Fluorescent labeling of transfer RNA and study of protein synthesis
Provided are methods for labeling transfer RNA comprising replacing the uracil component of a dihydrouridine of said transfer RNA with a fluorophore. The disclosed methods may comprise fluorescent labeling of natural tRNAs (i.e., tRNAs that have been synthesized in a cell, for example, in a bacterium, a yeast cell, or a vertebrate cell) at dihydrouridine (D) positions, or fluorescent labeling of synthetic tRNAs. In another aspect, the present invention provides methods for assessing protein synthesis in a translation system comprise providing a tRNA having a fluorophore substitution for the uracil component of a dihydrouridine in a D loop of the tRNA; introducing the labeled tRNA into the translation system; irradiating the translation system with electromagnetic radiation, thereby generating a fluorescence signal from the fluorophore; detecting the fluorescence signal; and, correlating the fluorescence signal to one or more characteristics of the protein synthesis in the translation system. The disclosed methods are useful in single molecule as well as in ensemble settings.
1. A method for labeling a transfer RNA molecule comprising:
providing a substitution comprising a fluorophore bearing a hydrazide functional group at the dihydrouracil component of a dihydrouridine of said transfer RNA by incubating said transfer RNA in a reaction medium comprising at least 40 mM of said fluorophore.
2. The method according to claim 1 , wherein the transfer RNA has at least one uridine in its D loop, and further comprising converting said uridine to dihydrouridine prior to substituting at said uridine component with said fluorophore.
3. The method according to claim 1 further comprising loading onto the 3′ end of said transfer RNA an amino acid corresponding to a triplet nucleotide sequence that base-pairs to the anticodon sequence of said transfer RNA.
4. The method to claim 1 further comprising subjecting said transfer RNA to conditions effective to load onto the 3′ end of said transfer RNA an amino acid corresponding to a triplet nucleotide sequence that base-pairs to the anticodon sequence of said transfer RNA.
5. The method of claim 1 wherein dihydrouridine is subjected to reducing conditions.
6. The method of claim 5 wherein the reducing conditions comprise reacting with borohydride.
7. A nucleic acid composition comprising a transfer RNA molecule including a fluorophore substitution at the dihydrouracil component of a dihydrouridine in a D loop of the transfer RNA, the fluorophore hearing a hydrazide functional group, and the fluorophore-labeled transfer RNA having been prepared by incubating said transfer RNA in a reaction medium comprising at least 40 mM of said fluorophore.
8. The composition of claim 7 wherein the fluorophore substitution is at the dihydrouracil component of a dihydrouridine that is at a position U16, U17, U20, or U20b on said transfer RNA.
9. A method of assessing protein synthesis in a translation system that comprises a living cell comprising:
providing a tRNA having a fluorophore substitution at the dihydrouracil component of a. dihydrouridine in a D loop of the tRNA, the fluorophore bearing a hydrazide functional group, and the fluorophore-labeled tRNA having been prepared by incubating said tRNA in a reaction medium comprising at least 40 mM of said fluorophore;
introducing the labeled tRNA into the translation system;
irradiating the translation system with electromagnetic radiation, thereby generating a fluorescence signal from said fluorophore;
detecting said fluorescence signal; and,
correlating said fluorescence signal to one or more characteristics of said protein synthesis in said translation system.
10. The method according to claim 9 wherein said fluorophore is a hydrazide comprising Cy3 hydrazide, Cy3.5 hydrazide. Cy5 hydrazide, Cy5.5 hydrazide, Alexa Fluor 488 hydrazide, Alexa Fluor 555 hydrazide, Alexa Fluor 568 hydrazide, Alexa Fluor 594 hydrazide, and Alexa :Fluor 647 hydrazide, Texas Red hydrazide, Lucifer yellow hydrazide, C5-DMB-ceramide, C6 -phosphatidylinositol 5-phosphate, Cascade Blue hydrazide, or ATTO dye.
11. The method according to claim 9 wherein the translation system comprises a cell-free system.
12. The method according to claim 9 wherein the translation comprises a living cell.
13. The method according to claim 9 further comprising providing at least one additional fluorescent labeled translation component.
14. The method according to claim 13 wherein said fluorescently labeled translation component comprises a ribosome, a ribosomal protein, an initiation factor, an elongation factor, a messenger RNA, or a ribosomal RNA.
15. The method according to claim 13 wherein the fluorophore of said labeled tRNA and the fluorophore of said at least one additional fluorescently labeled component comprise a FRET pair, and wherein said detecting said fluorescence signal comprises detecting energy transfer between the fluorophore of said labeled tRNA and the fluorophore of said at least one additional fluorescently labeled component.
16. The method according to claim 1 wherein said fluorophore is a hydrazide comprising Cy3 hydrazide, Cy3.5 hydrazide, Cy5 hydrazide, Cy5.5 hydrazide, Alexa Fluor 488 hydrazide, Alexa Fluor 555 hydrazide, Alexa Fluor 568 hydrazide, Alexa Fluor 594 hydrazide, and Alexa Fluor 647 hydrazide, Texas Red hydrazide, Lucifer yellow hydrazide, C5-DMB-ceramide, C6-phosphatidylinositol 5-phosphate, Cascade Blue hydrazide, or ATTO dye.
17. The method according to claim 9 wherein said fluorophore is a hydrazide comprising Cy3 hydrazide, Cy3.5 hydrazide, Cy5 hydrazide, Cy5.5 hydrazide, Alexa Fluor 488 hydrazide, Alexa Fluor 555 hydrazide, Alexa Fluor 568 hydrazide, Alexa Fluor 594 hydrazide, and Alexa Fluor 647 hydrazide, Texas Red hydrazide. Lucifer yellow hydrazide, C5-DMB-ceramide, C6-phosphatidylinositol 5-phosphate, Cascade Blue hydrazide, or ATTO dye.