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Abstract The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2′-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation.
We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA polymerase have been identified. INTRODUCTION A substitution of 2′-fluoro (2′-F) for the 2′-hydroxyl group in RNA does not substantially change the conformation of RNA but substantially increases its melting temperature, chemical stability and resistance to ribonuclease. These properties result in a longer survival time of 2′-F RNAs (or 2′-F DNAs if all the 2′ positions bear 2′-fluoro groups) in vitro and in vivo. 2′-F RNAs are used widely in studies of ribozymes, the selection of aptamers and in RNA interference. The most common 2′-F substitutions used in RNAs are 2′-F-dCMP and 2′-F-dUMP.
RNAs containing these substitutions are resistant to RNase A, the most common RNase contaminant, which recognizes the 2′-OH of pyrimidines for cleavage. 2′-F RNA can be synthesized enzymatically , and currently the standard enzyme for the preparation of 2′-F RNA is the bacteriophage T7 RNA polymerase Y639F in which tyrosine 639 is replaced with phenylalanine , commercialized by Epicentre as T7 R&DNA™ Polymerase and The DuraScribe® T7 Transcription Kit.
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The Y639F alteration in T7 RNA polymerase greatly reduces discrimination between non-canonical and canonical nucleoside triphosphates. However such discrimination is still substantial, especially when multiple 2′-modified NTPs or 2′-modified GTP (the strict initiation nucleotide for T7 RNA polymerase) are included in the reaction (,). Recently we characterized a single-subunit RNA polymerase from marine cyanophage Syn5 and described some of its advantages as a tool for in vitro transcription.
These advantages include product-3′-homogeneity, high processivity, flexible initiating nucleotide and tolerance to salt. In the current study we have investigated the ability of wild-type and altered Syn5 RNA polymerases to synthesize RNA containing 2′-F moieties. We have found that wild-type Syn5 RNA polymerase has an inherent low discrimination against 2′-F-dNTPs compared to T7 RNA polymerase. Syn5 RNA polymerase also retains high activity in the presence of manganese ions, which further decrease its discrimination against 2′-F-dNTPs. A single amino acid change in Syn5 RNA polymerase further reduces its ability to discriminate against 2′-F-dCTP and 2′-F-dUTP. We also describe an improved expression system for the overproduction of Syn5 RNA polymerase in Escherichia coli, characterize sequence elements in DNA templates that reduce the yield of RNA synthesized by Syn5 RNA polymerase, and describe ways to improve the yield in the presence of these DNA sequences. MATERIALS AND METHODS Materials Oligonucleotides were obtained from Integrated DNA Technology (oligonucleotides less than 60 nt were ordered at the 25 nmole scale and those longer than 60 nt were ordered at the 4 nmole Ultramer scale).
DNA purification kits and Ni-NTA resin were from Qiagen. Preparative Superdex 200 for gel filtration was from GE Healthcare. Restriction endonucleases, T4 DNA ligase, Vent R ® DNA Polymerase, Q5 ® Site-Directed Mutagenesis Kit, T7 RNA polymerase and E. coli inorganic pyrophosphatase were from New England Biolabs.
T7 R&DNA™ Polymerase (T7-Y639F RNA polymerase) was from Epicentre. DNA Clean & Concentrator™-5 kit was from ZYMO Research. RNaseOUT™ recombinant ribonuclease inhibitor was from Invitrogen.
NTPs were from USB and 2′-F-dNTPs were from Trilink. Protein expression and purification We have modified the original expression vector (,) to improve the expression of His-tagged Syn5 RNA polymerase by removing the internal Syn5 promoter sequence within the Syn5 RNA polymerase gene without changing the encoded amino acid (Supplementary Figure S1A). With this vector (Syn5 RNAP-NP-pET24) the synthesized Syn5 RNA polymerase will not initiate transcription from the internal promoter sequence, an event that would deplete the rNTP pools and inhibit the synthesis of the full-length mRNA for Syn5 RNA polymerase. E. coli cells harboring this vector grow significantly faster than those carrying the original plasmid and contain higher levels of overproduced protein (Supplementary Figure S1B and C). Y564F and Y574F mutations were introduced into the Syn5 RNA polymerase gene in the Syn5 RNAP-NP-pET24 vector by polymerase-chain-reaction mutagenesis. The purification procedure was modified from that previously described. E. coli BL21(DE3) cells containing the plasmid Syn5 RNAP-NP-pET24 were grown in 2 L of LB medium containing 50 μg/ml kanamycin at 37°C until they reached an OD 600 of 1.2.
