WO2021058145A1 - Promoteurs de phage t7 pour amplifier la transcription in vitro - Google Patents

Promoteurs de phage t7 pour amplifier la transcription in vitro Download PDF

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WO2021058145A1
WO2021058145A1 PCT/EP2020/066070 EP2020066070W WO2021058145A1 WO 2021058145 A1 WO2021058145 A1 WO 2021058145A1 EP 2020066070 W EP2020066070 W EP 2020066070W WO 2021058145 A1 WO2021058145 A1 WO 2021058145A1
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rna
sequence
positions
polynucleotide
promoter
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PCT/EP2020/066070
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Thomas Conrad
Sascha Sauer
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Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft
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Publication of WO2021058145A1 publication Critical patent/WO2021058145A1/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • the present invention relates to a DNA polynucleotide and a method for in vitro transcribing (IVT) RNA using said DNA polynucleotide.
  • the DNA polynucleotide comprises (i) a T7 promoter sequence comprising (1) a T7 core promoter sequence and (2) a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising three guanines at positions +1 to +3, an adenine or thymine at position +4, at least two adenines at positions +4 to +8 and/or a thymine at position +4, and at most one cytosine at positions +4 to +8, and downstream thereof (ii) a nucleotide sequence encoding the RNA to be transcribed.
  • the method comprises (a) providing said DNA polynucleotide as an IVT template, and (b) in vitro transcribing said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.
  • RNAs have been considered as the major determinant for converting genomic information from DNA into biological function.
  • research has been focusing on the transcriptome of cells including RNAs that are translated into protein as well as regulatory RNAs.
  • Unraveling RNA biology and the biology of diseases associated with RNA dysregulation requires detection, quantification, and characterization of different types of RNAs.
  • RNAs exist with strongly varying copy numbers per cell and many approaches require a minimum amount of a given target RNA for further investigation.
  • approaches are of interest for amplification of RNAs that maintain the relative abundances of different RNAs present in the original cell or tissue.
  • IVT using a T7 promoter has been applied for low-amount DNA sequencing using linear amplification via transposon insertion (LIANTI) that uses the Tn5 transposon to fragment the genome and simultaneously insert a T7 RNA promoter for in vitro transcription (IVT) (Chen, C.
  • CRISPRRNAs or (single) guide RNAs can be used for disrupting or restoring the sequence of target genes, and antisense RNAs and/or small- interfering RNAs can be used for translation inhibition of target mRNAs.
  • the T7 promoter refers to a 23 nucleotides long sequence comprising a transcription start site (TSS) denoted position +1.
  • TSS transcription start site
  • the T7 RNA polymerase binds the promoter DNA from nucleotide position -17 to -5 with high specificity, while the DNA double strand is melted from -4 to +2 to prime RNA synthesis from a GTP nucleotide at +1.
  • sequences of the T7 core promoter in particular the region from -17 to -1, and the first three transcribed nucleotides (+1 to +3) can affect the transcriptional activity, i.e. the promoter strength (defined by relative production of transcripts from a promoter; Ikeda, Biol. Chem. 267,11322-8, 1992) and that sequences upstream of the T7 core promoter positively affect T7 polymerase binding affinity to an IVT DNA template (Tang et al., Biol. Chem. 280,40707-13, 2005).
  • the +1 and +2 nucleotides as guanines (e.g.
  • the present application addresses the need for optimized T7 promoter sequences and methods using said promoter sequences for enhanced amplification of antisense RNA (aRNA) and/or improved in vitro transcription of RNAs by providing the embodiments as recited in the claims.
  • aRNA antisense RNA
  • the present invention relates in preferred aspects to a DNA polynucleotide, a kit comprising said DNA polynucleotide, and a method for in vitro transcribing (IVT) RNA using said DNA polynucleotide.
  • said DNA polynucleotide comprises (i) a T7 promoter sequence comprising (1) a T7 core promoter sequence and (2) a directly adjacent downstream flanking region, and downstream thereof (ii) a nucleotide sequence encoding the RNA to be transcribed.
  • the downstream flanking region of said DNA polynucleotide comprises eight bases (+1 to +8) comprising ⁇ three guanines at positions +1 to +3 (GGG) ⁇ ,
  • the method comprises the steps of (a) providing said DNA polynucleotide as an IVT template, and (b) in vitro transcribing the RNA from said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.
  • the present invention relates in a further preferred aspect to a method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene, wherein the method comprises the steps of (a) reverse transcribing an RNA to the first strand of a cDNA comprising annealing a DNA polynucleotide according to the present invention, and (b) transcribing the cDNA into RNA using a T7 polymerase.
  • T7 promoters comprising
  • An optimized T7 promoter according to the present invention can be readily applied in various IVT-based methods. It is especially advantageous when aiming at boosting linear amplification of nucleic acids.
  • the T7 promoters of the invention comprising a defined +1 to +8 downstream flanking region ensure efficient amplification of target polynucleotides in a sample while avoiding a bias in IVT efficiency between different target polynucleotides.
  • Optimized T7 promoters can be further used for increasing the efficiency of the synthesis of RNA in vitro , e.g. to produce large amounts of unmodified or modified RNA or recombinant proteins, preferably for therapeutic applications.
  • An optimized T7 promoter is also highly valuable for IVT-based RNA-sequencing methods such as CEL-seq2, inDrop and MARS-seq as especially linear amplification of antisense RNA (aRNA) can be boosted and/or performed more accurately by using a reverse transcription primer comprising the optimized T7 promoter according to the invention instead of a commercially available reverse transcription primer.
  • aRNA antisense RNA
  • employing an inventive T7 promoter provided herein in a single cell RNA-seq method allows to detect more gene transcripts, i.e. mRNA of lowly expressed genes, for example of important cell state determinants such as transcription factors and chromatin modifiers.
  • an inventive T7 promoter provided herein, which is especially advantageous, for example, in case of analyzing transcriptomes on a single-cell level and/or detecting polynucleotides in liquid biopsies, i.e. in case raw material such as circulating tumor cells (CTC) is scarce and/or precious (Lawson et al., Nat. Cell Biol. 20,1349- 1360 (2018).
  • an optimized T7 promoter is also highly valuable for IVT-based DNA- sequencing methods such as LIANTI, CHIL-Seq, Dam-seq (e.g. scDam&T-seq) or the sci-L3 method.
  • the present invention relates to a DNA polynucleotide, comprising a T7 promoter comprising a T7 core promoter sequence and a directly adjacent downstream flanking region, wherein the downstream flanking region comprises eight bases (+1 to +8) comprising ⁇ three guanines at positions +1 to +3 ⁇ ,
  • brackets i.e. “ ⁇ “[ ]” and “ ⁇ >”, are used to specify relationships and layers within rules defined herein, with terms and expressions being grouped within an opening and a closing bracket of the same type, e.g. “ ⁇ ”.
