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a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe <t>(Cy5)</t> (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor <t>Cy5</t> (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.
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a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe <t>(Cy5)</t> (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor <t>Cy5</t> (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.
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a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe <t>(Cy5)</t> (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor <t>Cy5</t> (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.
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a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe <t>(Cy5)</t> (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor <t>Cy5</t> (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.
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a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe <t>(Cy5)</t> (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor <t>Cy5</t> (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.
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a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe (Cy5) (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor Cy5 (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Recoding by N 1 -methylpseudouridine guides rational mRNA vaccine design

doi: 10.1038/s41467-026-77796-3

Figure Lengend Snippet: a Codon–anticodon stability. Drop-off of P-site peptidyl-tRNA Phe from the UUU C (gray) or m 1 Ψm 1 Ψm 1 Ψ C (maroon) codon and of P-site peptidyl-tRNA Ala from the GCA U codon (black). Shown are mean values with error bars representing SE from n = 3 independent biological replicates. Asterisks represent statistically significant differences between UUU C and m 1 Ψm 1 Ψm 1 Ψ C, according to a two-way repeated-measures ANOVA test. ** P = 0.0041 (F(1, 4) = 34.90; df = 1). Lines are single (black) or double (gray and maroon) exponential decay functions (“Methods” section). Exact values are summarized in Supplementary Table . b Schematic of smFRET experiment to monitor tRNA state in the P site. In the P/P state, peptidyl-tRNA Phe (Cy5) (purple star) resides far from L11-Cy3 (green star; FRET 0.2). Transition to the CHI state leads to an increase in FRET to 0.4. mRNA is shown as a solid line, with codons depicted as bold lines. c Static traces. Representative smFRET time trace of donor Cy3 (green) and acceptor Cy5 (purple) fluorescence intensities (F.I.), calculated FRET (blue), and hidden Markov modeling fit (HMM, black), corresponding to peptidyl-tRNA Phe in the P/P state (FRET 0.2). arb. units, arbitrary units. d Dynamic traces. Same as ( c ) for traces showing reversible transitions between FRET 0.2 (P/P) and FRET 0.35 states (CHI). e Percentage of traces showing stable FRET 0.2 (P/P; gray) and fluctuations between FRET 0.2 and FRET 0.35 (CHI) states (maroon) on UUU C and m 1 Ψm 1 Ψm 1 Ψ C codons. Shown are values of individual replicates (black circles) and the mean with error bars representing SE from n = 3 independent biological replicates, each with n = 10 technical replicates. Exact values are summarized in Supplementary Table . Number of traces ( N ): P/P, N = 671 for UUU C, N = 576 for m 1 Ψm 1 Ψm 1 Ψ C; CHI, N = 163 for UUU C, N = 324 for m 1 Ψm 1 Ψm 1 Ψ C. f FRET population distribution histogram of the POST complex on UUU and m 1 Ψm 1 Ψm 1 Ψ, showing the relative abundance of the P/P (gray shade) and CHI (maroon shade) states. Shown are data from n = 3 biological replicates, with n = 10 technical replicates each. Exact values are summarized in Supplementary Table . Source data are provided as a Source Data file.

Article Snippet: Phe-tRNA Phe labeling with Cy5 NHS ester (Cytiva, PA15101) was carried out as described .

Techniques: Stability, Fluorescence