WO2012170433A1 - Cassettes de terminaison des multiples arn polymérases pour une transcription efficace d'échantillons d'arn discrets - Google Patents

Cassettes de terminaison des multiples arn polymérases pour une transcription efficace d'échantillons d'arn discrets Download PDF

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WO2012170433A1
WO2012170433A1 PCT/US2012/040936 US2012040936W WO2012170433A1 WO 2012170433 A1 WO2012170433 A1 WO 2012170433A1 US 2012040936 W US2012040936 W US 2012040936W WO 2012170433 A1 WO2012170433 A1 WO 2012170433A1
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termination
class
rna
plasmid
trna
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Christopher J. Murray
Brian Young
Emily WILKES
James Rozzelle
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Sutro Biopharma, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • This invention provides for an improved and cost efficient means to transcribe circular plasmid DNA into RNA, either tRNA or mRNA.
  • the method involves the use of multiple DNA:RNA polymerase termination sequences to prevent wasted of high energy phosphates due to inefficient termination of the polymerases.
  • Protein synthesis is a fundamental biological process that underlies the development of polypeptide therapeutics, vaccines, diagnostics, and industrial enzymes.
  • rDNA recombinant DNA
  • RNA is mostly produced in vitro using either T7 RNA polymerase transcription or chemical synthesis, which is labor intensive and expensive.
  • T7 RNA polymerase transcription is a DNA plasmid-based method that requires a cloned DNA template located downstream of a phage promoter and a transcription terminator site at or near the 3' terminus of the desired transcript. The transcription reaction performed in reaction vessels and is scalable according to the desired need.
  • Another method is to transcribe recombinant RNA elements in vivo.
  • RNA polymerase directs a high level of transcription from a phage promoter in a multicopy DNA plasmid expressed in E.
  • RNA transcripts overexpressed in vivo are typically long and heterogenous due to terminator readthrough (Ponchon L and Dardel F, Nature Methods, 4: 571 -576; Studier et al., Methods Enzymol. 185:60-89 (1990)). In other cases, the method can also generate prematurely terminated products. Terminator readthrough and early termination are due to inefficiency by RNA polymerase at the transcriptional termination signal. Therefore, it remains possible to increase termination efficiency by improving the termination signal recognized by RNA polymerase.
  • RNA polymerases Two types have been shown to be used by RNA polymerases ⁇ see, e. g., MacDonald et al., J. Mol. Biol, 238: 145-158 ( 1994)).
  • the class 1 terminator domain exemplified by the late bacteriophage T7 terminator, encodes an RNA sequences that can form a stable stem-loop structure followed by a run of six U residues. Termination occurs at a 3'G residue just downstream of the U run.
  • the second type or class 2 terminator domain exemplified by the human preproparathyroid hormone (PTH) gene, encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure.
  • PTH human preproparathyroid hormone
  • This invention provides a method for the transcription of RNA such as mRNA and tRNA from a circular DNA plasmid comprising the steps of: (a) obtaining a circular DNA plasmid, wherein it comprises a phage promoter operably linked to a sequence encoding an RNA polymerase operably linked to a multiple terminator domain; and (b) transcribing the circular plasmid to produce tRNA.
  • the multiple terminator domain can have at least 95% termination efficiency, wherein the domain comprises at least three termination sequences selected from the group consisting of class 1 termination sequences and class 2 termination sequences.
  • RNA transcripts from DNA plasmids None of the existing methods of transcribing RNA transcripts from DNA plasmids have solved the problem of inefficient transcriptional termination. In the case of both in vitro and in vivo transcription of RNA from a DNA plasmid, termination efficiencies is less than 75% which results in termination readthrough and premature termination. This remains a major obstacle in producing large quantifies of RNA moieties. Therefore, there remains a need for an innovative method and a D A vector design for the transcription of RNA elements of interest. In order to solve this problem, the present invention describes a method for transcription of RNA transcripts from a DNA plasmid comprising multiple terminator domains.
  • the present invention provides a method for transcription of tRNA from a circular DNA plasmid comprising the steps of a) obtaining a circular DNA plasmid encodes the following RNA elements form 5' to 3', wherein a phage promoter is operably linked to a sequence encoding an RNA polynucleotide of interest operably linked to a multiple terminator domain; and b) transcribing the circular plasmid to produce tRNA.
  • the multiple terminator domain has at least a 95% termination efficiency, wherein the domain comprises at least three termination sequences selected from the group consisting of: class 1 termination sequences and class 2 termination sequences.
  • the class 1 termination sequences have a palindrome sequence of 7-20 bases followed by a poly U rich region of 5-12 bases and single termination efficiency of at least 30%; and the class 2 termination sequences include a length of 10 bases having the following motif: A U/C A C U/G G U having as ingle termination efficacy of at least 30%.
  • the phage promoter of the DNA plasmid is the bacteriophage T7 promoter.
  • the single termination efficiency of the termination sequences is at least 50%.
  • at least one of the class two termination sequences is from the human parathyroid gene.
  • at least one of the class one termination sequences is selected from a group comprising of the pBR322 terminator or phi terminator.
  • the class two termination sequence is AAUCUGUU.
  • the multiple termination domain comprises 5 termination sequences in the following 5' to 3' order: i) a class I termination sequence operably linked to; ii) a class 1 termination sequence operably linked to; iii) a class 2 termination sequence operably linked to; iv) a class 2 termination sequence operably linked to; and v) a class 1 termination sequence.
  • the RNA polynucleotide of the present invention is a tRNA.
  • the RNA polymerase is a mRNA.
  • transcription of the circular plasmid to produce RNA occurs within a bacterial cell.
  • transcription occurs in a reaction vessel wherein the vessel comprises ribonucleotide triphosphates and a phage RNA polymerase able to transcribe the DNA plasmid.
  • the present invention describes a circular DNA plasmid encoding the following RNA elements form 5' to 3', wherein a phage promoter is operably linked to a sequence encoding an RNA polynucleotide of interest operably linked to a multiple terminator domain having at least a 95% termination efficiency, wherein the domain comprises at least three termination sequences selected from the group consisting of: class 1 termination sequences and class 2 termination sequences.
  • the class 1 termination sequences have a palindrome sequence of 7-20 bases followed by a poly U rich region of 5-12 bases and single termination efficiency of at least 30%; and the class 2 termination sequences include a length of 10 bases having the following motif: A U/C A C U/G G U having as ingle termination efficacy of at least 30%.
  • the phage promoter of the DNA plasmid is the bacteriophage T7 promoter.
  • the single termination efficiency of the termination sequences is at least 50%.
  • the class two termination sequence comprises
  • the multiple termination domain comprises 5 termination sequences in the following 5' to 3' order: i) a class 1 termination sequence operably linked to; ii) a class 1 termination sequence operably linked to; iii) a class 2 termination sequence operably linked to; iv) a class 2 termination sequence operably linked to; and v) a class 1 termination sequence.
  • the RNA polynucleotide is either a mRNA or a tRNA.
  • Figure 1 depicts the Multiple Termination Domain incorporated into a tRNA expression cassette, containing a T7 promoter and DNA elements encoding S.cerevisiae tRNA phe with an amber stop anticodon and the hepatitis delta virus (HDV) ribozyme.
  • the constituent elements of the multiple termination domain include phi T7 terminators, PTH terminators, and a pBR322 terminator.
  • Figure 2 shows the Fractogel TMAE ion exchange elution profile of PNK-treated transcription purifications in the absence (A) and presence (B) of a tRNA refolding step prior to chromatography. Purification fractions were analyzed by 10% TBE/Urea PAGE.
  • Figure 3 shows the tRNA yield from MTD plasmid transcription and single terminator plasmid transcription.
  • A The elimination of large RNA byproducts and increased tRNA yield from MTD plasmid transcription compared to transcription from single terminator plasmid.