The gene for Syn5 RNA polymerase was induced by the addition of 0.5 mM IPTG at 25°C and incubation was continued for 4–8 h. The cells were harvested, resuspended in 50 mM sodium phosphate, pH 8.0, 100 mM NaCl, and lysed by three cycles of freeze-thaw in the presence of 0.5 mg/ml lysozyme. Solid NaCl was added to the lysed cells to a final concentration of 2 M and then the cleared lysate was collected after centrifugation. 5 ml Ni-NTA agarose was added to the clear lysate and gently mixed at 4°C overnight. The resin was loaded and collected in a column and washed with 60 ml of Wash Buffer (50 mM sodium phosphate, pH 8.0, 2 M NaCl and 10 mM imidazole).
Syn5 RNA polymerase was eluted from the column using 30 ml Elution Buffer (50 mM sodium phosphate, pH 8.0, 2 M NaCl and 100 mM imidazole). Eluted protein was concentrated to 1 ml using an Amicon Ultra-15 Centrifugal Filter Units (Millipore). The concentrated sample was loaded directly onto a 200 ml preparative Superdex 200 column. The gel filtration buffer contained 20 mM Tris-HCl pH 7.5, 2 M NaCl, 0.5 mM DTT and 0.5 mM EDTA. Fractions were analyzed on SDS-PAGE gels and those fractions that contained homogenous Syn5 RNAP were pooled.
The pooled fractions were concentrated by Amicon Ultra-15 Centrifugal Filter followed by dialysis against Dilution Buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM β-ME, 1 mM EDTA, 50% glycerol and 0.1% Triton® X-100) and stored at −20°C. Dilutions for enzyme assays were carried out using Dilution Buffer. The yield of Syn5 RNA polymerase following this procedure was greater than 20 mg protein per gram of wet cells, with the majority of the soluble Syn5 RNA polymerase (80%) retained in the flow-through and wash fractions of the Ni-NTA step. Repetition of this procedure using the flow-through fraction of the Ni-NTA column generates similar amounts of purified protein.
Syn5 RNA polymerase mutants Y564F and Y574F were purified following the same procedure resulting in a similar yield. DNA templates for transcription assays DNA templates were constructed by annealing the complementary synthesized oligonucleotides listed below. Only the non-template strands are shown, listed 5′-3′, with the promoters shown in bold and the nucleotides corresponding to residues 1–12 of the RNA products underlined. 2′-F-dNMP incorporation into transcripts synthesized by Syn5 RNA polymerase on various DNA templates. Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. (A) Efficiency of 2′-F-dNMP incorporation by Syn5 RNA polymerase is correlated to the sequence at the 5′ end of the RNA.
Transcription reactions were carried out using Syn5 RNA polymerase on templates T1 (lanes 1–6), T2 (lanes 7–13) and T3 (lanes 14–20). In some reactions some of the four NTPs were replaced by the corresponding 2′-F analogs, as indicated at the top of the gel. The first 12 nucleotides at the 5′-end of the transcripts produced on each template are shown below each gel. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Efficiency of 2′-F-dNMP incorporation by Syn5 RNA polymerase on template T4.
Normal NTPs were replaced by the corresponding 2′-F analogs as indicated at the top of each lane. The first 12 nucleotides at the 5′-end of the transcripts produced by template T4 are shown below each gel.
The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. 2′-F-dNMP incorporation into transcripts synthesized by Syn5 RNA polymerase on various DNA templates. Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. (A) Efficiency of 2′-F-dNMP incorporation by Syn5 RNA polymerase is correlated to the sequence at the 5′ end of the RNA. Transcription reactions were carried out using Syn5 RNA polymerase on templates T1 (lanes 1–6), T2 (lanes 7–13) and T3 (lanes 14–20). In some reactions some of the four NTPs were replaced by the corresponding 2′-F analogs, as indicated at the top of the gel.
The first 12 nucleotides at the 5′-end of the transcripts produced on each template are shown below each gel. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Efficiency of 2′-F-dNMP incorporation by Syn5 RNA polymerase on template T4. Normal NTPs were replaced by the corresponding 2′-F analogs as indicated at the top of each lane.
