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GSJ: Received May 19, 2007:
http://wbabin.net/saba/saba77.htm
Discovery of Drugs Targeting the Ribosome
James Saba
The Ribosome is a target for several antibiotics (1, 2).
Herein is described materials and methods of screening for molecules which inhibit protein synthesis, preferably but not necessarily via binding to the ribosome.
One embodiment of the invention involves affixing the mRNA or ribosome to a support as delineated in Figure 1. Notice various types to labels other than fluorescent dyes, such a radio isotopes, could be used.
As an alternative to labeled amino acids one could utilize a labeled antibody or other molecule which binds the synthesized polypeptide.
Another embodiment of the invention is to use site-specific ribosomal incorporation of two unnatural amino acids (3) each having one of two fluorescence resonance energy transfer (FRET) dyes (4,5). As Figure 2 outlines, only when translation proceeds does the incorporation of the FRET pair and fluorescence occur. No affixing the mRNA or ribosome, or washing is required in this method.
Notice this process can be used in vivo to monitor protein synthesis, such as to optimize incorporation of unnatural amino acids.
Numerous screening formats are possible. Particularly attractive is the use of a biochip microarray; or microwells with and without the use of beads (6); or beads alone.
If it should be that the above invention is indeed novel any patentable rights I may have, I freely give away.
It is hoped that others will honor the invention as delineated above and by the following claims.
Claims
2) The method of claim 1 where the label is a fluorescent dye(s).
3) The method of claim 1 where the label is radioactive isotope.
4) A method of monitoring in vitro or in vivo protein synthesis which involves the use of fluorescent dye labeled amino acids (or tRNAs charged with such amino acids) and FRET.
5) A method of in vitro screening for inhibitors of protein synthesis which involves the use of fluorescent dye labeled amino acids (or tRNAs charged with such amino acids) and FRET.
6) The method of claim 1 or 5 which also utilizes a microarray biochip.
7) The method of claim 1 or 5 which also utilizes an array of microwells.
8) The method of claim 7 wherein beads are positioned in the microwells.
9) A kit for monitoring in vitro or in vivo protein synthesis which comprises labeled amino acids (or tRNAs charged with such amino acids) and a support-affixed mRNA or ribosome.
10) A kit for screening for inhibitors of protein synthesis which comprises labeled amino acids (or tRNAs charged with such amino acids) and a support-affixed mRNA or ribosome.
11) A kit for screening for inhibitors of protein synthesis which comprises amino acids labeled with FRET fluorescent dyes (or tRNAs charged with such labeled amino acids).
2)
Ribosomal Crystallography: Peptide Bond Formation, Chaperone
Assistance and Antibiotics Activity.
Yonath Mol Cells. 2005 Aug
31;20(1):1-16
3)
Expanding the genetic code.
Wang L, et al Annu Rev Biophys Biomol
Struct. 2006;35:225-49
4) Fluorescence resonance energy transfer
5)
Fluorescence resonance energy transfer between unnatural amino acids
in a structurally modified dihydrofolate reductase.
Anderson, et al J
Am Chem Soc. 2002 Aug 21;124(33):9674-5
6)
Nucleic acid sequencing using microsphere arrays.
Chee, et al US Patent Appl 20050191698 September 1, 2005
It has been recognized that the polypeptide product could could itself be the reporter. For example it could be fluorescent such as green fluorescent protein (GFP) or could be an enzyme such as a protease or peroxidase.
While such reporters are generally much larger than the peptides needed in the prior described labeling schemes, the need for labeled amino acids is obviated.
Furthermore there is not the requirement that the protein product need be retained by the ribosome as in Figure 1.
Claims
13) Claim 12, wherein the reporter protein is fluorescent.
14) Claim 12, wherein the reporter protein is an enzyme.