  • brackets are to be understood as auxiliary means only for visually simplifying specific relationships and layers within rules defined herein.
  • polynucleotide refers to a polynucleotide of at least 13 nucleotides covalently bonded in a chain and thus, representing a sequence of nucleotides.
  • nucleotide refers to deoxyribonucleotides in case of DNA and cDNA (complementary DNA) molecules and to ribonucleotides in case of RNA molecules.
  • Nucleotides consist of a nitrogenous base (also known as nucleobase), a five-carbon sugar (ribose in case of RNA or deoxyribose in case of DNA and cDNA), and at least one phosphate group.
  • Nucleotides comprising as the respective nitrogenous base adenine, guanine, cytosine, thymine, and uracil are referred to as A, C, G, T and U nucleotides, respectively. Said nucleotides are preferred, though the term nucleotides can further comprise nucleotide analogues, such as peptide nucleic acids, morpholinos and/or locked nucleic acids, and/or modified nucleotides. Modified nucleotides include for example nucleotides comprising nucleobases that are methylated, formylated or hydroxylated nucleobases.
  • modified or analogous nucleobases include for example 5-methyl-cytosine, 5-hydroxymethyl- cytosine, N6-methyladenine, 7-methylguanine, 2-aminopurine, pyrrolo-dC, 5-bromouracil, and hypoxanthine.
  • polynucleotide refers to single- or double-stranded DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecule built up of A, C, G, T and/or U nucleotides and/or modified versions thereof.
  • RNA molecules include, but not limited to mRNA, IncRNAs, circular RNAs, miRNAs, snoRNAs, tRNAs, snRNAs, siRNAs, crRNAs and sgRNAs.
  • adenine refers not only to the respective nucleobase, but also to the respective nucleotide or nucleoside.
  • an “adenine”, “A” or “adenosine” refers to an “adenine”, “adenosine”, “adenosine triphosphate” (“ATP”), “adenosine diphosphate” (“ADP”) or “adenosine monophosphate” (“AMP”).
  • the polynucleotide of the invention is a polynucleotide, preferably a DNA polynucleotide, more preferably a double-stranded DNA polynucleotide.
  • a single-stranded polynucleotide consists of one nucleotide sequence and a double-stranded polynucleotide of two complementary nucleotide sequences.
  • the term “complementary” refers to nucleotides, the nitrogenous bases of which can naturally bind to each other by hydrogen bonds, i.e. A and T nucleotides, A and U nucleotides as well as C and G nucleotides. Both, a nucleotide and a nucleotide sequence have a 5’ (five prime) end and a 3’ (three prime) end based on the five carbon sites of sugar molecules in adjacent nucleotides.
  • a nucleotide sequence is read herein from its respective 5’ to its respective 3’end, wherein the term “downstream” refers to a nucleotide which is in 3’ direction of a reference nucleotide.
  • the term “upstream” refers to a nucleotide which is in 5’ direction of a reference nucleotide within a respective nucleotide sequence.
  • a nucleotide which is located downstream of a reference nucleotide within a respective nucleotide sequence can be directly, i.e. covalently, linked to the 3’ end of said reference nucleotide, which is referred to herein as “directly adjacent”.
  • a nucleotide which is located upstream of a reference nucleotide within a respective nucleotide sequence can be directly, i.e. covalently linked to the 5’ end of said reference nucleotide, which is also referred to herein as “directly adjacent”.
  • the polynucleotide, preferably the DNA polynucleotide, according to the present invention comprises a T7 promoter sequence comprising a T7 core promoter sequence and a directly adjacent downstream flanking region.
  • promoter and “promoter sequence” are used interchangeably herein and refer to a nucleotide sequence required for initiation of transcription of a target nucleotide sequence by DNA-binding enzymes such as RNA polymerase, transcription factors and/or epigenetic modifiers, e.g. histone acetylases, histone methylases or DNA-methylases.
  • DNA-binding enzymes such as RNA polymerase, transcription factors and/or epigenetic modifiers, e.g. histone acetylases, histone methylases or DNA-methylases.
  • the promoter is a T7 bacteriophage (Escherichia virus T7) promoter
  • the respective enzyme(s) binding to it preferably comprise a T7 RNA polymerase.
  • the T7 promoter and the T7 RNA polymerase are derived from a T7 bacteriophage and thus, may comprise the sequence of the naturally occurring T7 bacteriophage core promoter (e.g. SEQ ID NO:l) and RNA polymerase, respectively, and/or may comprise modifications compared to their respective naturally occurring sequence.
  • the T7 RNA polymerase may also be a recombinant protein and/or comprise altered post-translational modifications.
  • T7 promoter is used interchangeably with the term “T7 promoter sequence” and refers to a nucleotide sequence comprising a transcription start site (TSS) which is denoted as position +1. Positions downstream the TSS are denoted herein with a positive sign (“+”) and positions upstream of the TSS with a negative sign (“-”).
  • TSS transcription start site
  • the T7 promoter sequence comprises at least a T7 core promoter sequence and a directly adjacent downstream flanking region that is directly, i.e. covalently, linked to the 3’ end of said T7 core promoter sequence.
  • T7 core promoter sequence refers to the nucleotide sequence upstream of the TSS, more specifically to the nucleotide sequence from positions -1 to -17 and has the nucleotide sequence TAATACGACTCACTATA (SEQ ID NO:l).
  • the downstream flanking region comprises eight positions (positions +1 to +8 of the T7 promoter sequence) that have a nucleotide sequence composed according to certain rules.
  • the downstream flanking region comprises
  • the TSS is preferably a G nucleotide.
  • the two nucleotides at positions +2 and +3 are preferably G nucleotides.
  • the number and concrete position of certain nucleotides at positions +4 to +8 influence the activity of the T7 promoter.
  • at least three adenines or thymines at positions +4 to +8 are advantageous for enhancing the activity of the T7 promoter.
  • the nucleotides at positions +4 to +7 and especially at position +4 have a strong impact on the performance of the T7 promoter.
  • the absence of a cytosine is preferred within positions +4 to +7, in particular within positions +4 to +6 and/or in particular when the cytosine directly follows a thymine and/or directly precedes a guanine.
  • sequences of covalently linked thymines e.g. TT, TTT or TTTT
  • the combination of rules can result in an increased effect compared to sequences according to only one of the respective rules.
  • a negative effect of sequences of covalently linked thymines on T7 promoter activity is reduced in the absence of a guanine or cytosine at positions +4 to +8 or the presence of an adenine at position +4.