  • B Relative tRNA peaks in HPLC chromatograms of MTD plasmid transcription and single terminator plasmid transcription.
  • Expensive nucleotide triphosphates are used to generate RNA polynucleotides from DNA templates with DNA:RNA polymerase under in vitro conditions.
  • the RNA product often extends beyond the desired termination point and this results in a waste of the expensive nucleotide triphosphate reagents.
  • This invention solves this problem by dramatically improving the termination efficiency of naturally occurring DNA-RNA polymerases.
  • nucleic acid refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • Polynucleotides can also include modified nucleotides. Nucleic acid sequences include both DNA strand sequences, and RNA sequences, e.g., mRNA (for a coding sequence), rRNA, or tRNA. Polynucleotides can include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • a recombinant expression vector can include sequence elements that are not found in functional proximity to each other in a non-recombinant cell.
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. ##
  • a "vector” refers to a carrier DNA molecule into which a nucleic acid sequence can be inserted for introduction into a new host cell where it will be replicated, and in some cases expressed.
  • Vectors can be derived from plasmids, bacteriophages, plant, animal viruses, etc.
  • An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • host cell or "recombinant host cell”, as used herein, refers to a cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector, and includes not only the particular subject cell but also the progeny thereof. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • tRNA or "transfer RNA” refers to small RNA molecules that are capable of transferring a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation.
  • mRNA or "messenger RNA” refers to an RNA molecule that is transcribed from a DNA template and carries coding information to the ribosomes for protein synthesis.
  • An mRMA molecule may be processed, edited, and transported within a cell prior to translation.
  • tRNA expression cassette in the context of the present invention refers to
  • terminal efficiency refers to the efficiency at which RNA polymerase terminates transcription.
  • the value can be calculated using a mathematical equation, such as (termination product)/ (termination product + run-off product).
  • the "termination product” is the amount of properly terminated RNA product obtained from a transcription reaction
  • "run-off product” is the amount of improperly terminated RNA product obtained from the same transcription reaction.
  • transcription reactions are performed in a volume of lOul of GHT buffer (30 mM -HEPES, pH 7.8, lOOmM potassium glutamate, 15 mM Mg(OAc) 2 , 0.25 mM EDTA, I mM dithiothreitol, 0.05% Tvveen-20) containing 0.5 mM ATP, CTP, GTP and UTP; 2 ⁇ of [ ⁇ - 32 ⁇ ] ATP (specific activity of 6,000 Ci/mmol) or 4 ⁇ of y- 32 P]GTP (specific activity of 6,000 Ci/nmol); 10-20 ng of RNA polymerase, l ug plasmid template of 50 nM synthetic DNA template, and 4 units of RNasin.
  • GHT buffer 30 mM -HEPES, pH 7.8, lOOmM potassium glutamate, 15 mM Mg(OAc) 2 , 0.25 mM EDTA, I mM dithiothreitol,
  • Reactions are incubated at 37°C for 15 min, and the products were analyzed by electrophoresis in polyacrylamide gels containing 7 M urea.
  • the radioactivity in each RNA species resolved by electrophoresis was quantified by exposing the gel to a phosphorimager, scanner and
  • homogeneous 5' ends or “homogeneous 3' ends” refers to RNA transcripts with the same ribonucleotide sequence at either the start (5') or end (3') of the RNA molecule.
  • transcription by RNA polymerase can produce RNA transcripts all with the same 5' and/or 3' ribonucleotide sequence.
  • heterogeneous 5' ends or “heterogeneous 3' ends” refers to RNA transcripts with different ribonucleotide sequences at either the start (5') or end (3') of the RNA molecule.
  • transcription by RNA polymerase can produce RNA transcripts all with the same 5' and/or 3' ribonucleotide sequence.
  • hepatitis delta virus ribozyme or "HDV ribozyme” refers to a RNA element that can spontaneously fold into an autocatalytic enzyme that cleave RNA immediate 5' to its own sequence, leaving homogenous 3'ends.
  • S. cerevisiae tRNA Phe with an amber stop anticodon refers to a suppressor tRNA from yeast that can recognize the amber stop codon but readthrough it with canonical amino acids such as phenylalanine (Phe).
  • codon refers to a group of 3 consecutive nucleotides in a nucleic acid template that specify a particular naturally occurring amino acid, non-native amino acid, or translation stop (polypeptide chain termination) signal. Due to the degeneracy of the genetic code, an amino acid can be specified by more than one codon.
  • amber codon refers to a polypeptide chain-termination sequence UAG in RNA that acts to terminate polypeptide translation in most organisms.
  • the amber codon can also encode the proteinogenic amino acid pyrrolysine if the appropriate tRNA is charged by its cognate aminoacyl-tRNA synthetase.
  • the present invention provides a method for efficient transcription of RNA molecules such as tRNA or mRNA from circular plasmid DNA. Also provided by the invention is a new plasmid containing a novel arrangement of multiple DNA:RNA polymerase termination sequences to prevent inefficient termination of the polymerases. In some embodiments the multiple terminator domains have a high termination efficiency and comprise at least three termination sequences. The method of the present invention is useful for reducing the amount of unincorporated high energy phosphates during transcription.
  • the invention involves routine techniques in the field of recombinant technology, such as but not limited to the expression of RNA molecules encoded in DNA plasmids, in vitro transcription of RNA, in vivo transcription of RNA.
  • Routine molecular biology and recombinant DNA techniques known to those skilled in the art are used in the construction of a circular DNA plasmid of the present invention. These techniques include but are not limited to restriction enzyme digestion, ligation, polymerase chain reaction, plasmid DNA transformation into E.
  • coli and plasmid DNA extraction, for the use and construction of a circular DNA plasmid comprising an origin of replication, a selectable marker cassette, a phage promoter, a sequence encoding an RNA polynucleotide and multiple termination domains.
  • Basic texts disclosing the general methods for cloning and using this invention include Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001 ); riegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al. , eds., 1994-1999).
  • Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods.
  • Such manufacturers include SIGMA (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia L B Biotechnology (Piscataway, N.J.), Clontech Laboratories, Inc. (Palo Alto, Calif.), Aldrich Chemical Company (Milwaukee, Wis.), Invitrogen (San Diego, Calif.), Applied Biosystems (Fosters City, Calif.), as well as many other commercial sources known to one of skill in the art.
  • PCR amplification methods are well known in the art and are described, for example, in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc. San Diego, Calif., 1990.
  • An amplification reaction typically includes the DNA that is to be amplified, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.
  • dNTPs deoxynucleotide triphosphates
  • reaction buffer typically magnesium.
  • a desirable number of thermal cycles is between 1 and 25.
  • the PCR primers may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into specific restriction enzyme sites in a vector. If restriction sites are to be added to the 5' end of the PCR primers, it is preferable to include a few (e.g., two or three) extra 5' bases to allow more efficient cleavage by the enzyme.
  • One aspect of the present invention provides a novel DNA plasmid vector comprising a phage promoter, a sequence encoding a RNA polynucleotide of interest, and multiple termination domains.
  • circular DNA plasmids capable of replicating in a prokaryotic host cell e.g., Escherichia coli
  • a prokaryotic host cell e.g., Escherichia coli
  • ori origin of replication
  • Examples of a DNA plasmid vector commonly used for cloning and containing an ori and an antibiotic resistance marker include pBR322, pUC 18, pUC 19 and pACYCl 84.
  • the novel DNA plasmid vector of the present invention comprises a phage promoter (e.g., T3, T7 or SP6 promoter) wherein it provides a DNA region which is recognized by its specific phage RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase).
  • a phage promoter e.g., T3, T7 or SP6 promoter
  • the phage promoter can be the promoter from
  • the phage promoter can be cloned into a circular DNA plasmid using recombinant molecular techniques described herein.
  • the phage promoter can be obtain using methods that are known to those of skill in the art. Suitable nucleic acids including DNA can be made using standard recombinant or synthetic techniques. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning techniques are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd ed.) Vol.
