Reliable semi-synthesis of hydrolysis-resistant 3'-peptidyl-tRNA conjugates containing genuine tRNA modifications

Abstract

The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.

Figure 1.

Hydrolysis-resistant 3′-peptidyl-tRNA conjugates. (a) Crucial structural feature of an exemplary target: The amide linkage (highlighted in red). (b) Cartoon presentation of a 3′-peptidyl-tRNA conjugate. Typical positions of natural chemical modifications are shown in red (PDB coordinates 1EVV: S. cerevisiae tRNA^Phe). The 3′-terminal sequences (18 nt) of tRNAs are generally unmodified (blue); arrow (cyan) indicates the intended site of tRNA cleavage (by using DNA enzymes) and the site of ligation (to synthetic 3′-peptidylamino-RNA conjugates); (c) concept for the semi-synthesis of non-hydrolyzable 3′-peptidyl-tRNA conjugates (for explanation see main text). Modified nucleosides, N; cyclophosphate, cp; phosphate, p.

Figure 2.

Example for the cleavage of a natural tRNA by DNA enzymes. (a) Secondary structure of E. coli tRNA^Val 1 and 10–23 DNA enzyme 2. The desired tRNA 5′-fragment 3 contains all modified nucleosides and possesses a 2′,3′-cyclophosphate terminus. (b) Anion-exchange HPLC analysis of the cleavage reaction (88% yield according to peak area integration) and the purified fragment; reaction conditions: c_DNAzyme = 80 µM; c_tRNA = 40 µM; 50 mM Tris–HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, temperature cycles (25–90°C); anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. (c) LC-ESI MS analysis of 3: m.w. (calcd) = 19113, m.w. (found) = 19112 ± 10. For structures and abbreviations of modified nucleosides, see Supplementary Data.

Figure 3.

Example for enzymatic dephosphorylation of the 2′,3′-cyclophosphate at the tRNA 5′-fragment. (a) Structure of the 5′-fragment from E. coli tRNA^Phe 2′,3′-cyclophosphate 3 and product 5 after exposure to T4 PNK. (b) The PNK reaction was monitored by anion-exchange HPLC analysis. The difference in retention time between 3 and 5 is marginal; reaction conditions: T4 PNK (0.5 U/µl; c_RNA = 15 µM; 70 mM Tris–HCl (pH 7.6), 10 mM MgCl₂, 5 mM DTT, 37°C. (c) LC-ESI MS analysis of 5: m.w. (calcd) = 19045, m.w. (found) = 19046 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data.

Figure 4.

Example for enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. (a) Structures of the 5′-fragment from E. coli tRNA^Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. (b) The ligation reaction was monitored by anion-exchange HPLC analysis: 83% yield was achieved after 3 h; reaction conditions: T4 RNA ligase (0.5 U/µl; c_RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Purified 3′-peptidyl-tRNA; (d) LC-ESI MS analysis of 8: m.w. (calcd) = 25030, m.w. (found) = 25029 ± 10. Anion-exchange HPLC: for conditions see ‘Materials and Methods’ section. For structures and abbreviations of modified nucleosides see Supplementary Data.

Figure 5.

Example for splint-assisted enzymatic ligation of fully modified tRNA 5′-fragments to synthetic 3′-peptidylamino-RNA conjugates. (a) Structures of the 5′-fragment from S. cerevisiae tRNA^Phe 5 and the dipeptide-RNA conjugate 6 to form a preligation complex that allows T4 RNA ligation of the full-length tRNA-peptide conjugate 8. (b) Without splint 7 only marginal amounts of product 8 were formed; reaction conditions: T4 RNA ligase (0.5 U/µl; c_RNA = 40 µM each strand; donor/acceptor = 1/1), 50 mM HEPES–NaOH (pH 8.0), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 0.1 mg/ml BSA, 37°C. (c) Ligation promoted by splint 7 resulted in 75% yield of 8. The reaction was monitored by anion-exchange HPLC (for conditions see ‘Materials and Methods’ section); an unidentified, unreactive impurity is marked by an asterisk; reaction conditions: T4 RNA ligase (0.25 U/µl; c_RNA = 40 µM each strand; c_DNA = 40 µM; donor/acceptor/splint = 1/1/1), buffer as in (b) and 0.5 mM ATP, 37°C. For structures and abbreviations of modified nucleosides see Supplementart Data.