  • a negative effect of a cytosine within positions +4 to +6 on T7 promoter activity is reduced when at least three adenines are present at positions +4 to +8.
  • a guanine at position +5 has a positive effect on T7 promoter activity in combination with an adenine or thymine at position +4 and/or an adenine at position +6.
  • GGGAGA and GGGTGA at positions +1 to +6 can have positive effects on T7 promoter activity despite the presence of a guanine within positions +4 to +8.
  • the downstream flanking region further comprises at least two adenines at positions +4 to +8.
  • downstream flanking region further comprises
  • the downstream flanking region does not comprise three consecutive thymines.
  • downstream flanking region does not comprise three consecutive thymines within positions +4 to +8. In certain embodiments, the downstream flanking region does not comprise
  • the downstream flanking region does not comprise two consecutive thymines within positions +4 and +7.
  • the downstream flanking region does not comprise a thymine followed by a cytosine within positions +4 and +7.
  • the downstream flanking region does not comprise a cytosine within positions +4 to +6 when less than three adenines are present at positions +4 to +8.
  • the downstream flanking region does not comprise a cytosine within positions +4 to +6.
  • the downstream flanking region does not comprise a cytosine followed by a guanine within positions +4 to +7.
  • the downstream flanking region comprises ⁇ [at least three adenines or thymines at positions +4 to +8] and/or
  • cytosine or guanine is present at positions +4 to +8> or ⁇ a thymine, cytosine or guanine is present at position +4> ] ⁇ ,
  • downstream flanking region further comprises an adenine at position +4.
  • the downstream flanking region has the sequence GGGAGAGT.
  • the downstream flanking region further comprises at most one guanine or cytosine at positions +4 to +8.
  • an adenine at position +4 improves the T7 promoter strength compared to any other nucleotide at this position. It was further found, that downstream sequences are advantageous that do not comprise a guanine within positions +4 to +8 except in the case the nucleotide is covalently linked to an adenine in the context of an AGA (or TGA) nucleotide sequence within positions +4 to +6. Thus, more than one guanine or cytosine within positions +4 to +8 is preferably avoided for increasing the activity of the T7 promoter.
  • downstream flanking region comprises
  • cytosine or guanine is present at positions +4 to +8> or ⁇ a thymine, cytosine or guanine is present at position +4> ] ⁇ ,
  • downstream flanking region further comprises (an adenine at position +4 ⁇ and (at most one guanine or cytosine at positions +4 to +8 ⁇ .
  • the downstream flanking region is selected from the group consisting of GGGAAATA, GGGAAAAT, GGGAATAT, GGGATAAT, GGGAGAAT, GGGAATAC, GGGAAGTA, GGGAGATT, GGGAGATA, GGGAAATG, GGGAAAAC and GGGAAAGT.
  • position +8 has the smallest effect on the T7 promoter activity among positions +4 to +8.
  • the downstream flanking region is selected from the group consisting of GGGAAATN, GGGAAAAN, GGGAATAN, GGGATAAN, GGGAGAAN, GGGAATAN, GGGAAGTN, GGGAGATN, GGGAGATN, GGGAAATN, GGGAAAAN and GGGAAAGN; wherein N denotes A, C, G, T or U.
  • the downstream flanking region comprises three guanines at positions +1 to +3 ⁇ ,
  • ⁇ a cytosine or guanine is present at positions +4 to +8> or
  • the downstream flanking region comprises ⁇ three guanines at positions +1 to +3 ⁇
  • the downstream flanking region does not comprise a thymine at position +5 followed by a guanine at position +6.
  • the downstream flanking region does not comprise two consecutive thymines within positions +4 to +8.
  • the downstream flanking region does not comprise ⁇ a thymine at position +5 followed by a guanine at position +6 ⁇ and ⁇ two consecutive thymines within positions +4 to +8 ⁇ .
  • downstream flanking region comprises
  • downstream flanking region further comprises
  • the downstream flanking region is selected from the group consisting of GGGAAATA, GGGAAAAT, GGGAATAT, GGGATAAT, GGGAGAAT, GGGAATAC and GGGAAGTA.
  • the downstream flanking region is selected from the group consisting of GGGAAATN, GGGAAAAN, GGGAATAN, GGGATAAN, GGGAGAAN, GGGAATAN and GGGAAGTN; wherein N denotes A, C, G, T or U.
  • the downstream flanking region comprises ⁇ three guanines at positions +1 to +3 ⁇ ,
  • the downstream flanking region comprises (three guanines at positions +1 to +3 ⁇ ,
  • the downstream flanking region further comprises an adenine at position +4, three to four adenines at positions +4 to +8, and no guanines or cytosines at positions +4 to +8.
  • downstream flanking region comprises
  • downstream flanking region further comprises ⁇ an adenine at position +4 ⁇ and
  • downstream flanking region does not comprise
  • downstream flanking region further comprises
  • downstream flanking region has the sequence GGGATAAT.
  • the downstream flanking region has a sequence selected from the group consisting of GGGADDDN, wherein D denotes A, G or T/U, and wherein N denotes A, C, G, T or U.
  • the sequence of the downstream flanking region is selected from the group consisting of GGGADDWH, preferably from GGGAWWWW, wherein D denotes an A, G or T/U, wherein W denotes A or T/U, and wherein H denotes A, C or T/U.
  • the downstream flanking region comprises three guanines at positions +1 to +3, an adenine at position +4, three to four adenines at positions +4 to +8, no guanine or cytosine at positions +4 to +8, and no consecutive thymines within positions +4 and +8.
  • the downstream flanking region has the sequence GGGAAATA (see the T7 promoter sequence as set forth in SEQ ID NO:21), GGGAAAAT (see the T7 promoter sequence as set forth in SEQ ID NO:23), GGAATAT (see the T7 promoter sequence as set forth in SEQ ID NO:25), GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17), or GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19).
  • the downstream flanking region has the sequence GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17) or GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19), more preferably GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17).
  • the downstream flanking region is selected from the group consisting of GGGAAATN, GGGAAAAN, GGAATAN, GGGATAAN, or GGGAGAGN; wherein N denotes A, C, G, T or U.
  • the length of the primer may be as small as possible.
  • the T7 promoter of the invention may be used for improving a pre-existing protocol, it may be preferable to modify the pre-existing protocol as little as possible while profiting from the enhanced T7 promoter activity according to the invention.
  • the downstream flanking region of the inventive T7 promoter may overlap with the sequencing adapter further downstream thereof.
  • the pre-existing sequencing adapter may be part of the downstream flanking region, i.e. at positions +6 to +8 or +7 to +8.
  • the downstream flanking region further comprises the sequence AGT at positions +6 to +8, or the sequence AG at positions +7 to +8, preferably AGT at positions +6 to +8.