The first 12 nucleotides at the 5′-end of the transcripts produced by template T4 are shown below each gel. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. Impact of initial sequence of transcript on the yield of transcripts synthesized by Syn5 RNA polymerase. (A) Influence of different sequences at various positions in the first 12 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 RNA polymerase. The DNA template used in each reaction is indicated at the top of each lane. All of the templates were derived from template T4, from which the first 12 nucleotides at the 5′ end of the RNA product are shown at the top of the gel.
The variations in the sequence of RNA products from template T4 synthesized on each template are color coded; variations in the first three nucleotides are in orange, nucleotides 4–6 in blue, nucleotides 7-9 in purple and nucleotides 10–12 in green. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Influence of different sequences at various positions in the first 10 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 and T7 RNA polymerases. Transcription reactions were carried out using Syn5 RNA polymerase (lanes 1–7, 13 and 14) and T7 RNA polymerase (lanes 8–12). The templates used for each reaction and the sequence of the first 10 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel. Variations in RNA products encoded by each template are in blue background.
(C) Influence of NTP concentration on the yield of products synthesized by Syn5 RNA polymerase. Transcription reactions were carried out on templates T32 (lanes 1–3), T2 (lanes 4–6) and T33 (lanes 7–9).
The first three nucleotides of the transcript synthesized on each of these templates are UGA (T32), GGG (T2) and GGG (T33), as indicated at the bottom of the gel. The concentration of the NTP being varied in each reaction mixture is shown at the top; the concentrations of the other three NTPs were fixed at 4 mM. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (D) and (E) Incorporation of 2′-F-dCMP and/or 2′-F-dUMP into transcripts synthesized by Syn5 and T7 RNA polymerases. The templates used for each reaction (T18, T17, T28 and T4) and the sequence of the first 12 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel.
The NTP analog present in each reaction is indicated at the top of the gel where ‘C/U’ corresponds to reactions carried out in the presence of both 2′-F-dCTP and 2′-F-dUTP. Impact of initial sequence of transcript on the yield of transcripts synthesized by Syn5 RNA polymerase. (A) Influence of different sequences at various positions in the first 12 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 RNA polymerase. The DNA template used in each reaction is indicated at the top of each lane. All of the templates were derived from template T4, from which the first 12 nucleotides at the 5′ end of the RNA product are shown at the top of the gel. The variations in the sequence of RNA products from template T4 synthesized on each template are color coded; variations in the first three nucleotides are in orange, nucleotides 4–6 in blue, nucleotides 7-9 in purple and nucleotides 10–12 in green.
The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Influence of different sequences at various positions in the first 10 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 and T7 RNA polymerases. Transcription reactions were carried out using Syn5 RNA polymerase (lanes 1–7, 13 and 14) and T7 RNA polymerase (lanes 8–12). The templates used for each reaction and the sequence of the first 10 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel. Variations in RNA products encoded by each template are in blue background. (C) Influence of NTP concentration on the yield of products synthesized by Syn5 RNA polymerase.
Transcription reactions were carried out on templates T32 (lanes 1–3), T2 (lanes 4–6) and T33 (lanes 7–9). The first three nucleotides of the transcript synthesized on each of these templates are UGA (T32), GGG (T2) and GGG (T33), as indicated at the bottom of the gel. The concentration of the NTP being varied in each reaction mixture is shown at the top; the concentrations of the other three NTPs were fixed at 4 mM. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (D) and (E) Incorporation of 2′-F-dCMP and/or 2′-F-dUMP into transcripts synthesized by Syn5 and T7 RNA polymerases. The templates used for each reaction (T18, T17, T28 and T4) and the sequence of the first 12 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel.
The NTP analog present in each reaction is indicated at the top of the gel where ‘C/U’ corresponds to reactions carried out in the presence of both 2′-F-dCTP and 2′-F-dUTP. Effect of Mn 2+ ions on 2′-F-dNMP incorporation by Syn5 RNA polymerase.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. (A) Incorporation of 2′-F-dCMP and 2′-F-dUMP by Syn5 RNA polymerase in the presence of Mg 2+ or Mn 2+. Transcription reactions were carried out by Syn5 RNA polymerase on the template T1; the sequence of the first nine nucleotides of the 37 nt transcript produced on this template is shown at the bottom of the figure, with the C and U residues in gray background. The metal ion present in each reaction and its concentration are shown at the top of the gel. The reaction mixtures that contained both 2′-F-dCTP and 2′-F-dUTP (C/U) are also indicated.