  • the recombinant DNA of the invention is synthesized from oligonucleotides in vitro.
  • Synthetic DNA regions may be synthesized using microchip-based technology for multiplex gene synthesis as described by Richmond et al., Nucleic Acids Res, 32, 501 1-5018 (2004); Tian et al, Nature, 432, 1050-1054 (2004); Zhou et al, Nucleic Acids Res., 32, 5409-5417 (2004).
  • the desired nucleic acid sequence may be obtained by an amplification reaction, e.g., PCR.
  • the DNA plasmid may already contain a phage promoter region ⁇ e.g., T3 promoter, T7 promoter and SP6 promoter).
  • a phage promoter region ⁇ e.g., T3 promoter, T7 promoter and SP6 promoter. Examples include pGEM
  • pPCRII (Life Technologies)
  • pBluescript (Agilent Technologies).
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression of RNA in prokaryotic cells or in vitro in a reaction vessel may be used.
  • the novel DNA plasmid vector of the present invention further comprises desired RNA sequences ⁇ e.g., transfer RNA (tRNA) and messenger (mRNA)).
  • desired RNA sequences e.g., transfer RNA (tRNA) and messenger (mRNA)
  • tRNA transfer RNA
  • mRNA messenger
  • Native and synthetic tRNA genes can be encoded in the DNA plasmid of the present invention and transcribed by in vitro transcription or in transformed host cells.
  • the methods of the present invention find use in the efficient expression of a variety of tRNA and mRNA molecules.
  • the novel DNA plasmid vector of the present invention comprises a nucleic acid sequence encoding the desired tRNA.
  • Wild-type and synthetic tRNA genes can be encoded in the DNA plasmid of the present invention and transcribed by in vitro transcription or in vivo in transformed host cells. Examples of wild-type tRNAs include but are not limited to prokaryotic tRNAs, eukaryotic tRNAs, mitochondrial tRNAs, as well as others known in the art..
  • Examples of synthetic tRNAs include amber suppressor tRNAs (i.e., tRNA that binds to an amber codon) and spacer tRNAs, as well as others known in the art.
  • DNA plasmid of the present invention can contain DNA sequences encoding an RNA element such as hepatitis delta virus (HDV) ribozyme.
  • RNA element such as hepatitis delta virus (HDV) ribozyme.
  • HDV hepatitis delta virus
  • This non-coding RNA is known to those skilled in the art to process RNA transcripts in a self-cleavage reaction. It has not specific sequence requirements upstream of the cleavage site and generates 2', 3'-cyclic phosphate ends (see, e.g., Handbook of RNA Biochemistry: Student Edition, edited by R. K. Hartmann, A. Bindereif, A. Schon and E. Westhof,, Wiley-VCH Verlag GmbH & Co., GaA,
  • mRNAs transcribed by the DNA plasmid of the present invention can encode hormones, receptors, antibodies and antibody fragments, enzymes and structural proteins.
  • hormones include but are not limited to gonadotropins, glycoprotein hormones (such as, chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH)), and members of the integrin family, as well as others known in the art.
  • gonadotropins such as, chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH)
  • CG chorionic gonadotropin
  • TSH thyrotropin
  • LH lutropin
  • FSH follitropin
  • Receptors can include any receptor that can be expressed on the cell surface of a cell. Examples include but are not limited to nuclear hormone receptor superfamily of receptors, Her (EGF/erbB) family of protein receptors, transforming growth factor family of receptors, G- protein coupled receptors (GPCR), gamma-aminobutyric acid type B (GABA(B)) receptors, GABA receptors, lipoprotein receptors, interleukin receptors, tyrosine receptor kinases, cytokine receptors, TNF receptors, insulin receptor (alpha2 beta2), and immunoglobulin proteins (such as T-cell receptors and MHC molecules), as well as others known in the art.
  • GPCR G- protein coupled receptors
  • GABA(B) gamma-aminobutyric acid type B
  • Antibodies and antibody fragments can include monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multi-specific antibodies, and fragments thereof, regardless of how they are produced (i.e., using immunization, recombinant, synthetic methods).
  • Enzymes include protein that can catalyze chemical reactions by converting substrates into enzymatic products.
  • Examples of enzymes include but are not limited to polymerases, synthetases, endonucleases, exonucleases, recombinases, Iigases, hydrolases, reductases, kinases, phosphorylases, oxireductases, transferases, lyases, and isomerases, as well as others known in the art.
  • Structural proteins can include but are not limited to collagen, fibronectin, laminin, actin, actinin, cadherin, elastin, fibrinogen, heparin, mucin, myelin associated glycoprotein, spectrin, tropomysin, troponin, tubulin, vimentin and vitronectin, as well as others known in the art.
  • the RNA of interest can be cloned into the DNA plasmid of the present invention using recombinant techniques described herein.
  • sequence encoding for the RNA polynucleotide of interest can be constructed by assembling different overlapping oligonucleotides generated by automated oligonucleotide synthesizers or by amplification (e.g., PCR or cloning) from other plasmids encoding the RNA of interest.
  • the desired nucleotide region can be cloned into the DNA plasmid using standard cloning techniques such as but not limited to restriction enzyme digestion, DNA ligation, transformation into prokaryotic host cells, and plasmid purification (see, e.g., Sambrook et al., Molecular Cloning— A Laboratory Manual (2nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY ( 1989)).
  • RNA polymerase termination domains Transcription by RNA polymerase terminates at discrete termination domains (also referred to as terminators) located in nascent and synthetic DNA. Terminators cause realse of the RNA and dissociation of the transcription complex. Two main types of termination domains (Class 1 and Class 2) have been found to cause pausing or termination by RNA polymerase. [00S5] Class 1 termination domains are thought to involve the formation of stem-loop structures in the nascent RNA transcripts. The typical class 1 termination signal for
  • bacteriophage RNA polymerase encodes an RNA sequence that can form a stable, GC-rich stem-loop followed by a string of U residues.
  • class 1 termination domains include but are not limited to the ⁇ 3- ⁇ terminator and the termination signal indentified in pBR322 at the end of the P4 transcription region (pBR322- P4 terminator; see, e.g., Lynakhov et al., J. Mol. Bio., 280, 201 -213 (1998); Stuber D and Bujard H, Proc. Natl. Acad. Sci. USA, 78, 167-171 (1981 )).
  • a class 1 terminator domain is composed of a sequence located on the template strand of the DNA and consists of a region containing a sequence that is then repeated a few base pairs away in the inverted sequence (e.g., palindrome sequence).
  • the sequence is a G/C-rich stretch of nucleotides, followed by an A/T-rich stretch of nucleotides.
  • the weak A-U base pairing in the DNA:RNA duplex allows the release of the complex, thereby terminating transcription (see, e.g., Enzymology Primer for Recombinant DNA Technology , ed. Hyone-Myong Eun, Academic Press, Inc., San Diego, (1996); Dunn JJ and Studier FW, J. Mol. Bio., 166, 477-535 (1983)).
  • class 2 termination domains do not encode RNAs with an apparently consistent secondary structure, rather, they share a common DNA sequence.
  • Examples of such a sequence are ATCTGTT, in the non-template strand; HATCGTT (H designating A, C or T) in the nontemplate strand; ans HATCTGTTTT in the non-template strand.
  • Bacteriophage RNA polymerases can also recognize class 2 termination domains such as but not limited to the human preparathyroid hormone terminator, the vesicular stomatitus virus terminator, the rrnB Tl terminator at the end of the ribosomal operator, the rrnC terminator, and the concatemer junction of replication T7 DNA.
  • Class II termination domains share a common sequence, HATCGTT (H designating A. C. or T), followed by a T-rich sequence on the non-template strand located 7 bp upstream of the site of termination (see, e.g., He et al. , J. Biol.