  • this rule may be combined with other rules for the composition of the downstream flanking region, as provided herein, as far as there is no conflict.
  • T7 promoters with such a downstream flanking region i.e. with AGT at positions +6 to +8) may be particularly useful in some embodiments of the inventive sequencing methods provided herein.
  • the downstream flanking region has the sequence GGGAAAGT, GGGTAAGT, GGGATAGT, GGGTGAGT, or GGGAGAGT, preferably GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19).
  • the downstream flanking region has the sequence GGGAAAAG, GGGAATAG, GGGATAAG, GGGTGAAG, GGGTAAAG, GGGAGAAG, GGGTGTAG, GGGAGTAG, GGGATTAG.
  • the downstream flanking region has the sequence GGGAAATA (see the T7 promoter sequence as set forth in SEQ ID NO:21), GGGAAAAT (see the T7 promoter sequence as set forth in SEQ ID NO:23), GGAATAT (see the T7 promoter sequence as set forth in SEQ ID NO:25), GGGATAAT (see the T7 promoter sequence as set forth in SEQ ID NO: 17), GGGAGAGT (see the T7 promoter sequence as set forth in SEQ ID NO: 19), GGGAAAGT, GGGTAAGT, GGGATAGT or GGGTGAGT, preferably GGGAGAGT.
  • the T7 promoter of the invention is selected primarily based on the 5’RACE-seq rank (Table 1).
  • the sequence of the downstream flanking region is selected from the group consisting of the “Pos. +1 to +8” sequences in Table 1 of ranks 1 to 510, ranks 1 to 176, ranks 1 to 132 or ranks 1 to 66, preferably ranks 1 to 66.
  • the sequence of the downstream flanking region is selected from the group consisting of the “Pos. +1 to +8” sequences in Table 1 of ranks 1 to 64, ranks 1 to 48, ranks 1 to 32, ranks 1 to 24, or ranks 1 to 12.
  • sequence of the downstream flanking region does not have the sequence GGGGTTCA, GGGAGTTC or GGGATACC. In some embodiments, the sequence of the downstream flanking region does not have the sequence GGGTAGAT.
  • nucleotide sequences comprising AATT, in particular GAATT, directly adjacent upstream of the T7 core promoter further enhance the T7 promoter strength, especially in combination with a downstream flanking region according to the present invention.
  • Further increasing the strength of an optimized T7 promoter sequence comprising a downstream flanking region as shown above by combining it with an upstream flanking region according to the present invention is particularly advantageous for approaches based on scarce and/or precious raw material (e.g. liquid biopsies) such as in case of in vitro transcription (IVT) when the concentration of the IVT template is low, e.g. very low as described herein, and/or analyses are performed, for example, on a single-cell level, or on less than 100 cells, preferably less than 10 cells, very preferably a single cell.
  • upstream flanking region refers to the sequence directly adjacent upstream of the T7 core promoter sequence and thus, covalently bound to its 5’ end.
  • the upstream flanking region comprises 4 or 5 nucleotides, preferably 5 nucleotides.
  • the upstream flanking region has the sequence AATT, GAATT, GATTT or GAAAT, preferably AATT or GAATT, even more preferably GAATT (see e.g. the T7 promoter sequences as set forth in SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26).
  • the T7 promoter further comprises four bases consisting of adenines and/or thymines directly upstream of the T7 core promoter.
  • it further comprises a guanine directly upstream of said adenines and/or thymines.
  • the T7 promoter comprises a downstream flanking region of the invention and an upstream flanking region of the invention.
  • the T7 promoter further comprises the sequence AATT directly upstream of the T7 core promoter sequence.
  • the T7 promoter further comprises the sequence GAATT directly upstream of the T7 core promoter sequence.
  • the DNA polynucleotide according to the present invention can further comprise downstream of the T7 core promoter sequence a nucleotide sequence encoding an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPRRNA.
  • a nucleotide sequence encoding an mRNA or non-coding RNA preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPRRNA.
  • the terms “encoding” or “encode” refer to genetic information comprised in a DNA sequence that can be transcribed into an RNA molecule, i.e. that can be used as a template for synthesis of an RNA molecule.
  • nucleotide sequence preferably refers to the strand that comprises the coding strand.
  • a coding strand comprises a sequence identical to the RNA it encodes.
  • a sequence is identical to another sequence if each nucleotide at each position is the same, except that a thymine can be in place of an uracil and an uracil can be in place of a thymine according to the nucleotides comprised in DNA and RNA, respectively.
  • the encoded RNA can be an in-vitro transcribed RNA according to the invention, in particular an mRNA or a non-coding RNA.
  • mRNA molecule and “mRNA” are used interchangeably and refer to a class of RNA molecules, preferably single-stranded sequences built up of A, C, G, and U nucleotides that contain one or more coding sequences. Said one or more coding sequences can be used for example as a translation template during synthesis of an amino acid sequence and thus, for converting genomic information stored in an mRNA molecule into an amino acid sequence.
  • mRNA should be understood to mean any RNA molecule which is suitable for the expression of an amino acid sequence or which is translatable into an amino acid sequence such as a protein.
  • an mRNA comprises preferably a 5’ UTR, a coding sequence and a 3’ UTR.
  • the 5’ end of the 5’ UTR is defined by the TSS and its 3’ end is followed by the coding sequence.
  • the coding sequence is delineated by the start and the stop codon, i.e. the first and the last three nucleotides of the mRNA that can be translated, respectively.
  • the 3’ UTR starts after the stop codon of the coding sequence and can be followed by a poly A tail.
  • the 5’ UTR generally comprises at least one ribosomal binding site (RBS) such as the Shine- Dalgarno sequence in prokaryotes and the Kozak sequence or translation initiation site in eukaryotes.
  • RBS ribosomal binding site
  • the activity of a given RBS can be optimized by varying its length and sequence as well as its distance to the start codon.
  • the 5’ UTR comprises internal ribosome entry sites or IRES.
  • a 5’ UTR can comprise one or more additional regulatory sequences such as a binding site for an amino acid sequence that enhances the stability of the mRNA, a binding site for an amino acid sequence that enhances the translation of the mRNA, a regulatory element such as a riboswitch, a binding site for a regulatory RNA molecule such as an miRNA, and/or a nucleotide sequence that positively affects initiation of translation.
  • additional regulatory sequences such as a binding site for an amino acid sequence that enhances the stability of the mRNA, a binding site for an amino acid sequence that enhances the translation of the mRNA, a regulatory element such as a riboswitch, a binding site for a regulatory RNA molecule such as an miRNA, and/or a nucleotide sequence that positively affects initiation of translation.