Lanes 1 contains only the DNA template as a marker. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Incorporation of 2′-F-dNMPs by Syn5 RNA polymerase in the presence of a mixture of Mg 2+ and Mn 2+ ions. Transcription reactions were carried out by Syn5 RNA polymerase on the template T1; the sequence of the first nine nucleotides of the 37 nt transcript produced on this template is shown at the bottom of the gel, with the C and U residues in gray background. (C) Incorporation of 2′-F-dNMPs into a 37 nt transcript and a 2700 nt transcript by Syn5 RNA polymerase in the presence of a mixture of Mg 2+ and Mn 2+. Transcription reactions were carried out using either template T22, which encodes a 37 nt transcript (lanes 1–6), or T31, which encodes a 2700 nt transcript (lanes 7 and 8).
The sequence of the first nine nucleotides of each transcript is shown at the bottom of the gel, with the C and U residues in gray background. The metal ions present in each reaction and their concentrations are shown at the top of the gel; a mixture of 10 mM Mg 2+ and 5 mM Mn 2+ were present in the reaction mixtures in lanes 4–8. Also the reaction mixtures that contained both 2′-F-dCTP and 2′-F-dUTP or all four 2′-F-dNTPs are shown at the top of the gel. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. Effect of Mn 2+ ions on 2′-F-dNMP incorporation by Syn5 RNA polymerase. Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide.
(A) Incorporation of 2′-F-dCMP and 2′-F-dUMP by Syn5 RNA polymerase in the presence of Mg 2+ or Mn 2+. Transcription reactions were carried out by Syn5 RNA polymerase on the template T1; the sequence of the first nine nucleotides of the 37 nt transcript produced on this template is shown at the bottom of the figure, with the C and U residues in gray background. The metal ion present in each reaction and its concentration are shown at the top of the gel.
The reaction mixtures that contained both 2′-F-dCTP and 2′-F-dUTP (C/U) are also indicated. Lanes 1 contains only the DNA template as a marker.
The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Incorporation of 2′-F-dNMPs by Syn5 RNA polymerase in the presence of a mixture of Mg 2+ and Mn 2+ ions. Transcription reactions were carried out by Syn5 RNA polymerase on the template T1; the sequence of the first nine nucleotides of the 37 nt transcript produced on this template is shown at the bottom of the gel, with the C and U residues in gray background. (C) Incorporation of 2′-F-dNMPs into a 37 nt transcript and a 2700 nt transcript by Syn5 RNA polymerase in the presence of a mixture of Mg 2+ and Mn 2+.
Transcription reactions were carried out using either template T22, which encodes a 37 nt transcript (lanes 1–6), or T31, which encodes a 2700 nt transcript (lanes 7 and 8). The sequence of the first nine nucleotides of each transcript is shown at the bottom of the gel, with the C and U residues in gray background. The metal ions present in each reaction and their concentrations are shown at the top of the gel; a mixture of 10 mM Mg 2+ and 5 mM Mn 2+ were present in the reaction mixtures in lanes 4–8.
Also the reaction mixtures that contained both 2′-F-dCTP and 2′-F-dUTP or all four 2′-F-dNTPs are shown at the top of the gel. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. Incorporation of 2′-F-dNMPs by wild-type, Y564F and Y574F Syn5 RNA polymerases.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. (A) Incorporation of 2′-F-dNMPs into 37 nt and 2700 nt transcripts using wild-type Syn5, Syn5-Y564F and Syn5-Y574F RNA polymerases. Transcription reactions were carried out using either template T22, which encodes a 37 nt transcript (lanes 1–6), or T31, which encodes a 2700 nt transcript (lanes 7–12). The sequence of the first nine nucleotides of each transcript is shown at the bottom of the gel, with the C and U residues in gray background. The RNA polymerase used for each reaction is indicated at the top of the gel. The reaction mixtures that contained only unmodified NTPs (‘-’) or both 2′-F-dCTP and 2′-F-dUTP (‘C/U’) are indicated at the top of the gel. Mg 2+ was used as the only metal ion in all reactions.
