t7 dna template  (New England Biolabs)

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Schematic of 21 tRNAs synthesis by the tRNA array method. The 21 tRNA genes are divided into five groups and encoded in a single DNA template. After transcription, five transcripts are processed into mature-sized tRNAs through HDVR self-cleavage and RNase P digestion. (B) Schematic of the supplementation assay. The purified 21 tRNAs (240 ng/μL) synthesized by the tRNA array method and an individually-synthesized additional tRNA mixture (PIEN: 40 ng/μL each; others: 4.7 ng/μL each), corresponding to one of the five groups, were used for translation of three reporter proteins (firefly luciferase, sfGFP, and GUS) in a tRNA-free PURE system (tfPURE; composition A, see Tables S5 and S6) containing reporter-encoding DNA template (1 nM) and T7 RNA polymerase (0.42 U/μL) at 30 °C for 16 h. (C) Luciferase activities. (D) sfGFP fluorescence. (E) GUS activities. GUS activity was quantified by an enzymatic assay after the reaction, and the slope of fluorescence increase over time was used as a measure of activity. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Purification, Synthesized, Luciferase, Fluorescence, Activity Assay, Enzymatic Assay

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A, C, E) Minimum free energy (MFE) secondary structures of tRNA Ile (A), tRNA Asn (C), and tRNA Pro (E) variants predicted using ViennaRNA . (B, D, F) Translation activity assays of tRNA Ile (B), tRNA Asn (D), and tRNA Pro (F) variants. Translation reactions were performed in the tfPURE system (composition A) containing a 20-tRNAs mixture (500 ng/μL) lacking the target tRNA, one of the target tRNA variants (tRNA Ile or tRNA Asn at 13 ng/μL, tRNA Pro at 25 ng/μL), a DNA template encoding luciferase (1 nM), and T7 RNA polymerase (0.42 U/μL). Reactions were incubated at 30 °C for 16 h and luciferase activity was measured. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Activity Assay, Luciferase, Incubation

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Schematic of the HDVR self-cleaving assay of PIEN group constructs. Transcription was performed using templates with different tRNA orders in the absence of RNase P, and the resulting transcripts were analyzed by urea-PAGE. (B) Urea-PAGE analysis of transcripts from PIEN group constructs, followed by SYBR Green II staining. Predicted bands corresponding to transcripts containing HDVR and those lacking HDVR are indicated by blue and yellow arrowheads, respectively. (C) Schematic of the experimental workflow for the RNase P digestion assay. Prepared pre-tRNAs (250 nM) lacking HDVR were used as substrates and incubated with RNase P (500 nM M1 RNA and 750 nM C5 protein) in buffer R (see Methods). (D) Urea-PAGE analysis of the digestion products, followed by SYBR green II staining. Individually prepared PIEN tRNA mixtures were included as size controls. Undigested pre-tRNA species are indicated by red arrows.

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Construct, SYBR Green Assay, Staining, Incubation

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Design of the improved PIEN construct (L_PIEN_v2), in which tRNA Pro , tRNA Ile , and tRNA Asn were replaced with the higher-activity variants (tRNA Pro _G1C_U40A_C16A, tRNA Ile _G16A, and tRNA Asn _C17G, respectively) and a leader sequence was inserted upstream of tRNA Pro . (B) Urea-PAGE analysis of transcripts generated from the previous construct (PIEN_v1) and L_PIEN_v2 templates, followed by SYBR green II staining. Predicted bands corresponding to the uncleaved transcript containing HDVR and the transcript after self-cleavage of HDVR are indicated by yellow and blue arrows, respectively. (C) Schematic of translation assay using PIEN tRNAs. Reactions were performed in tfPURE (composition B) supplemented with the tRNA PIEN array template DNA (25 nM), luciferase template DNA (1 nM), RNase P (1 μM M1 RNA and 1.5 μM C5 protein), T4 polynucleotide kinase (0.094 U/μL), T7 RNA polymerase (1.7 U/μL), and 17 IVS tRNAs that lack PIEN tRNAs. After incubation at 30 °C for 24 h, luciferase activity was measured. (D) Measured luciferase activities. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Construct, Activity Assay, Sequencing, Generated, SYBR Green Assay, Staining, Luciferase, Incubation