  • the mRNA may optionally comprise a 3’ UTR.
  • the 3’ UTR may comprise one or more regulatory sequences such as a binding site for an amino acid sequence that enhances the stability of the mRNA molecule, a binding site for a regulatory RNA molecule such as a miRNA, and/or a signal sequence involved in intracellular transport of the mRNA.
  • the coding sequence of an mRNA comprises codons that can be translated into an amino acid sequence.
  • the coding sequence can contain the codons of a naturally occurring coding sequence or it can be a partially or completely synthetic coding sequence. Alternatively, the coding sequence can be a partly or fully codon optimized sequence derived from the natural sequence to be used. Most of the amino acids are encoded by more than one codon, i.e. three consecutive nucleotides of an mRNA that can be translated into an amino acid. Codons exist that are used preferentially in some species for a given amino acid.
  • codons can enhance the amount of amino acid sequences translated based on a given mRNA molecule compared to the same mRNA molecule but comprising comparably rare codons.
  • amino acid sequence encompasses any kind of amino acid sequence, i.e. chains of two or more amino acids which are each linked via peptide bonds and refers to any amino acid sequence of interest.
  • the encoded amino acid sequence is at least 5 amino acids long, more preferably at least 10 amino acids, even more preferably at least 50, 100, 200 or 500 amino acids.
  • amino acid sequence covers short peptides, oligopeptides, polypeptides, fusion proteins, proteins as well as fragments thereof, such as parts of known proteins, preferably functional parts.
  • the function of the encoded amino acid sequence in the cell or in the vicinity of the cell is needed or beneficial, e.g. an amino acid sequence the lack or defective form of which is a trigger for a disease or an illness, the provision of which can moderate or prevent a disease or an illness, or an amino acid sequence which can promote a process which is beneficial for the body, in a cell or its vicinity.
  • the encoded amino acid sequence can be the complete amino acid sequence or a functional variant thereof.
  • the encoded amino acid sequence can act as a factor, inducer, regulator, stimulator or enzyme, or a functional fragment thereof, where this amino acid sequence is one whose function is necessary in order to remedy a disorder, in particular a metabolic disorder or in order to initiate processes in vivo such as the formation of new blood vessels, tissues, etc.
  • functional variant is understood to mean a fragment which in the cell can undertake the function of the amino acid sequence whose function in the cell is needed or the lack or defective form whereof is pathogenic.
  • such an amino acid sequence is advantageous with respect to applications in supplemental or medical purposes to generate or regenerate physiological functions caused by suboptimal amino acid sequence biosynthesis and thus also to favorably influence directly or indirectly the course of diseases.
  • a preferred coding sequence downstream of the T7 core promoter encodes a transposase or a genome editing enzyme.
  • a transposase refers to an enzyme that binds to the end of a transposon and catalyzes its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism.
  • Preferred transposases include Sleeping Beauty transposases and PiggyBac transposases.
  • a genome editing enzyme refers to an enzyme which recognizes a specific nucleotide sequence, preferably a DNA sequence, and cuts the nucleotide sequence at or nearby the recognition position.
  • Some genome editing enzymes in particular CRISPR associated proteins, such as, but not limited to Cas9, require a polynucleotide, for example, a CRISPR RNA or guide RNA, which guides them to the specific target sequence.
  • Some genome editing enzymes, in particular ZNFs and TALENs recognize the specific target sequence by themselves.
  • Preferred genome editing enzymes include zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and CRISPR-associated proteins such as Cas9, Casl2b or Cpfl, preferably Cas9.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector-based nucleases
  • CRISPR-associated proteins such as Cas9, Casl2b or Cpfl, preferably Cas9.
  • a CRISPR-associated protein is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence.
  • Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can target specific desired DNA sequences, which enables zinc-finger nucleases to target unique sequences even within complex genomes.
  • the DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs.
  • the non-specific cleavage domain from the type IIs restriction endonuclease Fokl is typically used as the cleavage domain in ZFNs.
  • Transcription activator-like effector nucleases are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA- binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands).
  • Transcription activator-like effectors can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations.
  • the DNA binding domain contains a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition.
  • RVD Repeat Variable Diresidue
  • non-coding RNA refers preferably to regulatory RNA such as microRNA (miRNA), small-interfering RNA (siRNA), antisense RNA, CRISPR RNA (crRNA), single guide RNA (sgRNA), guide RNA (gRNA), antagoNAT, or a precursor thereof such as pri-miRNA, pre-miRNA or short-hairpin RNA (shRNA).
  • miRNA microRNA
  • siRNA small-interfering RNA
  • crRNA CRISPR RNA
  • sgRNA single guide RNA
  • gRNA guide RNA
  • antagoNAT antagoNAT
  • a precursor thereof such as pri-miRNA, pre-miRNA or short-hairpin RNA (shRNA).
  • a microRNA refers to a small non-coding RNA molecule (comprising about 22 nucleotides) that functions in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function via base-pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules cleaved, destabilized by shortening the polyA tail of the mRNA and/or less efficiently translated into proteins by ribosomes. Hence, microRNA molecules can reduce the amount of protein produced by a respective mRNA.
  • small interfering RNA also known as short interfering RNA or silencing RNA refer to double-stranded RNA molecules of 20 to 25 nucleotides in length. siRNA molecules are involved in the degrading mRNA after transcription, thus preventing their translation into proteins.
  • An antisense RNA refers to a single stranded RNA that is complementary to another polynucleotide.
  • an antisense RNA is a non-coding RNA.
  • an antisense RNA can hybridize to the complementary polynucleotide.
  • the complementary polynucleotide is an RNA, preferably an mRNA.
  • the antisense RNA is used for inhibiting the translation of a complementary mRNA into protein by hybridization of the two RNAs. Preferably, the inhibition occurs within a living cell.
  • the antisense RNA is used for detecting a further polynucleotide by hybridization of the RNA to the polynucleotide.
  • the polynucleotide to be detected is an RNA, preferably an mRNA.
  • the hybridization can occur outside or within a cell.
  • said cell is fixed.
  • An antisense RNA which is used for detecting another polynucleotide is also termed “probe”.
  • the antisense RNA is an amplified antisense RNA which is generated in the linear amplification method according to the invention.
  • the amplified antisense RNA may be transcribed from a double-stranded DNA.
  • the amplified antisense RNA is transcribed from a cDNA.
  • the cDNA has been reverse-transcribed from an RNA, preferably an mRNA, by using the DNA polynucleotide of the invention as reverse transcription primer.
  • RNA refers to an amplified antisense RNA.
  • An antagoNAT refers to a single stranded oligonucleotide which can inhibit a naturally occurring antisense RNA and thus, can be used for increasing gene expression, in particular by antagonizing said antisense RNA.