The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Incorporation of 2′-F-dNMPs by Syn5 and Syn5-Y564F RNA polymerases.
Transcription reactions were carried out using template T8, which encodes a 37 nt transcript. Reactions were carried out using either wild-type Syn5 RNA polymerase (lanes 1–6) or Syn5-Y564F RNA polymerase (lanes 7–14). Each of the four NTPs was individually replaced by the corresponding 2′-F analog, as indicated at the top of the gel.
In the reactions carried out in lanes 6 and 12, both CTP and UTP were replaced by 2′-F-dCTP and 2′-F-dUTP. In the reactions carried out in lanes 7 and 14, all four NTPs were replaced by 2′-F-dNTPs. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. Incorporation of 2′-F-dNMPs by wild-type, Y564F and Y574F Syn5 RNA polymerases.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. (A) Incorporation of 2′-F-dNMPs into 37 nt and 2700 nt transcripts using wild-type Syn5, Syn5-Y564F and Syn5-Y574F RNA polymerases.
Transcription reactions were carried out using either template T22, which encodes a 37 nt transcript (lanes 1–6), or T31, which encodes a 2700 nt transcript (lanes 7–12). The sequence of the first nine nucleotides of each transcript is shown at the bottom of the gel, with the C and U residues in gray background. The RNA polymerase used for each reaction is indicated at the top of the gel. The reaction mixtures that contained only unmodified NTPs (‘-’) or both 2′-F-dCTP and 2′-F-dUTP (‘C/U’) are indicated at the top of the gel.
Mg 2+ was used as the only metal ion in all reactions. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Incorporation of 2′-F-dNMPs by Syn5 and Syn5-Y564F RNA polymerases. Transcription reactions were carried out using template T8, which encodes a 37 nt transcript. Reactions were carried out using either wild-type Syn5 RNA polymerase (lanes 1–6) or Syn5-Y564F RNA polymerase (lanes 7–14). Each of the four NTPs was individually replaced by the corresponding 2′-F analog, as indicated at the top of the gel. In the reactions carried out in lanes 6 and 12, both CTP and UTP were replaced by 2′-F-dCTP and 2′-F-dUTP.
In the reactions carried out in lanes 7 and 14, all four NTPs were replaced by 2′-F-dNTPs. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. Synthesis of 2′-F RNAs by wild-type and Y564F Syn5 RNA polymerases on three different templates. Transcription reactions were carried out using either wild-type Syn5 RNA polymerase (lanes 1–6) or Syn5-Y564F RNA polymerase (lanes 7–9). Reactions were carried out in the presence of 20 mM Mg 2+ (lanes 1–3 and 7–9) or a mixture of 10 mM Mg 2+ and 5 mM Mn 2+ (lanes 4–6).
The reaction mixtures that contained only normal NTPs (lanes 1, 4 and 7), both 2′-F-dCTP and 2′-F-dUTP (lanes 2, 5 and 8), or all four 2′-F-dNTPs (lanes 3, 6 and 9) are shown at the top of the gel. Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. The template used for the top gel is T22, which encodes a 37 nt transcript. The template used for the middle gel is T29, which encodes a 54 nt transcript. The template used for the bottom gel is T30, which encodes a 2700 nt transcript.
For each template, the sequence of the first nine nucleotides of the transcript is shown at the right of the gel, with the C and U residues in gray background. Synthesis of 2′-F RNAs by wild-type and Y564F Syn5 RNA polymerases on three different templates. Transcription reactions were carried out using either wild-type Syn5 RNA polymerase (lanes 1–6) or Syn5-Y564F RNA polymerase (lanes 7–9). Reactions were carried out in the presence of 20 mM Mg 2+ (lanes 1–3 and 7–9) or a mixture of 10 mM Mg 2+ and 5 mM Mn 2+ (lanes 4–6). The reaction mixtures that contained only normal NTPs (lanes 1, 4 and 7), both 2′-F-dCTP and 2′-F-dUTP (lanes 2, 5 and 8), or all four 2′-F-dNTPs (lanes 3, 6 and 9) are shown at the top of the gel.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. The template used for the top gel is T22, which encodes a 37 nt transcript. The template used for the middle gel is T29, which encodes a 54 nt transcript.
