Quantitative analysis of recombination in YFP and CFP gene of FRET biosensor induced by lentiviral or retroviral gene transfer.

Transcription

1 Supplementary Information: Quantitative analysis of recombination in and gene of FRET biosensor induced by lentiviral or retroviral gene transfer. Akira T. Komatsubara, Michiyuki Matsuda,, and Kazuhiro Aoki 3,* Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto , Japan Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto , Japan 3 Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto , Japan

2 Supplementary Figure S FIGURE S Multiple alignment of nucleotide sequences in and genes. DNA sequences of e0ypet, h0ypet, nturquoise-gl, and TFP were aligned to highlight their sequence identity with the boxed area.

3 Supplementary Figure S FIGURE S Comparison of fluorescence intensities among chimeric GFP variants. (A) An alignment of amino acid sequences in YPet and nturquoise-gl. (B) Fluorescence intensities of h0ypet, chimeric GFP variants and were measured in HeLa cells transiently expressing the indicated biosensors (Left). Fluorescence intensities of channel were normalized by fluorescence intensities of channel. The averaged / ratios are represented with S.E. (N > 0 cells).

4 (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) (a.u) Supplementary Figure S3 Gene delivery: HIV-based lentivirus, Cell: HeLa A B C D h0 h7-e h0-e0 h-e (a.u.) 0 00 (a.u.) 0 00 (a.u.) 0 00 (a.u.) E F G H e0 e7-h e0-h0 e-h (a.u.) 0 00 (a.u.) 0 (a.u.) 0 00 (a.u.) Gene delivery: MuLV-based retrovirus, Cell: HeLa I J K L h0 h7-e h0-e0 h-e (a.u.) (a.u.) (a.u.) (a.u.) M N O P e0 e7-h e0-h0 e-h (a.u.) (a.u.) (a.u.) (a.u.) FIGURE S3 Recombination between and genes by lentiviral or retroviral gene transfer in HeLa cells. HeLa cells were infected with lentivirus (A-H) or retrovirus (I-P) encoding 8 different FRET biosensors as shown in the upper panel. At least 4 days after infection, the cells were imaged with an epi-fluorescence microscope. The average fluorescence intensities of and are represented as a log-log plot. Each dot corresponds to a HeLa cell. Three hundred cells were analyzed from two independent experiments. Red lines are the fitted line with the e0ypet data. Orange and cyan arrowheads indicate the T03Y and Y66W mutations, respectively.

5 (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) (a.u.) Supplementary Figure S4 Gene delivery: Lipofection with lentivirus plasmid, Cell: HeLa A B C D h0 h7-e h0-e0 h-e (a.u.) 0 00 (a.u.) 0 00 (a.u.) 0 00 (a.u.) E F G H e0 e7-h e0-h0 e-h (a.u.) 0 00 (a.u.) 0 00 (a.u.) 0 00 (a.u.) Gene delivery: Lipofection with retrovirus plasmid, Cell: HeLa I J K L h0 h7-e h0-e0 h-e (a.u.) (a.u.) (a.u.) (a.u.) M N O P e0 e7-h e0-h0 e-h (a.u.) (a.u.) (a.u.) (a.u.) FIGURE S4 Transient expression of and in HeLa cells with lipofection. HeLa cells were transfected with lentiviral (A-H) or retroviral (I-P) vector plasmids encoding 8 different FRET biosensors as shown in the upper panel. At least days after transfection, the cells were imaged with an epi-fluorescence microscope. The average fluorescence intensities of and are represented as a log-log plot. Each dot corresponds to a HeLa cell. Three hundred cells were analyzed from two independent experiments. Red lines are the fitted line with the e0ypet data. Orange and cyan arrowheads indicate the T03Y and Y66W mutation, respectively.

6 Counts Supplementary Figure S A h0ypet ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG 0 Sequence Data ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG 0 nturquoise-gl ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG 0 h0ypet GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTTCTATGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTA 00 Sequence Data GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGCTTCTATGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGGGCTA 00 nturquoise-gl GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGTCCTG 00 h0ypet 0 CGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC 300 Sequence Data 0 CGGCCTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC 300 nturquoise-gl 0 GGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC 300 h0ypet 30 TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG 400 Sequence Data 30 TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG 400 nturquoise-gl 30 TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG 400 h0ypet 40 ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAA 00 Sequence Data 40 ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAA 00 nturquoise-gl 40 ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCGGGAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAA 00 h0ypet 0 GATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC 600 Sequence Data 0 GATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC 600 nturquoise-gl 0 GATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC 600 h0ypet 60 TACCTGAGCTACCAGTCCGCCCTGTTCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCCTGACCGCCGCCGGGATCACTGAGGGCA 700 Sequence Data 60 TACCTGAGCACCCAGTCCGCCTTAAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCTTGACCGCCGCCGGGATCACTCTCGGCA 700 nturquoise-gl 60 TACCTGAGCACCCAGTCCGCCTTAAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCTTGACCGCCGCCGGGATCACTCTCGGCA 700 h0ypet 70 TGAACGAGCTGTAC 74 Sequence Data 70 TGGACGAGCTG 7 nturquoise-gl 70 TGGACGAGCTG 7 B YPet nturquoise-gl aa aa Length (bp) Counts F47L V68L F46L T6G Y66W (99-0 bp) N46I H48G T03Y (6-6 bp) H3F S08F D34N 38 aa 37 aa C 0 y = 0.039x R² = Length (bp) FIGURE S Verification of the recombination. (A) An example of results of DNA sequencing in recombined YPet and nturquoise-gl genes. A49 cells infected with h0ypet-carrying lentivirus were sorted by FACS depending on fluorescence, followed by genomic DNA extraction, PCR amplification of the recombined genes, and sequencing. Nucleotide sequences of wild-type h0ypet, a representative recombined DNA sequence, and wild-type nturquoise-gl are aligned with black lines, which enclose identical nucleotides. The red line encloses the region where the recombination has