  • Inefficient termination is a hallmark of both class 1 and class 2 termination domains located on bacteriophage DNA.
  • the ⁇ 7- ⁇ terminator has a 66% termination efficiency and the E. coll rrnC terminator has a 32% termination efficiency.
  • the pBR322 P4 terminator which is recognized as the most effective terminator of the plasmid has a 54% termination efficiency (see, e.g., Enzymology Primer for Recombinant DNA Technology , ed. Hyone-Myong Eun, Academic Press, Inc., San Diego, (1996)).
  • inefficient termination and read-through transcription via a phage termination domain may be required for phage survival, since the region of the phage genome that lies downstream of the terminator encoding essential functions is transcribed only by a polymerase that fails to terminate (see, e.g., Dunn JJ and Studier FW, J. Mol. Bio., 166, 477-535 (1983)).
  • the efficiency of termination is influenced, not only by the stem-loop and 3' U-rich region of the T7 terminator, but also sequence, stability, and length of the stem-loop, length and context of the U-rich region, promoter type, and distance between the promoter and termination site.
  • the DNA plasmid of the present invention further comprises a multiple terminator domain having at least a 95% termination efficiency where the domain comprises at least three termination sequences selection from the group consisting of class 1 termination sequences and classes 2 termination sequences wherein the class 1 termination sequences have a palindrome sequence of 7-20 bases followed by a poly U rich region of 5-12 bases and a single termination efficiency of at least 30%; and, said class 2 termination sequences include a length of 10 bases have the following motif: AU/CUCU/GGUU and having a single termination efficiency of at least 30%.
  • the class 1 termination sequence can be selected from a group comprising the pBR322-P4 terminator or the phi terminator.
  • the class two termination sequence can be selected from a group comprising the human parathyroid gene terminator.
  • the class 2 termination sequences may include a sequence of ten bases with the following motif: AAUCUGUU.
  • the multiple termination domain of the present invention comprises 5 termination sequences in the following 5' to3' order: a class 1 termination sequence operably linked to; a class 1 termination sequence operably linked to; a class 2 termination sequence operably linked to; a class 2 termination sequence operably linked to; and a class 1 termination sequence.
  • class 1 termination domains include but are not limited to the ⁇ 3- ⁇ terminator, ⁇ 7- ⁇ terminator and the pBR322-P4 terminator.
  • class 2 termination domains include but are not limited to the human preparathyroid hormone terminator, the vesicular stomatitus virus terminator, the rrnB Tl terminator at the end of the ribosomal operator, the rrnC terminator, and the concatemer junction of replication T7 DNA.
  • the multiple termination domain can be cloned into the DNA plasmid of the present invention using recombinant techniques described herein.
  • sequence encoding for the class 1 and class 2 termination sequences can be constructed by assembling different overlapping oligonucleotides generated by automated oligonucleotide synthesizers or by amplification ⁇ e.g., PCR or cloning) from other plasmids encoding the termination sequences.
  • plasmids comprising a class 1 or class 2 termination sequence include pDL44, pDL75, pBR322 (Lynakhov et al, J. Mol. Bio., 280, 201 -213 ( 1998)).
  • the desired nucleotide region can be cloned into the DNA plasmid using standard cloning techniques such as but not limited to restriction enzyme digestion, DNA ligation, transformation into prokaryotic host cells, and plasmid purification ⁇ see, e.g., Sambrook et al., Molecular
  • the plasmids of the present invention can be expressed in a variety of bacterial host cells, including E. coli (e.g., DH5 , DH 10B, HBl Ol ). Plasmid DNA can be introduced into chemically competent bacterial host cells by heat-shock. In other instances, plasmid DNA can be introduced by electroporation. Methods known by those skilled in the art can be used to transform bacterial host cells ⁇ see, e.g., Sambrook et al., Molecular Cloning— A Laboratory Manual (2nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY ( 1989)).
  • the plasmids of the invention can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for ?, coli and calcium phosphate treatment.
  • bacterial host cells including, but are not limited to, DH5a, DH 10B, JM 101 , JM 1 10, XLl -Blue, Topl O, Topl OF' and HBl Ol E. coli cells.
  • Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the ampicillin, tetracycline, chloramphenicol, blasticidin, neomycin, and hygromycin genes.
  • the transformed cells can be cultured in an appropriate nutrient medium to a desired density, the cells can be harvested, and a lysate can be prepared by conventional means, e.g., agitation with glass beads, or phenol-based extraction methods. Methods known to those skilled in the art can used to purify the DNA plasmid, such as, but not limited to, ion exchange column chromatography. The plasmid may be further processed by dialysis, or even concentrated.
  • the DNA plasmid of the present invention comprises a phage promoter, a sequence encoding an RNA of interest, and a multiple termination domain can be in vitro transcribed in a reaction vessel, wherein the reaction vessel contains ribonucleotide triphosphates and phage RNA polymerase.
  • an in vitro transcription reaction in a reaction vessel comprises components, such as but not limited to, a purified DNA plasmid, a specific phage RNA polymerase that recognizes the phage promoter in the plasmid, RNA polynucleotides, spermidine, pyrophosphatase, RNase A inhibitor, a reducing agent (e.g., dithiothreitol or Tris(2-carboxyethyl)phosphine) and a buffer solution.
  • a reducing agent e.g., dithiothreitol or Tris(2-carboxyethyl)phosphine
  • transcription from plasmid DNA can be performed in a reaction vessel containing 40mM Tris- HC1, pH 8.0, I mM spermidine, 0.05mg/ml BSA, 20mM MgCl 2 , 5mM DTT, 0.01 % TritonX-100, 4mM each NTP, 0.005 ⁇ / ⁇ inorganic pyrophosphatase, l U/ul ribonuclease inhibitor and 25U RNA polymerase.
  • the reaction is incubated at 37°C for 1 hour.
  • the DNA plasmid used in the transcription reaction can be linearized by restriction enzyme digestion and purified to remove contaminant which can inhibit transcription.
  • DNA plasmid of the present invention can contain DNA sequences encoding an RNA element such as hepatitis delta virus (HDV) ribozyme.
  • This non-coding RNA is known to those skilled in the art to process RNA transcripts in a self-cleavage reaction. It has not specific sequence requirements upstream of the cleavage site and generates 2', 3'-cyclic phosphate ends (see, e.g., Handbook of RNA Biochemistry: Student Edition, edited by R. K. Hartmann, A. Bindereif, A. Schon and E. Westhof,, Wiley-VCH Verlag GmbH & Co., KGaA, Weinheim,Germany, (2009)).
  • HDV hepatitis delta virus
  • In vitro transcription of DNA plasmid can be performed using recombinant proteins such as porcine RNase A inhibitor, S. cerevisiae pyrophosphatase and T7 RNA polymerase.
  • Inorganic pyrophosphatase from yeast such as S. cerevisiae is known by those skilled in the art to catalyze the hydrolysis of inorganic pyrophosphate to two orthophosphates, thereby increasing the yield of RNA synthesis by in vitro transcription.
  • Ribonuclease inhibitors such as, but not limited to, RNase A inhibitors, protect against RNA degradation by binding RNases and preventing their activity.
  • Recombinant proteins of the in vitro transcription reaction such as inorganic pyrophosphatases, ribonuclease inhibitors or phage RNA polymerases can be purchased from various vendors, such as but not limited to, Fermentas (part of Thermo Fisher Scientific, Waltham, MA), Life Technologies Corporation (Carlsbad, CA), Roche Applied Science (Indianapolis, IN), New England Biolabs, Inc. (Ipswich, MA).
  • recombinant proteins can be synthesized and purified using methods known to those skilled in the art (see, e.g., Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et ah, eds., 1994-1999)). Briefly, DNA plasmids containing protein expression cassette are transformed and expressed in prokaryotic host cells.
