Surface based translation system
View Patent ↗Translationally competent ribosomes are bound to a solid substrate through a specific attachment site. A spatial array of such immobilized ribosomes may be produced on a planar substrate, microbeads, on fiber optics. One or more components of the ribosome complex are preferably labeled, e.g. with a fluorescent dye. Ribosomal RNAs, including mRNA and tRNA; and/or ribosomal proteins may be labeled at specific positions, and arrays of immobilized ribosomes may comprise a panel of different labels and positions of labels. Fluorescence detection can then be used to monitor conformational dynamics and translation rates.
1. A translationally competent isolated bacterial ribosome complex bound to a solid surface at a specific attachment site on an RNA of said ribosome complex wherein said RNA is a rRNA; wherein said specific attachment site comprises one member of a specific binding pair bound to said solid surface, and a polynucleotide linker which is (a) attached to said solid surface through a complementary binding pair member which is covalently attached to said linker and (b) hybridized to a surface accessible hairpin loop present on said rRNA.
2. The ribosome complex of claim 1 , wherein said surface hairpin loop is naturally occurring.
3. The ribosome complex of claim 2 , wherein said naturally occurring surface accessible hairpin loop is expanded by from about 8-18 nucleotides.
4. The ribosome complex according to claim 1 , wherein said surface accessible hairpin loop is selected from the group consisting of the 16S rRNA loop H6, 16S rRNA loop H10, 16S rRNA loop H26, 16S rRNA loop H33a, 16S rRNA loop H39, 6S rRNA loop H44, 23SrRNA loop H9, 23SrRNA loop H68 and 23SrRNA loop H101.
5. A solid surface comprising an array of translationally competent ribosome complexes according to claim 1 .
6. The array of claim 5 , wherein said solid surface is quartz.
7. The ribosome complex of claim 1 , wherein said ribosome complex further comprises a fluorescent label.
8. The ribosome complex of claim 7 , wherein said ribosome complex comprises two distinct fluorescent labels, wherein said labels are a donor/acceptor pair.
9. The ribosome complex of claim 7 , wherein the fluorescent label is present on one or more of: tRNA, rRNA, mRNA, and ribosomal protein.
10. The ribosome complex of claim 9 , wherein said tRNA is directly labeled.
11. The ribosome complex of claim 9 , wherein said ribosomal protein is directly labeled.
12. The ribosome complex of claim 11 , wherein said ribosomal protein is S21 or S5.
13. The ribosome complex of claim 9 , wherein said rRNA is genetically modified to comprise a peptide binding site, and wherein a fluorescently labeled peptide is bound to said modified rRNA.
14. The ribosome complex of claim 13 , wherein said fluorescently labeled peptide is BIV Tat.
15. The ribosome complex of claim 13 , wherein said fluorescently labeled peptide is HIV-Rev.