Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-03
2002-12-03
Borin, Michael (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S110000, C422S068100
Reexamination Certificate
active
06489102
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to the fabrication of biomolecular or cellular arrays on metal surfaces for use in the study of interactions between large molecules, between cells and large molecules, and between cells, such as nucleic acid-protein interactions or cellular interactions with antigens.
DESCRIPTION OF THE PRIOR ART
The binding of proteins to DNA plays a pivotal role in the regulation and control of gene expression, replication and recombination. In addition, enzymes that recognize and modify specific oligonucleotide sequences are critical components of biological nucleic acid manipulation and repair systems. An enhanced understanding of how these proteins recognize certain oligonucleotide sequences would aid in the design of biomedical systems which could, for example, be used to regulate the expression of therapeutic proteins. For this reason, the study of protein-nucleic acid interactions (i.e., protein-DNA and protein-RNA interactions) is a rapidly growing area of molecular biology, aided in part by recent advances in NMR and X-ray structural determination methods. At the same time, the explosive increase in the amount of available genomic and extra-genomic (i.e., ribosomal) sequence information obtained from large-scale nucleic acid sequencing efforts creates a need to survey this vast amount of new sequence data for protein binding sites. The present invention addresses this need by using surface plasmon resonance (SPR) imaging techniques as a rapid and efficient method for screening the sequence or structure-specific binding of proteins to large arrays of nucleic acid molecules immobilized at chemically-modified metal surfaces.
Arrays of DNA molecules attached to planar surfaces are currently employed in hybridization adsorption experiments to sequence DNA, Pease et al. (1994)
Proc. Natl. Acd. Sci. USA
91:5022-5026; to screen for genetic-mutations, Winzeler et al. (1998)
Science
281:1194-1197: and in DNA computing applications, Frutos et al. (1997)
Nucleic Acids Res.
25:4748-4757; and Frutos et al (1998)
J. Am. Chem. Soc.
120:10277-10282. These arrays are exposed to solutions containing fluorescently labeled complementary DNA sequences, rinsed, and then “read-out” using fluorescence imaging methods.
The technique of surface plasmon resonance (SPR) is a surface-sensitive, optical detection method well suited to the monitoring of reversible, protein-nucleic acid interactions. The commercially successful “BIAcore” SPR instrument (Biacore AB, Uppsala, Sweden) has been used previously, for example, to study the interaction of DNA molecules with various enzymes. Although powerful, the “BIAcore” instrument has no imaging capabilities. This severely limits the number of DNA sequences that can be screened in a single experiment.
Surface plasmon resonance (SPR) is a surface optical technique which is sensitive to the thickness and index of refraction of material at the interface between a free electron metal (e.g. gold, silver, copper, cadmium, aluminum) and a bulk medium, such as air or water. Surface plasmon resonance may be achieved by using the evanescent wave which is generated when a laser beam linearly polarized parallel to the plane of incidence impinges onto a prism coated with a thin metal film. The metal may also be coated onto a thin transparent substrate such as glass, and this glass brought into optical contact with the prism. SPR is most easily observed as a reduction of the total internally reflected light just past the critical angle of the prism. This angle of minimum reflectivity (denoted as the SPR angle) shifts to higher angles as material is adsorbed onto the metal layer. The shift in the angle can be converted to a measure of the thickness of the adsorbed or added material by using complex Fresnel calculations and can be used to detect the presence or absence of materials on top of the metal layer.
In using SPR to test for biological, biochemical, or chemical substances, a beam of light from a laser source is directed through a prism onto a biosensor consisting of a transparent substrate, usually glass, which has one external surface covered with a thin film of a noble metal, which in turn is covered with an organic film that interacts strongly with an analyte, such as a biological, biochemical, or chemical substance. The organic film can contain substances, such as antibodies or antigens, which can bind with an analyte in a sample to cause an increased thickness which will shift the SPR angle. By monitoring either the position of the SPR angle or the reflectivity at a fixed angle near the SPR angle, the presence or absence of an analyte in the sample can be detected.
Various types of equipment for using SPR with a biosensor for biological or biochemical or chemical substances are described by the Liedberg et al. article found in “Sensors and Actuators,” Vol. 4, 1983, page 299. See also European Patent Application 0 305 108 and U.S. Pat. No. 5,374,563.
The use of conventional SPR as a testing tool offers several advantages and disadvantages. For example, it is relatively fast, it requires no labeling, and it can be performed on site. However, as noted above, commercially-available devices, such as the “BIAcore” instrument, offer no imaging capabilities. Additionally, to achieve the high through-put demanded by large-scale users, there is a need for a simple, practical biosensor which can be readily modified or adapted to test a wide variety of compounds simultaneously.
In SPR imaging, a light source (typically a HeNe laser) is used to illuminate a prism/thin gold film sample assembly at an incident angle that is near the SPR angle, and the reflected light is detected at a fixed angle with a CCD camera to produce an SPR image. The SPR image arises from variations in the reflected light intensity from different parts of the sample; these variations are created by any changes in organic film thickness or index of refraction that occur upon adsorption onto the modified gold surface. Since SPR imaging is sensitive only to molecules in close proximity to the surface (within ~200 nm), unbound molecules remaining in solution do not interfere with in situ measurements.
The formation of robust, reproducible arrays of oligonucleotides tethered to metal-coated surfaces (most often gold) is an essential requirement for SPR imaging of protein-nucleic acid binding interactions. To use SPR imaging techniques, it is essential that the nucleic acid array be constructed on a noble metal surface, and for this reason DNA arrays on glass supports from commercially available sources such as Affymetrix (Santa Clara, Calif.) are not a viable option. Using self-assembled monolayers of substituted alkanethiols as a starting point, others have previously developed schemes to attach single-stranded DNA molecules to chemically modified gold surfaces. See, for instance, U.S. Pat. No. 5,629,213). In the subject invention, however, UV photopatterning and microcontact printing techniques are brought to bear to allow alkanethiols to be assembled in a site-directed manner on the metal surface, thereby enabling the creation of multi-component arrays. A combination of these processing techniques along with novel surface chemical reactions enables the manufacture of nucleic acid arrays as described herein.
SUMMARY OF THE INVENTION
Disclosed is a multi-step chemical modification procedure to create biomolecule and/or cellular arrays on metal substrates, the arrays being specifically tailored for the study of biomolecular and cellular interactions using surface plasmon resonance imaging. Arrays fabricated by this procedure meet three specific requirements, namely (i) the biomolecules are covalently attached to the surface and remain active and accessible to hybridization and protein binding; (ii) the array background is, at first, sufficiently hydrophobic so as to allow for the “pinning” of aqueous solutions of biomolecules or cells at specific array locations; and (iii) the final array background acts to inhibit the non-specific binding of protein molecules
Brockman Jennifer M.
Corn Robert M.
Frutos Anthony G.
Borin Michael
DeWitt Ross & Stevens S.C.
Leone, Esq. Joseph T.
Wisconsin Alumni Research Foundation
Zhou Shubo
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