  • CRISPR refers to a “clustered regularly interspaced short palindromic repeats” sequence, and in particular to a CRISPR RNA (crRNA) and a single-guide RNA (sgRNA).
  • crRNA CRISPR RNA
  • sgRNA single-guide RNA
  • a crRNA comprises a guide RNA which is an approximately 20 nucleotides long sequence and which guides genome editing enzymes such as CRISPR associated proteins to the respective nucleotide sequence to be edited which is preferably comprised in a double-stranded genomic DNA molecule.
  • CRISPR associated proteins further require a trans-activating CRISPR RNA (tracrRNA), which can be provided as a single guide RNA (sgRNA) and thus a fusion product comprising at least one crRNA and a trans-activating CRISPR RNA (tracrRNA).
  • the DNA polynucleotide according to the present invention can comprise downstream of the T7 core promoter sequence a nucleotide sequence encoding an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA or CRISPR RNA, wherein the said nucleotide sequence is not, partially, or completely overlapping with the downstream flanking region directly adjacent to the T7 core promoter sequence.
  • said nucleotide sequence is not or partially overlapping with the downstream flanking region directly adjacent to the T7 core promoter sequence.
  • the DNA polynucleotide according to the present invention can further comprise a polyT- stretch, wherein the polyT-stretch is a nucleotide sequence of 9 to 35 consecutive thymines, preferably 22 to 27 consecutive thymines, more preferably 24 consecutive thymines, preferably followed by an adenine, cytosine or guanine, preferably at the 3’ end of the DNA polynucleotide.
  • polyT-stretch refers to a nucleotide sequence consisting of consecutive, i.e. covalently linked thymines. Said polyT-stretch is especially advantageous for using the DNA polynucleotide as a reverse transcription primer for reverse transcription of an RNA, e.g. an mRNA, into a cDNA.
  • a “primer”, as used herein, refers to a poly- or oligonucleotide which comprises a sequence complementary to a sequence comprised in another poly- or oligonucleotide.
  • a primer can be used for example for PCR, reverse transcription and/or the synthesis of the complementary strand of a DNA molecule.
  • the DNA polynucleotide according to the present invention comprises downstream of the T7 core promoter sequence a polyT-stretch.
  • Embodiments comprising the DNA polynucleotide and a polyT-stretch are especially advantageous for use as a reverse transcription primer.
  • the DNA polynucleotide according to the present invention can further comprise a polyT, and a sequencing adapter, wherein the sequencing adapter is a nucleotide sequence comprising a nucleotide sequence comprised in a library preparation primer, wherein the library preparation primer comprises a flow cell binding sequence, which is complementary to an oligo- or polynucleotide present at the surface of the flow cell of a next-generation-sequencing device, and wherein the sequencing adapter does not comprise more than eight consecutive thymines and/or only thymines.
  • a sequencing adapter refers to a polynucleotide which functions as a binding sequence for a polymerase chain reaction (PCR) primer and/or library preparation primer.
  • a library preparation primer comprises a flow cell binding sequence (FCBS) and a sequence which is identical or highly similar to a sequence comprised in the sequencing adapter, such that the LPP can bind to the complementary sequence of the sequencing adapter.
  • FCBS flow cell binding sequence
  • a flow cell binding site refers to an oligo- or polynucleotide which binds to a complementary sequence immobilized at the surface of a flow cell.
  • a flow cell is a part of a sequencing device that can be loaded with the material to be sequenced, wherein a sequencing device refers to a device for determining the sequence of a polynucleotide.
  • the sequencing adapters are sequencing adapters used in next- generation sequencing methods comprising library preparation methods and in particular Illumina library preparation protocols.
  • a preferred sequencing adapter sequence is a sequence comprised in an Illumina library preparation primer such as PEI, PE2 or PE2-N6 and suitable flow cell binding sequences are for example comprised in Illumina P5 and P7 oligo- or polynucleotides.
  • an oligonucleotide as used herein, the same applies as it has been described in the context of a polynucleotide, except its length.
  • a polynucleotide can comprise more than two, preferably 24 or more, or even up to 250 nucleotides
  • an oligonucleotide is to be understood as a sequence of two to 500, preferably 2 to 250 covalently linked nucleotides.
  • the other features of such an oligonucleotide can be as described above.
  • the DNA polynucleotide according to the present invention comprises a T7 promoter sequence and a sequencing adapter. This is especially advantageous for use of the DNA polynucleotide as a reverse transcription primer in approaches that comprise a sequencing step following reverse transcription of a nucleotide sequence encoding for example an mRNA, sgRNA, guide RNA or CRISPR RNA.
  • the sequencing adapter can optionally at least partially overlap with the T7 promoter sequence and/or the polyT-stretch.
  • the sequencing adapter is directly adjacent downstream of the T7 promoter sequence.
  • the DNA polynucleotide according to the present invention further comprises a barcode and/or a unique molecular identifier (UMI).
  • a barcode refers to an oligonucleotide which can be used to uniquely label e.g. an RNA of a specific sample, in particular of one specific cell.
  • An UMI refers herein to an oligonucleotide which can be used to specifically label one specific RNA molecule.
  • the DNA polynucleotide comprises a sequencing adapter downstream of the T7 core promoter and a polyT-stretch downstream of the sequencing adapter and not overlapping with the sequencing adapter.
  • the present invention relates to a method for in vitro transcribing (IVT) RNA, said method comprising the steps of (a) providing a DNA polynucleotide according to the present invention as an IVT template and downstream thereof a nucleotide sequence encoding the RNA to be transcribed, and (b) in vitro transcribing said IVT template in the presence of a T7 polymerase and ribonucleotide triphosphates.
  • IVTT in vitro transcribing
  • IVT refers to a process, wherein RNA is produced in vitro and thus, in a system that is neither a cell nor comprised in a cell.
  • the term “cell” refers to a living organism or a unit of a multicellular organism or an explant thereof which can preferably be cultured.
  • In vitro transcription requires an IVT template comprising a promoter and a template sequence downstream thereof, as well as ribonucleotide triphosphates and an RNA polymerase.
  • IVT can be used for linear amplification of a polynucleotide.
  • linear amplification refers to increasing the amount of a polynucleotide over time in any relationship which resembles more a linear relationship than an exponential relationship at least for a certain period of time, for example 1 min, 10 min, 0.5 h, 1 h, 5 h, 10 h or 1 day.
  • Linear amplification is advantageous over exponential amplification methods such as PCRfor certain applications, for example, but not limited to single-cell sequencing, in particular by reducing the amplification bias. Reducing an amplification bias is especially important when the starting material is scarce, such as the material obtained from a few cells or a single cell.