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Schematic of translation assays using 21 tRNAs prepared by the translation-uncoupled method. Twenty-one tRNAs were simultaneously expressed using the tRNA array method from a 21-tRNA array version 2, in which the PIEN group was replaced with version 2 variants. The resulting 21-tRNA mixture (240 ng/μL) was purified and added to tfPURE (composition A) together with a reporter gene (luciferase, sfGFP, or GUS)–encoding DNA template (1 nM) and T7 RNA polymerase (0.42 U/μL), followed by incubation at 30 °C for 16 h before activity measurements. (B) Luciferase activities. (C) sfGFP fluorescence. (D) GUS activity. (E) Schematic of translation-coupled in situ synthesis of all 21 tRNAs using tRNA array version 2. Reactions were performed in tfPURE (composition C) supplemented with the 21 tRNA template (50 nM), a reporter gene (luciferase, sfGFP, or GUS)–encoding DNA(1 nM), T7 RNA polymerase (3.4 U/μL), T4 polynucleotide kinase (0.094 U/μL), and RNase P (4 μM M1 RNA and 6 μM C5 protein). When using individually prepared 21 IVS tRNAs (IVS), the concentration was set to 600 ng/μL. After incubation at 30 °C for 24 h, translation activities were measured. (F) Luciferase activities. (G) sfGFP fluorescence. (H) GUS activity. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Purification, Luciferase, Incubation, Activity Assay, Fluorescence, In Situ, Concentration Assay

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Schematic of 21 tRNAs synthesis by the tRNA array method. The 21 tRNA genes are divided into five groups and encoded in a single DNA template. After transcription, five transcripts are processed into mature-sized tRNAs through HDVR self-cleavage and RNase P digestion. (B) Schematic of the supplementation assay. The purified 21 tRNAs (240 ng/μL) synthesized by the tRNA array method and an individually-synthesized additional tRNA mixture (PIEN: 40 ng/μL each; others: 4.7 ng/μL each), corresponding to one of the five groups, were used for translation of three reporter proteins (firefly luciferase, sfGFP, and GUS) in a tRNA-free PURE system (tfPURE; composition A, see Tables S5 and S6) containing reporter-encoding DNA template (1 nM) and T7 RNA polymerase (0.42 U/μL) at 30 °C for 16 h. (C) Luciferase activities. (D) sfGFP fluorescence. (E) GUS activities. GUS activity was quantified by an enzymatic assay after the reaction, and the slope of fluorescence increase over time was used as a measure of activity. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Purification, Synthesized, Luciferase, Fluorescence, Activity Assay, Enzymatic Assay

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A, C, E) Minimum free energy (MFE) secondary structures of tRNA Ile (A), tRNA Asn (C), and tRNA Pro (E) variants predicted using ViennaRNA . (B, D, F) Translation activity assays of tRNA Ile (B), tRNA Asn (D), and tRNA Pro (F) variants. Translation reactions were performed in the tfPURE system (composition A) containing a 20-tRNAs mixture (500 ng/μL) lacking the target tRNA, one of the target tRNA variants (tRNA Ile or tRNA Asn at 13 ng/μL, tRNA Pro at 25 ng/μL), a DNA template encoding luciferase (1 nM), and T7 RNA polymerase (0.42 U/μL). Reactions were incubated at 30 °C for 16 h and luciferase activity was measured. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Activity Assay, Luciferase, Incubation