  • the protein expression cassette may contain DNA elements encoding affinity tags or amino acid purification tags (e.g., hexahistidine, FLAG, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST)) or peptide linkers (e.g. , Gly-Gly-Ser, Gly-Gly or Gly-Ser).
  • affinity tags or amino acid purification tags e.g., hexahistidine, FLAG, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST)
  • GST glutathione-S-transferase
  • the recombinant proteins expressed by the host cells are extracted and purified by methods such as affinity chromatography.
  • the circular DNA plasmid of the present invention encodes a RNA element.
  • the RNA polynucleotide is a tRNA.
  • the plasmid will encode tRNA (e.g., S. cerevisiae tRNA Phe ) and HDV ribozyme.
  • HDV ribozyme processes RNA transcripts in a self-cleavage reaction to generate 2', 3'-cyclic phosphate ends (see, e.g., Handbook of RNA Biochemistry: Student Edition, edited by R. . Hartmann, A. Bindereif, A. Schon and E. Westhof,, Wiley-VCH Verlag GmbH & Co., KGaA,
  • T4 PNK phage T4 polynucleotide kinase
  • T4 PNK can be purchased from vendors (e.g., Fermentas (part of Thermo Fisher Scientific, Waltham, MA), Life Technologies Corporation (Carlsbad, CA), Roche Applied Science (Indianapolis, IN), New England Biolabs, Inc.
  • the PNK-treated transcription reactions may be exposed to thermal cycling to facilitate proper folding of tRNA products.
  • the PNK-treated transcription mixtures may be incubated in a 70°C water bath in the presence of l OmM MgCl 2 for 15-20 minutes, and then slow cooled to room temperature. This procedure may be repeated one or more times. Thermal cycling improves the recovery of pure tRNA molecules generated by in vitro transcription.
  • the refolded PNK-treated transcription mixtures may be purified by centrifugation, filtration and ion exchange chromatography.
  • tRNA- containing fractions eluted from an ion exchange column may be analyzed on a gel
  • the tRNA may be precipitated, washed to remove excess salt, and resuspended in storage buffer containing 10 mM HEPES (pH 8) and 0.1 mM EDTA. tRNA can be stored at - 80°C.
  • transcription of the DNA plasmid of the present invention can be performed in vivo.
  • the RNA of interest can be purified from the cell lysate by chromatography ⁇ see, e.g., Meinnel et al, Nucleic Acids Res., 16, 8095-8096 ( 1988); Ponch L and Dardel F, Nature Methods, 4, 571 -576 (2007)).
  • the plasmid can be transformed into E. coli cells, such as but not limited to BL21 (DE3) using conventional chemical transformation techniques (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.; Sambrook and Russell (2001)).
  • Transformed bacterial host cells can be used to inoculated small scale and large scale cultures.
  • a small culture is inoculated with a transformed bacterial colony, grown to saturation at 37 °C, shaking, and then used to inoculate a larger, commercial scale culture in a fermentor.
  • the culture may be grown until the optical density at 600nm is about 12 or higher.
  • the transformed bacterial cells can be harvested by centrifugation and then the total tRNA can be extracted from the cells. Methods known to those skilled in the art can be used to isolate total tRNA such as phenol extraction followed by isopropanol precipitation (see, e.g., (Raczniak et a!. , J. Biol. Chem.
  • the total tRNA may be passed through an ion exchange column to further purify.
  • chaplet column chromatography can be used (Suzuki T and Suzuki T, Methods Enzymol .425:231-9 (2007); Kaneko, T et al , Methods Enzymol., 425:231 -9 (2007)). 30-mer synthetic 3' biotinylated DNA oligos complementary to the 3' end S.
  • cerevisiae tRNA phe can be synthesized (Eurofins MWG Operon, Huntville, AL) and used to charge Streptavidin Sepharose HP resin columns (GE Healthcare, Piscataway, NJ). S. cerevisiae tRNA Phe will selectively bind the DNA oligo and be immobilized on the column. After a series of wash steps, the tRNA of interest can be eluted and collected. The purified tRNa of interest can be precipitate, concentrated and washed. It can be resuspended in 10 mM HEPES (pH 8.0). To optimize the proper folding of tRNA, it can be heated to 70 °C in the presence of l OmM MgCl 2 and cooled slowly to room temperature.
  • Example 1 Design of Multi-terminator domain (MTD) plasm id.
  • T7 terminators were placed in series ( Figure 1 ) to produce a multi-termination domain (MTD). If each terminator acts independently at 70% efficiency, five terminators should collectedly block the extension of 99.75% of the transcripts (0.30 readthrough * 0.30 readthrough * 0.30 readthrough* 0.30 readthrough * 0.30 readthrough ⁇ 0.0024 overall readthrough).
  • phi terminators occupied the first two positions (positions 1 and 2) in the multi- terminator domain. Since phi terminators are strongly sequence context dependent ⁇ see, e.g., Macdonald LE, Zhou Y, and McAllister WT, ./. Mol. Biol,. 232, 1030-1047 (1993); Macdonald LE, Durbin RK, Dunn JJ, McAllister WT, J. Mol Bio. , 238, 145-158 (1994)), each terminator domain contains 104 base pairs upstream and 1 1 base pairs downstream of the termination base sequence. Since multiple repeated sequences can result in recombinational sequence loss when plasmids are passaged in E. coli, no other phi terminators were used.
  • PTH terminators were placed in positions 3 and 4, downstream of the phi terminators. It has been shown that a PTH terminator has about 70% termination efficiency when the termination domain is restricted to 43 base pairs upstream and 7 bases downstream of the stop base, (see, e.g., Macdonald, LE, Zhou Y, and McAllister WT, J Mol. Biol,. 232, 1030-1047 (1993)). Hence, PTH in this sequence context was used in tandem positions. As above, to limit recombination, only two PTH terminators were used. Finally, a pBR322 P4 terminator with 38 base pairs upstream and 20 base pairs downstream of the stop base was place in the position 5 of the multi- termination domain. The P4 terminator has been shown to have about 54% termination efficiency.
  • a tRNA expression cassette incorporating the MTD domain described above was constructed via gene synthesis (DNA2.0, Menlo Park, CA). Briefly, the cassette contained the fol lowing elements from 5' to 3' direction: the T7 promoter, S. cerevisiae tRNA phe with an amber stop anticodon, the hepatitis delta virus (HDV) ribozyme, and the MTD domain ( Figure 1 ).
  • the HDV element serves to improve the functionality of the tRNA since in vitro transcribed tRNA has heterogenous 3' ends that can abrogate its function (Ellman J et a., Methods Enzymol. 202:301 -36 (1991 )).
  • HDV elements spontaneously fold into autocatalytic enzymes that cleave RNA immediately 5' to their own sequences, thereby leaving homogenous tRNA 3' ends.
  • the tRNA expression cassette was subcloned between EcoRI and Xho I sites of the Sutro expression vector pYD318 (a variant of pYD317 with Xhol site introduced immediately 3' of the Sail site by PCR mutagenesis ⁇ see, JUNHAO pYD317 PATENT; Sambrook et al., Molecular Cloning- A Laboratory Manual (3nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY (2001)).
  • this plasmid construct is referred to as the MTD plasmid
  • Example 2 Measuring termination efficiency.
  • This example illustrates a method for measuring and calculating the termination efficiency of terminator domains following in vitro transcription.
  • a typical protocol entails constructing an expression plasmid bearing putative terminator domains, performing in vitro transcription reactions with radioisotope labeled rNTP.
  • a expression plasmid is constructed contain a T7 promoter, an expression cassette and the putative terminator domain for T7 RNA polymerase.
  • the potential terminator domain can be cloned between a T7 promoter and a blunt end restriction site and spaced at an appropriate distance.
  • the plasmid can be linearized by conventional restriction enzyme digestion of DNA and isolated by gel purification following gel electrophoresis ⁇ see, e.g., Sambrook et al., Molecular Cloning— A Laboratory Manual (3nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY (2001 )).