The template used for the bottom gel is T30, which encodes a 2700 nt transcript. For each template, the sequence of the first nine nucleotides of the transcript is shown at the right of the gel, with the C and U residues in gray background. Effect of Mn 2+ ions on 2′-F-dNMP incorporation by Syn5-Y564F RNA polymerase.
Transcription reactions were carried out by Syn5-Y564F RNA polymerase. The metal ion present in each reaction and its concentration are shown at the top of the gel. All the reaction mixtures contained ATP, GTP, 2′-F-dCTP and 2′-F-dUTP.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. The position of the migration of the DNA templates and the transcripts are marked. The template used for the top gel is T22, which encodes a 37 nt transcript. The template used for the bottom gel is T30, which encodes a 2700 nt transcript.
Effect of Mn 2+ ions on 2′-F-dNMP incorporation by Syn5-Y564F RNA polymerase. Transcription reactions were carried out by Syn5-Y564F RNA polymerase.
The metal ion present in each reaction and its concentration are shown at the top of the gel. All the reaction mixtures contained ATP, GTP, 2′-F-dCTP and 2′-F-dUTP. Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. The position of the migration of the DNA templates and the transcripts are marked. The template used for the top gel is T22, which encodes a 37 nt transcript.
The template used for the bottom gel is T30, which encodes a 2700 nt transcript. RESULTS AND DISCUSSION Influence of initiation sequences on 2′-F-dNMP incorporation and transcription yield by Syn5 RNA polymerase Currently, most of the applications for 2′-F RNA involve the incorporation of 2′-F-dCMP and 2′-F-dUMP into RNAs used for siRNA, RNA aptamers and ribozymes. Thus we initially focused on the incorporation of these analogs into small RNAs (0.8 μg/μl reaction). With Mg 2+, Mn 2+ and wild-type Syn5 RNA polymerase, all four rNMPs can be substituted with 2′-F-dNMPs at a yield of ∼0.1 μg/μl reaction. The use of wild-type Syn5 RNA polymerase with just Mg 2+ ions is effective only when the small RNA product has no U and one C in the first nine nucleotides of the transcript (Figure middle gel, lane 2); however, this standard reaction mixture does provide a simple and robust method of transcription of such small RNAs containing 2′-fluoro substitutions. For the production of long 2′-F RNAs, Syn5-Y564F RNA polymerase is the most efficient enzyme on templates that contain an optimized initiation region (Figure, lane 10 versus Figure, lane 8). We also examined Syn5-Y564F RNA polymerase on a very difficult template that produces a transcript starting with GGG and contains two U's in the first nine nucleotides of the transcript (template T30).
Although the yield on this template is low (Figure lane 13), a prolonged incubation to 8 h significantly increases the yield (Figure bottom gel, lane 1), allowing us to compare various methods to optimize the amount of product synthesized. Again Syn5-Y564F RNA polymerase was superior to wild-type Syn5 RNA polymerase for the incorporation of 2′-F-dCMP and 2′-F-dUMP (Figure bottom gel, lane 8 versus lanes 5 and 2). However, for the incorporation of all four 2′-F-dNMPs into the transcript produced on this template, the only conditions that produced any full-length product were those using wild-type Syn5 RNA polymerase with a mixture of Mg 2+ and Mn 2+ ions. Even under these conditions the efficiency was very low (Figure bottom gel, lane 6). We tested the effect of Mn 2+ on the 2′-F RNA synthesis catalyzed by Syn5-Y564F RNA polymerase and found that the addition of Mn 2+ did not improve the yield of a small 2′-F RNA (37 nt) with 2′-F-dCMP and 2′-F-dUMP substitutions as it does for wild-type enzyme (Figure ) at all conditions tested (Figure upper gel, lanes 4–12 versus 2). However, in many reactions containing Mn 2+, the yield of a long 2′-F RNA (2700 nt) with 2′-F-dCMP and 2′-F-dUMP substitutions is significantly improved compared to that with Mg 2+ only (Figure bottom gel, lanes 5, 6, 8–11 versus 2).