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Schematic of the HDVR self-cleaving assay of PIEN group constructs. Transcription was performed using templates with different tRNA orders in the absence of RNase P, and the resulting transcripts were analyzed by urea-PAGE. (B) Urea-PAGE analysis of transcripts from PIEN group constructs, followed by SYBR Green II staining. Predicted bands corresponding to transcripts containing HDVR and those lacking HDVR are indicated by blue and yellow arrowheads, respectively. (C) Schematic of the experimental workflow for the RNase P digestion assay. Prepared pre-tRNAs (250 nM) lacking HDVR were used as substrates and incubated with RNase P (500 nM M1 RNA and 750 nM C5 protein) in buffer R (see Methods). (D) Urea-PAGE analysis of the digestion products, followed by SYBR green II staining. Individually prepared PIEN tRNA mixtures were included as size controls. Undigested pre-tRNA species are indicated by red arrows.

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Construct, SYBR Green Assay, Staining, Incubation

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Design of the improved PIEN construct (L_PIEN_v2), in which tRNA Pro , tRNA Ile , and tRNA Asn were replaced with the higher-activity variants (tRNA Pro _G1C_U40A_C16A, tRNA Ile _G16A, and tRNA Asn _C17G, respectively) and a leader sequence was inserted upstream of tRNA Pro . (B) Urea-PAGE analysis of transcripts generated from the previous construct (PIEN_v1) and L_PIEN_v2 templates, followed by SYBR green II staining. Predicted bands corresponding to the uncleaved transcript containing HDVR and the transcript after self-cleavage of HDVR are indicated by yellow and blue arrows, respectively. (C) Schematic of translation assay using PIEN tRNAs. Reactions were performed in tfPURE (composition B) supplemented with the tRNA PIEN array template DNA (25 nM), luciferase template DNA (1 nM), RNase P (1 μM M1 RNA and 1.5 μM C5 protein), T4 polynucleotide kinase (0.094 U/μL), T7 RNA polymerase (1.7 U/μL), and 17 IVS tRNAs that lack PIEN tRNAs. After incubation at 30 °C for 24 h, luciferase activity was measured. (D) Measured luciferase activities. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Construct, Activity Assay, Sequencing, Generated, SYBR Green Assay, Staining, Luciferase, Incubation

Journal: bioRxiv

Article Title: Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

doi: 10.64898/2026.01.06.697847

Figure Lengend Snippet: (A) Schematic of translation assays using 21 tRNAs prepared by the translation-uncoupled method. Twenty-one tRNAs were simultaneously expressed using the tRNA array method from a 21-tRNA array version 2, in which the PIEN group was replaced with version 2 variants. The resulting 21-tRNA mixture (240 ng/μL) was purified and added to tfPURE (composition A) together with a reporter gene (luciferase, sfGFP, or GUS)–encoding DNA template (1 nM) and T7 RNA polymerase (0.42 U/μL), followed by incubation at 30 °C for 16 h before activity measurements. (B) Luciferase activities. (C) sfGFP fluorescence. (D) GUS activity. (E) Schematic of translation-coupled in situ synthesis of all 21 tRNAs using tRNA array version 2. Reactions were performed in tfPURE (composition C) supplemented with the 21 tRNA template (50 nM), a reporter gene (luciferase, sfGFP, or GUS)–encoding DNA(1 nM), T7 RNA polymerase (3.4 U/μL), T4 polynucleotide kinase (0.094 U/μL), and RNase P (4 μM M1 RNA and 6 μM C5 protein). When using individually prepared 21 IVS tRNAs (IVS), the concentration was set to 600 ng/μL. After incubation at 30 °C for 24 h, translation activities were measured. (F) Luciferase activities. (G) sfGFP fluorescence. (H) GUS activity. Error bars indicate standard deviations (N = 3).

Article Snippet: The concentrations of tRNA template DNA, T7 RNA polymerase (Takara), T4 PNK (New England Biolab), and RNase P were adjusted depending on the experimental design and are specified in the corresponding figure legends.

Techniques: Purification, Luciferase, Incubation, Activity Assay, Fluorescence, In Situ, Concentration Assay