7 occurred. *, synonymous mutation. (B) Schematic representation of the difference between YPet and nturquoise-gl amino acids. (Upper) The dotted lines represent different amino acid residues between YPet and nturquoise-gl. (Lower) We analyzed chimeric GFP and variants, in which Y66 amino acid was conserved. Red regions indicate potential recombination site. Length indicates the number of identical DNA sequence for each potential recombination sites. Counts indicate the number of recombined GFP or variants that have been generated as a result of recombination in between the identical DNA sequence. (C) The number of recombined GFP or variants are plotted as a function of the number of each identical DNA sequence, in which the recombination has occurred.

8 Supplementary Figure S6 r : recombination rate (/bp) (, ) = (, ) (, ) = (0, ) (, ) = (0.08, 0) (, ) = (0., 0) (, ) = (0.9, 0) (, ) = (, 0) Y66W L68V N46T (, ) = (0.6, 0) H48G T03Y 00 0 (0, 0.08 ~) (, 0) 0 00 (, ) = (, ) 00 0 ave SD Noise 0 00 Slope ave ave SD ave SD FIGURE S6 Scheme of the mathematical model of recombination between and genes. (Upper panel) Schematic representation of the recombination of and genes. Red lines indicate the critical amino acid residues for the substitution of and from GFP, Y66W and T03Y, respectively. Black arrowheads indicate the position of recombination. Of note, fluorescence intensity of chimeric GFP genes is changed in accord with the recombination sites (Supplementary Fig. SB). (Lower panels) The distribution of and intensities are illustrated in a log-log plot. The blue, orange, and green areas represent cells expressing,, or both, respectively (left). To recapitulate the experimental data, the indicated 9 parameters were extracted from the experimental data set (right).

9 Supplementary Figure S7 Computer simulation, Gene delivery: HIV-based retrovirus, Cell: A49 A h0 B h7-e C h0-e0 D h-e7,000,000,000,000,000,000,000, ,000 0,000 0,000 0,000 E e0 F e7-h G e0-h0 H e-h7,000,000,000,000,000,000,000, ,000 0,000 0,000 0,000 Computer simulation, Gene delivery: HIV-based lentivirus, Cell: HeLa I h0 J h7-e K h0-e0 L h-e7,000,000,000,000,000,000,000, ,000 0,000 0,000 0,000 M e0 N e7-h O e0-h0 P e-h7,000,000,000,000,000,000,000, ,000 0,000 0,000 0,000 Computer simulation, Gene delivery: MuLV-based retrovirus, Cell: HeLa Q h0 R h7-e S h0-e0 T h-e7,000,000,000,000,000,000,000, ,000 0,000 0,000 0,000 U e0 V e7-h W e0-h0 X e-h7,000,000,000,000,000,000,000, ,000 0,000 0,000 0,000

10 FIGURE S7 Computer simulation of the recombination between and genes. The recombination of and genes in A49 cells infected with the indicated retrovirus (A-H) or HeLa cells infected with the lentivirus (I-P) or retrovirus (Q-X) was simulated by computer with the recombination rates, which showed maximal likelihood estimation (Table ). To reproduce the experimental data, 9 parameters were extracted from the experimental data set in Figure 3, and Supplementary Figure S3 (see Supplementary Figure S6 and the Methods for details). Red lines are the fitted line with the e0ypet data. Orange and cyan arrowheads indicate the T03Y and Y66W mutations, respectively.

11 Log likelihood Log likelihood Log likelihood Log likelihood Supplementary Figure S8 A Gene delivery: HIV-based lentivirus Cell: A49 B Gene delivery: MuLV-based retrovirus 4 Cell: A Recombination rate, r (/bp) Recombination rate, r (/bp) C -0.9 Gene delivery: HIV-based lentivirus 4 Cell: HeLa D Gene delivery: MuLV-based retrovirus - 4 Cell: HeLa Recombination rate, r (/bp) Recombination rate, r (/bp) FIGURE S8 Maximal log-likelihood estimation of recombination rate. Log-likelihood values were calculated as described in Methods, and plotted as a function of recombination rate (r) in A49 cells infected with lentivirus (A) or retrovirus (B), or in HeLa cells infected with lentivirus (C) or retrovirus (D). The red circles indicate maximal log-likelihood values.