  • In vitro transcription can be performed on blunt-end, linearized plasmid DNA under the following conditions: 20 mM Tris- HCL (pH7.9); 2 mM spermidine-HCl; 20 mM MgCl 2 ; 1 mM DTT; 0.5 mM ATP, GTP, CTP and UTP; [a- 32 P]rNTP (specific activity 0.2 Ci/mmol) (Perkin Elmer, Waltham, Massachusetts); 20 ng purified T7 RNA polymerase; and 1 ⁇ g linearized plasmid DNA template. Transcription reactions of a volume of 15 iL can be incubated for 15 min at 37°C.
  • Reactions can be stopped by the addition of an equal volume of reaction stop mix ( 1 78 mM Tris-borate pH 8.3, 5 mM EDTA, 8M urea, and 0.5% bromophenol blue and xylene cyanol).
  • Tripl icate samples can be run on 5% polyacrylamide/TBE/urea sequencing gels (5% polyacrylamide, 44 mM Tris-borate pH 8.3, 2.5 mM EDTA, and 7 M urea) and analyzed by autoradiography.
  • Radioactive dideoxy- mediated sequencing reactions can be run in parallel to determine if the termination sites resides within the putative termination sequence ⁇ see, e.g., Sambrook et al., Molecular Cloning— A Laboratory Manual (3nd ed.) Vol. 1 -3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY (2001 )). ImageQuant (GE Healthcare) densitometry can be used to measure the relative intensity of the terminator stop product (T) and the plasmid run-off product (RO).
  • Percent termination efficiency can be calculated using the following equation: (T/(T+R0))* 100. For more rigorous measurement, percent termination efficiency of a particular terminator domain should be calculated as the average of at least three independent samples.
  • Example 3 Production and purification of MTD plasmid at scale.
  • This example illustrates a method of propagating and purifying the MTD plasmid which can be used in transcription reactions.
  • the MTD plasmid was transformed into chemically competent DH5a E. coli cells (Invitrogen; Carlsbad, CA). Colonies were selected and used to inoculate 3 ml cultures of LB supplemented with 0.05 mg/ml kanamycin (LB-Kan). Overnight cultures were grown at 37°C in an orbital shaking incubator (Braun) at 250 rpm. The next morning, a 3 ml culture was used to inoculate 0.25 L of pre-warmed LB-Kan in a baffle flask and cultured for an additional 5 hours.
  • LB-Kan orbital shaking incubator
  • This culture was used to inoculate a 10 L fermenter containing growth media (such as Terrific Broth which consists of 96 g tryptone, 192 g yeast extract, 32 ml glycerol, 48 g ammonium sulfate, 46 g sodium chloride, 80mM potassium phosphate (pH -7.2) and 40mM magnesium sulfate per liter).
  • growth media such as Terrific Broth which consists of 96 g tryptone, 192 g yeast extract, 32 ml glycerol, 48 g ammonium sulfate, 46 g sodium chloride, 80mM potassium phosphate (pH -7.2) and 40mM magnesium sulfate per liter.
  • the culture was fermentation for 20 hours at 37°C with stirring at 600rpm until the final OD600 was 14. Cells were harvested by centrifugation (320g wet cell weight) and the cell pellets were stored at -80°C.
  • resuspension buffer 50mM Tris (pH 8.0), l OmM EDTA. Typically, 5 ml of resuspension buffer is used per gram of cells.
  • An equal volume of lysis buffer (0.2 M NaOH, 1% SDS) was added and the mixture was passed through a static mixer until cell lysis was complete.
  • This example illustrates expression plasmids that can be used to generate recombinant proteins. This example also demonstrates a method to express and purify proteins necessary for in vitro transcription reactions.
  • the plasmids for expression and purification of in vitro transcription protein reagents were designed and constructed to harbor amino acid linkers and hexahistidine tags to facilitate the purification of the recombinant proteins.
  • the plasmids bear DNA elements encoding either T7 phage RNA polymerase, porcine RNase a inhibitor or S. cerevisiae pyrophosphatase.
  • the parental plasmid pAR1219 Sigma Aldrich Catalog #T2076; St.
  • porcine RNAse A inhibitor expression plasmid the coding sequence of the porcine RNAse A inhibitor was fused to an N-terminal hexahistidine purification tag and a Gly-Gly-Ser linker by gene synthesis, and then subcloned into
  • pJexpress401 which contains a T5 promoter (DNA2.0; Menlo Park, CA).
  • Ipplp S. cerevisiae pyrophosphatase
  • the coding sequence of S. cerevisiae Ipplp was fused to a C-terminal Gly-Gly linker and a hexahistidine purification tag by gene synthesis, and then subcloned into pJexpress41 1 which contains a T7 promoter (DNA2.0, Menlo Park, CA).
  • the plasmids were introduced into E. coll, and proteins were expressed and purified by methods known to those skilled in the art. Briefly, plasmids were transformed into E. coli BL21 (DE3) cells. A few colonies from the transformation were selected and used to inoculate LB- Antibiotic. In certain embodiments, 200ml of LB containing 100 ⁇ ampicillin was used to culture the T7 RNA polymerase transformants; 200 ml LB containing 50 ⁇ g/ml kanamycin for the porcine RNase A inhibitor transformants; and 3 ml LB containing 50 ⁇ g/ml kanamycin for the S. cerevisiae pyrophosphatase transformants. The bacterial cultures were grown overnight at 37°C and then used as started cultures.
  • the 200 ml T7 RNA polymerase bacterial culture was used to inoculate a 10 liter Bioflow 3000 fermentor.
  • the culture was grown in LB-Amp at 37°C to an OD of approximately 2.0.
  • 1 mM Isopropyl ⁇ -D-l -thiogalactopyranoside (IPTG) was added and the bacteria was cultured for an additional 4-5 hours at 37°C.
  • 200 ml of bacteria expressing porcine RNase A inhibitor was used to inoculate a 10 liter Bioflow 3000 fermentor containing autoinduction media (0.025 M (NH 4 ) 2 S0 4 ; 0.050 M ⁇ 2 ⁇ 0 4 ; 0.050M NaHP0 4 ; 0.5% Glycerol; 0.05% glucose; 0.2% a-lactose; 1 mM MgS0 4 , 10 g/L N-Z-amine AS; 5 g/L yeast extract; 74 ⁇ FeCl 3 ; 14 ⁇ Na 2 Mo0 4 ; 0.027 g/L Choline Chloride; 0.024 g/ L Nicotinic Acid; 0.024 g/L PABA; 9 mg/L Pantothenic Acid; 1.35 mg/L Pyridoxine; 3.73 mg/L Riboflavin; 17 mg/L Thiamine; 0.1 1 mg/L Biotin; 8.32 ⁇ g/L Cyanocobalamin
  • the S. cerevisiae pyrophosphatase culture was used to inoculate 2 liters of autoinduction media supplemented with kanamycin. This culture was grown overnight at 37°C.
  • the cells were lysed with an Avestin C55 pressure homogenizer (Avestin; Ottawa, Ontario, Canada) at about 20,000 psi. Lysates were clarified by centrifugation in a JA-1 7 rotor (Beckman; Brea, CA) at 40,000 ⁇ 3 ⁇ 4 ⁇ for 30 min. The supernatant comprising recombinant protein were applied to Ni 2+ Sepharose 6 Fast Flow columns (GE Healthcare; Piscataway, NJ). In certain embodiments, columns for the T7 RNA polymerase protein were pre-equilibrated with 450 ml of buffer A; columns for porcine RNAse A inhibitor with 500 ml buffer A; and columns for S.
  • Avestin C55 pressure homogenizer Avestin; Ottawa, Ontario, Canada
  • Lysates were clarified by centrifugation in a JA-1 7 rotor (Beckman; Brea, CA) at 40,000 ⁇ 3 ⁇ 4 ⁇ for 30 min.