The highest yield was observed with a combination of 10 mM Mg 2+ and 5 mM Mn 2+ (Figure bottom gel, lane 8). However, the yield of 2′-F DNA with full 2′-F-dNMP substitutions by Syn5-Y564F RNA polymerase with any combination of Mg 2+ and Mn 2+ tested is still too low to be detected (Supplementary Figure S3). Finally we compared the Syn5-Y564F RNA polymerase to T7-Y639F RNA polymerase, the standard tool for synthesis of 2′-F RNA (2′-F DNA), with various initial sequences in the template (Figure ). When the transcripts start with GGA, both enzymes efficiently produce 2′-F RNA with 2′-F-dCMP and 2′-F-dUMP substitutions (Figure, lanes 2 and 5). Although the yield of natural RNA is similar for both enzymes (Figure, lane 1 versus 4), the yield of 2′-F RNA with 2′-F-dCMP and 2′-F-dUMP substitutions synthesized by T7-Y639F RNA polymerase is lower than that synthesized by Syn5-Y564F RNA polymerase (Figure, lane 2 versus 5). 2′-F DNA with full substitutions synthesized by Syn5-Y564F RNA polymerase (Figure, lane 3) is not observed with T7-Y639F RNA polymerase (Figure, lane 6), indicating that T7-Y639F RNA polymerase has stronger discrimination against 2′-F-dNTPs, especially 2′-F-dGTP and/or 2′-F-dATP than does Syn5-Y564F RNA polymerase.
When the transcripts start with CAG, Syn5-Y564F RNA polymerase showed a similar yield with that for transcripts starting with GGA (Figure, lanes 7–9). In the latter case the products synthesized by T7-Y639F RNA polymerase, even natural RNA, are barely detectable. Syn5-Y564F RNA polymerase is advantageous to synthesize 2′-F RNA (2′-F DNA) that is heavily modified and to synthesize transcripts containing initial sequences that are not synthesized effectively by T7 RNA polymerase. Synthesis of 2′-F RNAs by Syn5-Y564F and T7-Y639F RNA polymerases. Transcription reactions were carried out using either Syn5-Y564F RNA polymerase (lanes 1–3 and 7–9) or T7-Y639F RNA polymerase (lanes 4–6 and 10–12). The reaction mixtures that contained only normal NTPs (lanes 1, 4, 7 and 10), both 2′-F-dCTP and 2′-F-dUTP (lanes 2, 5, 8 and 11), or all four 2′-F-dNTPs (lanes 3, 6, 9 and 12) are shown at the top of the gel.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel.
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Dump File Tanaka T210
The template used for the experiments shown in the left gel is T1 (for Syn5-Y564F RNA polymerase) and T24 (for T7-Y639F RNA polymerase), which encode 37 nt transcripts of the same sequence starting with GGA. The template used for the right gel is T23 (for Syn5-Y564F RNA polymerase) and T27 (for T7-Y639F RNA polymerase), which encode 37 nt transcripts of the same sequence starting with CAG. For each template, the sequence of the first nine nucleotides of the transcript is shown at the bottom of the gel, with the first three residues in a gray background. Synthesis of 2′-F RNAs by Syn5-Y564F and T7-Y639F RNA polymerases. Transcription reactions were carried out using either Syn5-Y564F RNA polymerase (lanes 1–3 and 7–9) or T7-Y639F RNA polymerase (lanes 4–6 and 10–12). The reaction mixtures that contained only normal NTPs (lanes 1, 4, 7 and 10), both 2′-F-dCTP and 2′-F-dUTP (lanes 2, 5, 8 and 11), or all four 2′-F-dNTPs (lanes 3, 6, 9 and 12) are shown at the top of the gel.
Products of transcription reactions were separated by native gel electrophoresis and then stained with ethidium bromide. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. The template used for the experiments shown in the left gel is T1 (for Syn5-Y564F RNA polymerase) and T24 (for T7-Y639F RNA polymerase), which encode 37 nt transcripts of the same sequence starting with GGA. The template used for the right gel is T23 (for Syn5-Y564F RNA polymerase) and T27 (for T7-Y639F RNA polymerase), which encode 37 nt transcripts of the same sequence starting with CAG. For each template, the sequence of the first nine nucleotides of the transcript is shown at the bottom of the gel, with the first three residues in a gray background. We are grateful to Dr. Jiehua Zhou of Beckman Research Institute for suggestions to initiate the project and helpful discussions.
We thank Steven Moskowitz (Advanced Medical Graphics) for illustrations. We also thank Dr. Ya-Ming Hou for helpful discussions. FUNDING Funding for open access charge: Harvard University. Conflict of interest statement.
None declared.
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