  • the in vitro transcribed RNA is linearly amplified during IVT.
  • linear amplification of a polynucleotide comprises the amplification of the polynucleotide as RNA or DNA having the identical and/or complementary sequence of said polynucleotide.
  • IVT in vitro transcribing
  • the DNA polynucleotide of the present invention is used as the IVT template.
  • the IVT template the same applies as it has been described above in connection with the DNA polynucleotide according to the present invention.
  • the other features of such an IVT template can be as described above.
  • the DNA polynucleotide provided in step (a) comprises a T7 promoter sequence comprising a T7 core promoter sequence and a directly adjacent downstream flanking as defined above and a nucleotide sequence encoding the RNA to be transcribed, wherein said RNA is preferably an mRNA or non-coding RNA, preferably an mRNA, antisense RNA, siRNA, sgRNA, guide RNA and/or CRISPR RNA.
  • the nucleotide sequence encoding the RNA to be transcribed is an mRNA that encodes a transposase or a gene editing enzyme.
  • the transposase is a Sleeping Beauty transposase.
  • the genome editing enzyme is a CRISPR enzyme, for example, Cas9, Casl2b or Cpfl.
  • the DNA polynucleotide which is used as an IVT template is particularly suitable for large-scale synthesis of RNA in vitro.
  • the IVT template is preferably linear, doubled-stranded and/or purified, even more preferably double-stranded, linearized and purified.
  • the DNA nucleotide of the invention is comprised in a plasmid such as a cloning and/or an expression vector.
  • a linear IVT template can be obtained, for example, by linearizing a DNA plasmid comprising the IVT template by cutting the DNA plasmid using a restriction enzyme.
  • a plasmid or vector, as used herein, refers to a double-stranded DNA molecule which can replicate within a bacterial host, preferably an E. coli strain.
  • a plasmid or vector preferably comprises an origin of replication, a selection marker, such as a gene conferring resistance to an antibiotic, a cloning site such as restriction enzyme site, a recombination region, a promoter, an enhancer, a transcription termination site, a ribosomal binding site, a Shine Dalgarno and/or a Kozak sequence.
  • a selection marker such as a gene conferring resistance to an antibiotic
  • a cloning site such as restriction enzyme site, a recombination region, a promoter, an enhancer, a transcription termination site, a ribosomal binding site, a Shine Dalgarno and/or a Kozak sequence.
  • Suitable plasmids as backbone are, for example, but not limited to pT7 FLAG (Merck), pT7 MAT (Merck), GatewayTM pDESTTM (Thermo Fisher) and pRSET (Thermo Fisher) of derivatives thereof.
  • purification of the preferably linear and/or doubled- stranded IVT template is preferably performed using a spin-column such as a Monarch® PCR & DNA Cleanup Kit.
  • a spin-column such as a Monarch® PCR & DNA Cleanup Kit.
  • in vitro transcribing the IVT template is performed in the presence of ribonucleotide triphosphates which are preferably adenosine, uridine, cytidine and guanosine triphosphates or modified versions thereof.
  • the T7 polymerase is preferably a T7 polymerase that is capable of binding to the T7 promoter.
  • In vitro transcribing the IVT template comprises using the IVT template as a template during transcription, i.e. the synthesis of an RNA molecule.
  • said RNA has a nucleotide sequence that is identical to the sequence of the IVT template (identical to the coding strand and complementary to the template strand of the IVT template) except that instead of T nucleotides U nucleotides are comprised in the synthesized RNA molecule.
  • RNA is used as a template for the synthesis of a cDNA whereof the first strand has a sequence complementary to the sequence of the RNA and the second strand a sequence which is identical to the RNA except that instead of U nucleotides T nucleotides are comprised in the synthesized cDNA molecule.
  • the DNA polynucleotide which is used as an IVT template is present in a high concentration in step (b), wherein the term “a high concentration” refers to a concentration of more than 30 ng/m ⁇ , preferably of more than 40 ng/pl.
  • very low concentration refers to a concentration of less than 1 ng/pl, preferably of less than 100 pg/pl, e.g. less than 50 pg/pl, and even more preferably of less than 10 pg/pl, in particular less than 1 pg/pl, or even less than 0.1 pg/pl, e.g. 0.05 pg/pl.
  • a very low concentration is more than 0, e.g. at least 0.005 pg/pl, 0.01 pg/pl or 0.05 pg/pl.
  • the term “pg” refers to “picogram”, the term “ng” to “nanogram”, and the terms “pi” or “ul” to “microliter”.
  • the DNA polynucleotide which is used as an IVT template is present in a very low concentration in step (b), wherein the term “a very low concentration” refers to a concentration of less than 1 ng/pl, preferably of less than 100 pg/pl, e.g. less than 50 pg/pl and even more preferably of less than 10 pg/pl, in particular less than 1 pg/pl, or even less than 0.1 pg/pl, e.g. 0.05 pg/pl.
  • Said DNA polynucleotide or IVT template may be a mix of different DNA molecules, e.g.
  • each of said DNA molecules comprises the T7 promoter of the invention, i.e. the same T7 core promoter and the same inventive upstream and downstream flanking regions.
  • Said mix of DNA molecules may be obtained, for example, by reverse transcribing the mRNA from a few cells, i.e. less than 100 cells, preferably less than 10 cells, or, very preferably, from a single cell, and/or a liquid biopsy, with the reverse transcription primer comprising the inventive T7 promoter provided herein.
  • the resulting cDNA (IVT template) of the single cells (or different samples) may be pooled if the reverse transcription primers have a barcode as described herein, but the concentration may still be very low as defined herein.
  • the same principle applies to synthesizing the complementary strand of a target DNA polynucleotide, i.e. in a PCR reaction, from such few cells or a single cell and/or a liquid biopsy with a primer comprising the inventive T7 promoter and using the resulting library of DNA polynucleotides, i.e. double-stranded DNA molecules, which have incorporated said T7 promoter as IVT template, as described herein.
  • the primers used for the PCR reaction may be complementary to different target DNA molecules, and thus the resulting IVT template may be a mix of different DNA molecules comprising the same inventive T7 promoter, which may be present at a very low concentration, as described herein.
  • the IVT template comprising the T7 promoter of the invention may be obtained from at least one target polynucleotide that is present freely in a body fluid, such as blood, urine or cerebrospinal fluid.
  • a body fluid such as blood, urine or cerebrospinal fluid.
  • the IVT template may be obtained by reverse-transcription, and in case said target polynucleotide is a DNA, the IVT template may be obtained by synthesizing the complementary strand, i.e. in a PCR reaction, as described herein.