  • the supernatant comprising recombinant protein
  • cerevisiae pyrophosphatase with 20 ml buffer A. The columns were washed with at least 5 column volumes of buffer A. In another embodiment, T7 RNA polymerase and porcine RNase a inhibitor were eluted from the columns with buffer B (buffer A supplemented with 300 mM imidazole). In yet another embodiment, S. cerevisiae pyrophosphatase was eluted with a 10 column volume gradient of buffer A to buffer B. From each column eluted fractions containing protein were pooled, dialyzed in 2x storage buffer without glycerol, and then diluted 1 : 1 with glycerol.
  • recombinant T7 RNA polymerase protein was stored in buffer comprising 50 mM sodium phosphate (pH 7.9), 100 mM NaCl, 0.1% Triton X-100, 5 mM DTT, and 50% glycerol.
  • recombinant porcine RNAse A inhibitor protein was stored in buffer comprising 25 mM HEPES- KOH (pH 7.6), 50 mM KCl, 2 mM DTT, 1 mM EDTA, and 50% glycerol.
  • recombinant S was stored in buffer comprising 50 mM sodium phosphate (pH 7.9), 100 mM NaCl, 0.1% Triton X-100, 5 mM DTT, and 50% glycerol.
  • cerevisiae pyrophosphatase protein was stored in buffer comprising 20 mM Tris (pH8.0); 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 50% glycerol. These proteins were stored at -20°C.
  • This example illustrates a method for in vitro transcription of a plasmid comprising a promoter, a DNA sequence encoding an RNA element and a multi-termination domain.
  • a plasmid comprising a promoter, a DNA sequence encoding an RNA element and a multi-termination domain.
  • one liter in vitro transcription reactions were performed in RNAse-free vessels. The reactions contained the following: 40 mM Tris (pH 7.9); 20 mM NaCl; 10 mM MgCl 2 ; 1 mM DTT; 2.5 mM spermidine; 0.01 1 mg/ml S.
  • the S. cerevisiae pyrophosphatase, porcine RNase A inhibitor and T7 RNA polymerase proteins of the transcription reaction were expressed and purified as described above.
  • the Tris, NaCl and MgCl 2 buffers of the transcription reaction were made using in MilliQ-treated H 2 0 and preheated to 37°C, prior to the addition of the other reagents. Transcription reactions were incubated for 4 hours in a 37°C water bath. The reactions were then supplemented with additional T7 RNA polymerase to a final concentration of 0.048 mg/ml. The reactions incubated for another 4 hours at 37°C
  • Example 6 Removal of 2', 3' cyclic phosphate of tRNA produced using MTD plasmid.
  • This example illustrates removal of 2', 3' cyclic phosphate of tRNA generated by autocatalytic cleavage by HDV ribozyme.
  • In vitro transcription of tRNA typically generates heterogenous 3' ends that can hinder its function. However, these ends can be cleaved by HDV ribozymes to produce homogenous 3' ends.
  • the HDV element serves to improve the functionality of the tRNA since in vitro transcribed tRNA has heterogenous 3' ends that can abrogate its function (Ellman et ⁇ , Methods Enzymol. 202:301-36 (1991 )).
  • HDV elements spontaneously fold into autocatalytic enzymes that cleave RNA immediately 5' to their own sequences, thereby leaving homogenous tRNA 3' ends.
  • T4 PNK phage T4 polynucleotide kinase
  • T4 PNK To express and purify T4 PNK, the ORF was fused to an N-terminal hexahistidine purification tag and Gly-Gly linker by gene synthesis; this cassette was subcloned under control of the T7 promoter in Sutro expression vector pYD317 (DNA2.0, Menlo Park, CA). Expression and purification was similar to porcine RNAse A inhibitor described above (see Example 4) with the following exceptions. First, KC1 was substituted for NaCl in purification buffer A. Second, immediately after elution, T4 PNK was diluted to 1 .5 mg/ml before dialysis and glycerol dilution. Final T4 PNK storage buffer: (10 mM Tris(pH7.6), 50 mM KC1, 0.1 mM EDTA, 1 mM DTT, 50% glycerol).
  • Example 7 Refolding and additional HDV cleavage by thermal cycling
  • Example 7 for a description of the condition; modification of procedure includes, 110 ml column used and elution performed with a 20 Column Volume gradient from buffer MPC (350 mM NaCl in 50 mM Bis-Tris, pH 6.2, 10 mM MgCl 2 , 1 mM EDTA) to MPD (750 mM NaCl in 50 mM Bis-Tris, pH 6.2, 10 mM MgCl 2 , 1 mM EDTA)). Instead a large portion of tRNA from the reactions co-purified with precursor RNA and HDV cleavage byproducts present in the higher salt elution fractions (Fig. 2). This was determined by diluting 3 ⁇ elution fractions samples 1 : 1 with RNA Gel Loading Buffer II (Applied
  • This example illustrates the purification of tRNA by ion exchange chromatography following thermal cycling.
  • Refolded-PNK-treated mixtures from 1 1 transcription were clarified by centrifugation and 0.6 ⁇ filtered. Filtered mixtures were loaded onto a 400 ml Fractogel TMAE (EMD Chemicals; Gibbstown, NJ) column pre-equilibrated with buffer MPA (50mM Bis-Tris (pH6.2), l OmM MgCl 2 , I mM EDTA).
  • buffer MPA 50mM Bis-Tris (pH6.2), l OmM MgCl 2 , I mM EDTA).
  • 3 column volumes 0.1 M sodium hydroxide and 3 column volumes 3 M NaCl, 50 mM Bis-Tris washes were performed prior to pre-equilibration to remove residual RNA.
  • the column was washed with buffer MPB (50mM Bis-Tris (pH6.2), l OmM MgCl 2; I mM EDTA, 350 mM NaCl) until the UV detector returned to equilibration baseline.
  • tRNA was eluted with buffer Mobile Phase C (50mM Bis- Tris (pH6.2), lOmM MgCl 2 , I mM EDTA, 450 mM NaCl).
  • tRNA-containing fractions were confirmed by running 3 ⁇ samples diluted 1 : 1 with RNA Gel Loading Buffer II (Applied Biosystems; Austin, TX) on 10% TBE/UREA PAGE (Invitrogen; Carlsbad, CA) and staining gels with Sybr Green II (Molecular Probes, Eugene, OR).
  • RNA Gel Loading Buffer II Applied Biosystems; Austin, TX
  • TBE/UREA PAGE Invitrogen; Carlsbad, CA
  • Sybr Green II Molecular Probes, Eugene, OR
  • tRNA containing fractions were pooled and tRNA precipitated by addition of isopropanol to 50% followed by overnight incubation at -80°C tRNA was pelleted by centrifugation at 10,000xg for 60 minutes, washed with 70% ethanol, centrifuged at 10,000xg for 30 minutes, air dried briefly, then resuspended at ⁇ 20mg/ml in 10 mM HEPES (pH 8) 0.1 mM EDTA. For optimal activity, tRNA was refolded by heating to 70 °C, addition of 10 mM MgCl 2 and then slow cooling to room temperature. Transfer RNA was stored at -80°C.
  • HPLC hydrophobic interaction chromatography To quantitate tRNA produced, HPLC hydrophobic interaction chromatography (HIC) was performed. 10 ⁇ transcription reaction was diluted into 100 ⁇ buffer TA (50 mM potassium phosphate and 1.5 M ammonium sulfate pH 5.7) and injected onto a Wide Pore C5 HPLC Column (Sigma Aldrich Catalog # 567231 ; St. Louis, MO) pre- equilibrated in buffer TA. tRNA was separated from other RNA with a 55 minute linear gradient from buffer TA to buffer TB (50 mM potassium phosphate and 5% isopropanol); identity of tRNA peak was confirmed by pure tRNA reference samples. HPLC quantitation of tRNA in reactions indicated a 4-fold increase in yield for MTD plasmid transcription vs. single terminator plasmid transcription (Fig. 3B).
