Methods for the preparation of chemically misaminoacylated tRNA via protective groups
The present invention relates to methods for the preparation of chemically aminoacylated tRNAs for the purpose of introduction of markers into nascent proteins. The present invention also relates to methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system utilizing chemically aminoacylated tRNAs. tRNA molecules are misaminoacylated with non-radioactive markers which may be non-native amino acids, amino acid analogs or derivatives. Markers may comprise cleavable moieties, detectable labels, reporter properties wherein markers incorporated into protein can be distinguished from unincorporated markers, or coupling agents which facilitate the detection and isolation of nascent protein from other components of the translation system.
1. A method, comprising:
a) providing:
i) a markerless amino acid comprising an alpha amino group;
ii) at least one protective group; and
iii) an unprotected polynucleotide comprising x nucleotides, wherein x is an integer between 1 and 10;
b) attaching said protective group to said alpha amino group of said markerless amino acid to form a protected markerless amino acid;
c) converting said protected markerless amino acid so as to form a protected amino acid ester; and
d) reacting said protected markerless amino acid ester with said unprotected polynucleotide under conditions such that a markerless aminoacyl-polynucleotide conjugate comprising said protective group is formed; and
e) ligating said conjugate of step d) with a tRNA molecule lacking y nucleotides at the 3′ terminus, wherein y is an integer between 0 and 10, so as to form a markerless tRNA conjugate comprising said protective group attached to said alpha amino group.
2. The method of claim 1 , further comprising the steps:
f) removing said protective group of said markerless tRNA conjugate to as to form an unprotected markerless tRNA conjugate; and
g) attaching a marker moiety to said unprotected markerless tRNA conjugate so as to form a marker-tRNA conjugate.
3. The method of claim 1 , wherein said unprotected polynucleotide is a riboside.
4. The method of claim 1 , wherein said unprotected polynucleotide is deoxyribonucleotide.
5. The method of claim 1 , wherein said unprotected polynucleotide is an unprotected dinucleotide.
6. The method of claim 5 , wherein said unprotected dinucleotide is 5′-phospodeoxycytidyl-adnosine.
7. The method of claim 1 , wherein said markerless amino acid is a natural amino acid.
8. The method of claim 1 , wherein said markerless amino acid is an unnatural amino acid.
9. The method of claim 1 , wherein said protective group is selected from the group consisting of 1-(2-nitrophenyl-5-methyl)ethyloxycarbonyl (Npe), 1-(2-nitrophenyl-4,5-dimethoxy)ethyloxycabonyl (NVOC), 1-(2-nitrophenyl)ethyloxycarbonyl, 3,5-(dimethoxy) benzyloxycarbonyl, 2-nitrophenylsulfenyloxycarbonyl, biphenylylisopropyloxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), and pent-4-enoyl (Pent).
10. The method of claim 1 , wherein said protective group is photolabile.
11. The method of claim 1 , wherein said protective group is acid or base labile.
12. The method of claim 1 , wherein said protective group can be selectively removed.
13. The method of claim 2 , wherein said marker moiety is selected from photocleavable biotin, BODIPY-FL, BODIPY 558/568, fluorescein, NBD-X, phenyldiboronic acid (PDBA)-X, and cyanine 3 (Cy3).
14. The method of claim 1 , wherein said tRNA is an initiator tRNA.
15. The method of claim 1 , wherein said tRNA comprises any tRNA other than an initiator tRNA.