Directed evolution of a sequence-specific covalent protein tag for RNA labeling

Figures

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  Fig. 1.. Engineering RNA-reactive rHUH protein and directed evolution scheme. (A) Schematic of wild-type (WT) reaction of dHUH protein with ssDNA (the DNA recognition sequence, dRS), and desired conjugation between engineered rHUH protein and RNA (the RNA recognition sequence, rRS). (B) Crystal structure of WT HUH protein in complex with its target ssDNA sequence (dRS), from PBD ID 6WDZ (16). The nucleophilic Tyr96 (mutated to Phe in this structure) is red, the Mn2+ atom is purple, and the 12 residues we mutated over the course of evolution to rHUH are colored yellow. Zoom on Right shows multiple mutations proximal to -3 and -4 positions of bound dRS. (C) Expected active site chemistry of rHUH, showing nucleophilic attack by deprotonated Tyr96 on RNA backbone, leading to a covalent adduct between rHUH protein and the 3′ portion of the target RNA (rRS). M2+, divalent metal ion such as Mn2+ or Mg2+. (D) Yeast surface display selection scheme. To a library of HUH mutants (HUH*) is added biotinylated rRS or hybrid sequences [see (E)]. After washing, cells are stained with streptavidin-phycoerythrin (PE) to quantify probe labeling and anti-myc antibody to quantify HUH expression level. FACS…

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  Fig. 2.. Directed evolution results and characterization of top clones in yeast. (A) FACS comparison of original dHUH template and evolved clones G1–G5, illustrating progress of evolution. Yeast displaying the indicated constructs were labeled with 500 nM rRS for 1 h in the presence of 5 mM MgCl2 before streptavidin-PE/anti-myc staining and FACS. rCtrl is a scrambled version of rRS (sequence in Fig. 1E). (B) Quantification of data in (A) and two additional replicates (SI Appendix, Fig. S3A). Labeling of G1–G5 with DNA probes is shown in SI Appendix, Fig. S3 C and D. Mean ± SD, n = 3. (C) FACS comparison of evolved clones G5-G7 on yeast, using 5 nM rRS for 1 h in the presence of 5 mM MgCl2. The Y96F mutation inactivates G7. (D) Quantification of data in (C) and two additional replicates (SI Appendix, Fig. S3B). Mean ± SD, n = 3. (E) Time-dependent labeling of G7 on the yeast surface with 200 nM rRS or rCtrl, in the presence of 5 mM MgCl2. FACS plots in SI Appendix, Fig. S4A. Mean ± SD, n = 3. (F) Labeling of G7 on yeast at various dRS (Left) and rRS (Right) concentrations. Cells were labeled for 5 min in the presence of 50 μM MnCl2. FACS plots in SI Appendix, Fig. S5A. Mean ± SD, n = 3.…

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  Fig. 3.. In vitro characterization of purified rHUH (G7). (A) Gel shift assay showing reaction of purified rHUH with DNA and RNA probes. 5 µM rHUH was combined with 10 µM probe for 1 min in the presence of 1 mM MnCl2 and then analyzed by SDS-PAGE and Coomassie staining. Uncropped gel and additional replicates in SI Appendix, Fig. S7A. (B) Quantification of data in (A). Mean ± SD, n = 3. (C) Time-dependent labeling of purified rHUH with rRS. 5 µM rHUH was combined with 10 µM probe in the presence of 1 mM MnCl2. Uncropped gel and additional replicates shown in SI Appendix, Fig. S7B. (D) Quantification of data in (C). Mean ± SD, n = 3. (E) rHUH labeling in mammalian cell lysate. HEK 239 T cells expressing rHUH were lysed and incubated with 10 µM DNA and RNA probes as in (A), for 5 min in the presence of 1 mM MnCl2. rHUH protein was then detected by anti-myc western blotting.