-stranded or single-stranded.
In a further embodiment the nucleic acid is of natural character, modified, such as substituted with functional groups, non-modified or artificially generated.
In a still further embodiment the substrate is a single crystal surface or an amorphous surface.
More preferably the surface material is selected from the group comprising silicon oxides, glass, aluminum oxides, sapphire, perovskites, like SrTiO
3
, LaAlO
3
, NdGaO
3
, ZrO
2
and derivatives thereof and doped and/or stabilized derivatives thereof, for example using Yttrium as stabilizer.
In a further preferred embodiment microwave generated oxygen plasma producing atomic oxygen or mixtures of gases containing oxygen are used. Preferred gases for admixture are all noble gases.
Alternatively high-voltage generated and/or UV-light emitting source generated oxygen plasma producing atomic oxygen or mixtures of gases containing oxygen are used.
Still a further embodiment is characterized in that the substrate is treated with atomic oxygen plasma for about 0.1 to 10 minutes.
It is preferred that the atomic oxygen plasma treatment is carried out using an oxygen pressure in the range of about 0.1 to 1.0 mbar, preferably 0.2 to 0.8 mbar.
The immobilization of the nucleic acid to the substrate can be adjusted by changing the intensity and duration of the plasma treatment. For example, using s
Ford William E.
Harnack Oliver
Wessels Jurina
Frommer William S.
Frommer & Lawrence & Haug LLP
Megerditchian Samuel H.
Siew Jeffrey
Sony International (Europe) GmbH
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