  • tRNA production improvements were also measured by comparison of recovered yield from transcription purifications.
  • Small scale (250 ml) transcription reactions were performed, PNK treated, and refolded (see conditions: Examples 4-6) for both MTD plasmid and single terminator plasmid.
  • Ion exchange purification (Conditions: Example 7; 3 ml Fractogel TMAE column, 7.5 mL aliquot of transcription mix) of these samples showed a tRNA recovered yield from MTD plasmid of 2.6 g vs. 0.96 g for single terminator plasmid.
  • This ⁇ 3 fold increase in production was recapitulated in 1 liter scale transcriptional processing/purifications (Examples 4- 7). Absolute yield from 1 liter scale production was a striking 615 mg tRNA (average of two replicates.) This corresponds to -40% recovery of the theoretically possible tRNA yield.
  • Example 10 In vivo transcription and purification of tRNA using MTD plasmid
  • This example illustrates the expression of tRNA in prokaryotic cells and the purification of tRNA.
  • the plasmid can be transformed into BL21 (DE3) E. coli cells using conventional chemical transformation techniques (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.; Sambrook and Russell (2001 )).
  • a few colonies from the plate can be inoculated into 2 ml LB-KAN and grown to saturation at 37 °C; this culture can be diluted into 100 ml LB- KAN and grown to saturation at 37°C.
  • This entire culture can be used to inoculate 10 L of autoinduction media ⁇ see Example 4; Studier FW, Protein Expr. Purif., 41 :207-34 (2005)) supplemented with kanamycin in a Bioflow 3000 fermentor.
  • This culture can be grown for 18 hours at 37 °C until it reaches an OD of -12.
  • Cell can be harvested by centrifugations and frozen at -80 °C.
  • autoinduction fermentations yield ⁇ 200 g of cell pellet.
  • RNA can be precipitated by addition of sodium Acetate, pH 5.5, to 0.3 M and isopropanol to 50% with subsequent chilling to -80°C for 30 minutes. RNA can be pelleted by centrifugation at 17,500xg for 35 minutes (500 ml centrifugation tubes; Fiberlite Fl 0BA rotor).
  • RNA can then be dissolved in 200 mM Tris acetate, pH 8.5 and loaded onto a pre-equilibrated DEAE cellulose column (1 ml DE52 resin/ 100 A260 units of RNA).
  • the column can be washed with several column volumes of 10 mM sodium acetate, pH 5.5, 200 mM NaCl, and 10 mM MgCl 2 and eluted with 1 M NaCl.
  • An equal volume of isopropanol can be added to the eluate, the solution chilled to -80°C for 30 minutes and then tRNA pelleted by centrifugation for 35 minutes at 17500xg.
  • HEPES-KOH (pH 7.5), 5 mM EDTA) to 0.15 mM.
  • a Streptavidin Sepharose HP column of 1 ml/600 mg total tRNA can be packed and pre-equilibrated in buffer 1. The column can be charged by recirculating 1 ml 0.15 mM DNA oligo/1 ml resin over the column for 1 hour at room temperature. The column can then be equilibrated by washing with buffer pre-heated to 65 °C and loaded onto the column through a heat exchanger (0.75 mm stainless steel tubing formed into a spiral; catalog #5188-6466; Agilent Technologies, Santa Clara, CA) immersed in 65 °C water.
  • Equilibration can consist of 10 column volumes buffer I and 5 column volumes buffer BB ( 1 .2 M NaCl, 30 mM HEPES-KOH (pH7.5), 15 mM EDTA).
  • Total tRNA can be dissolved in buffer BB to a concentration of 4 mg/ml and pre-heated to 65°C.
  • This load fraction can be recirculated onto the column through the heat exchanger immersed in 65°C water for 30 minutes. The heat exchanger can be then immersed in room temperature water and the load recirculated through the column at room temperature for 80 minutes.
  • the column can be washed with buffer W (100 mM NaCL, 2.5 mM HEPES-KOH (pH7.5) and 1 .25 mM EDTA) preheated to 37 °C and loaded onto the column via the heat exchanger immersed in 37°C water. Wash can be continued until the UV absorbance of the wash flow through drops below 0.01 ⁇ 260 ⁇
  • the column can be eluted with 10 column volumes buffer E (20 mM NaCl; 0.5 mM HEPES-KOH (pH 7.5) and 0.25 mM EDTA) preheated to 65 ° C and loaded via 65 ° C heat exchanger.
  • tRNA 0.25 column volume fractions can be gathered and purified tRNA can be precipitated by addition of 1 /10 volume 3 M sodium acetate, and an equal volume of isopropanol. After 30 minute incubation at -80°C, tRNA can be concentrated by centrifugation at 20,000xg for 30 minutes, washed with 70% ethanol, air dried briefly, then resuspended at ⁇ 20mg/ml in 10 mM HEPES (pH 8.0). Refolding can be carried out by heating tRNA to 70 °C, adding MgCl 2 to 10 mM and then slow cooling to room temperature.

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Abstract

Cette invention concerne un moyen amélioré et peu coûteux pour transcrire un plasmide à ADN circulaire en ARN, soit ARNt ou ARNm. Le procédé met en jeu l'utilisation de séquences de terminaison de multiples ADN:ARN polymérases pour empêcher la perte de phosphates à haute énergie en raison d'une terminaison inefficace des polymérases.
PCT/US2012/040936 2011-06-08 2012-06-05 Cassettes de terminaison des multiples arn polymérases pour une transcription efficace d'échantillons d'arn discrets WO2012170433A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2014186334A1 (fr) * 2013-05-15 2014-11-20 Robert Kruse Traduction intracellulaire d'arn circulaire
US9445603B2 (en) 2013-03-15 2016-09-20 Monsanto Technology Llc Compositions and methods for the production and delivery of double stranded RNA

Citations (2)

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US6485934B1 (en) * 1997-04-23 2002-11-26 Nico Maurice August Corneel Mertens Regulatory system for inducible expression of genes with lambdoid promoters
US20100184135A1 (en) * 2009-01-12 2010-07-22 Sutro Biopharma, Inc. Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system

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Publication number Priority date Publication date Assignee Title
US6485934B1 (en) * 1997-04-23 2002-11-26 Nico Maurice August Corneel Mertens Regulatory system for inducible expression of genes with lambdoid promoters
US20100184135A1 (en) * 2009-01-12 2010-07-22 Sutro Biopharma, Inc. Mono charging system for selectively introducing non-native amino acids into proteins using an in vitro protein synthesis system

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Title
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HE ET AL.: "Characterization of an Unusual, Sequence-specific Termination Signal for T7 RNA Polymerase", J BIOL CHEM, vol. 273, no. 30, 24 July 1998 (1998-07-24), pages 18802 - 18881 *
MACDONALD ET AL.: "Characterization of Two Types of Termination Signal for Bacteriophage T7 RNA Polymerase", J MOL BIOL, vol. 238, no. 2, 29 April 1994 (1994-04-29), pages 145 - 158 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9445603B2 (en) 2013-03-15 2016-09-20 Monsanto Technology Llc Compositions and methods for the production and delivery of double stranded RNA
US10070652B2 (en) 2013-03-15 2018-09-11 Monsanto Technology Llc Compositions and methods for the production and delivery of double stranded RNA
US10757947B2 (en) 2013-03-15 2020-09-01 Monsanto Technology Llc Compositions and methods for the production and delivery of double stranded RNA
WO2014186334A1 (fr) * 2013-05-15 2014-11-20 Robert Kruse Traduction intracellulaire d'arn circulaire
US9822378B2 (en) 2013-05-15 2017-11-21 Ribokine, Llc Intracellular translation of circular RNA

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