  • the IVT template may be also obtained by attaching and/or integrating the DNA polynucleotide of the invention harboring the inventive T7 promoter to the target polynucleotide via a transposon system i.e. Tn5 mediation transposition, as described herein.
  • the IVT template may be present at a very low concentration as described herein.
  • a target polynucleotide e.g. a mRNA
  • a target polynucleotide or at least one target polynucleotide refers, in particular, to at least one polynucleotide to be analyzed in a sample, e.g. one or a few cells, a liquid biopsy, and/or a body fluid.
  • the individual molecules of the target polynucleotide are not necessarily fully identical although they comprise the same or a very similar nucleotide sequence to which the reverse transcription primer or primer comprising the T7 promoter of the invention can bind.
  • a very similar nucleotide sequence in this context may have at most 5, 4, 3, 2 or 1 nucleotide mismatches, e.g. due to SNPs, deletions and/or insertions, compared to the reference sequence which is complementary to a sequence comprised in the reverse-transcription primer or primer, and allows binding of the (reverse transcription) primer.
  • a target polynucleotide may refer to the mRNA molecules with a poly-A-tail in a sample, wherein the reverse transcription primer comprising a poly-T-stretch as provided herein binds to said poly-A-tail.
  • a target polynucleotide may also refer, for example, to a polynucleotide comprising a unique nucleic acid sequence, and the reverse-transcription primer or primer comprising the complementary sequence of said unique sequence binds to said unique sequence.
  • the target polynucleotide and the reverse-transcription primer or primer may be a mix of different polynucleotides, wherein the target polynucleotide is a plurality of polynucleotides which may comprise different unique nucleic acid sequences to which the reverse-transcription primer or primer mix binds.
  • the target polynucleotide refers to a genomic DNA sequence to which the DNA polynucleotide of the present invention comprising the inventive T7 promoter is attached and/or integrated (i.e. covalently linked via the DNA backbone).
  • said attachment and/or integration may be mediated by a transposase (transposon system), i.e a Tn5 transposase, e.g. as described in Harada (2019), Nat. Cell Biol. 21.
  • the DNA polynucleotide of the invention further comprises a transposase binding sequence, i.e.
  • Tn5 transposase binding sequence which may be also known in the context of the Tn5 transpose as a “mosaic end”.
  • Said transposase binding sequence is preferably downstream of the +1 to +8 downstream flanking region of the T7 promoter provided herein.
  • the individual molecules of the target polynucleotide may be structurally unrelated, wherein the target polynucleotide refers to genomic DNA sequences which are in proximity (e.g. within 5000 bp, 1000 bp, 500 bp, 200 bp or 100 bp, preferably within 200 bp) to a certain chromatin modification, e.g.
  • the DNA polynucleotide may be conjugated to a molecule (e.g. an antibody) which binds to a certain DNA binding molecule, as described herein.
  • a molecule e.g. an antibody
  • the DNA polynucleotide of the invention may be conjugated to an antibody, e.g.
  • ChIL-Seq is a suitable, non-limiting, example for using the T7 promoter of the invention for transposon-mediated attachement/integration to genomic DNA sites (target polynucleotides) (Harada (2019), Nat. Cell Biol. 21).
  • a T7 promoter sequence is fused at the 5’end to an Illumina adapter and a Tn5 recognition sequence and covalently coupled at the 3’end to an antibody with specificity for chromatin components i.e. specific histone modifications, and the Tn5 recognition sequence is loaded with Tn5 transposase.
  • the cells e.g. 100-1000 cells, or a single cell
  • containing the target polynucleotide of interest are then fixed, permeabilized and incubated with the Antibody-T7 promoter-Tn5 complex.
  • the DNA polynucleotide harboring the T7 promoter sequence Upon binding of the antibody to its target epitopes on the chromatin (DNA-binding molecules), the DNA polynucleotide harboring the T7 promoter sequence is covalently linked to the DNA backbone via Tn5 mediated transposition. The opposite DNA strand is repaired by incubation of the cells with DNA ligase, and DNA sequences adjacent to the integration sites (IVT template) are then in vitro transcribed into RNA by T7 polymerase. Conversion of the in vitro transcribed and amplified RNA into sequencing libraries followed by deep sequencing enables identification and/or quantification of the genomic antibody binding sites (i.e the target polynucleotide(s) in proximity to the DNA binding molecules that are recognized by the antibody).
  • a target polynucleotide or at least one target polynucleotide refers, in particular, to those polynucleotide molecules (e.g. in a sample) which are converted to an IVT template (comprising a T7 promoter according to the invention) as described herein (e.g. by reverse-transcription and/or synthesis of the complementary strand, or transposase-mediated integration/attachment). Therefore, the concentration, e.g. the very low concentration, of a target polynucleotide or at least one target polynucleotide, as used herein, refers, in particular, to the total concentration of those polynucleotide molecules (e.g. in a sample) which are converted to an IVT template.
  • the concentration of the IVT template is similar as the concentration of the corresponding target polynucleotide or at least one target polynucleotide, wherein “similar” may include a 5- fold, 3-fold, 2-fold, 1.5-fold, 1.2-fold, 1.1-fold or 1.05-fold difference, preferably a 2-fold or 1.5-fold difference.
  • a 2-fold difference is 10 pg/m ⁇ vs. 20 pg/m ⁇ , or 10 pg/m ⁇ vs. 5 pg/m ⁇ ; and a 1.5-fold difference is 10 pg/m ⁇ vs. 15 pg/m ⁇ , or 10 pg/m ⁇ vs. 6.66 pg/m ⁇ .
  • the DNA polynucleotide when the inventive DNA polynucleotide is present in a very low concentration, i.e. in step (b) of the inventive IVT method provided herein, the DNA polynucleotide comprises preferably an upstream flanking region as described above in the context of the upstream region of a DNA polynucleotide according to the present invention.
  • the in vitro transcribed RNA is a probe for hybridization with another RNA or DNA.
  • the probe is used for an in-situ hybridization method or southern blot.
  • the in-situ hybridization method is FISH.
  • the in vitro transcribed RNA is to be used as a FISH probe, it is transcribed in the presence of fluorescently labeled ribonucleotides.
  • a probe may be an antisense RNA which is complementary to the target oligo- or polynucleotide, for example an mRNA.
  • the RNA is in vitro transcribed in the presence of a capping and/or polyA-tailing enzyme.
  • a 5’ cap is added at the 5’ end and/or a 3’ polyA tail is added at the 3’ end of the in vitro transcribed RNA.
  • a 5’ -cap preferably refers to a 7-methyl guanosine (m7G) cap structure at the 5 ' end of an mRNA.
  • the capping may be performed by enzyme-based capping following the transcription reaction (posttranscriptional capping) and/or by incorporation of a cap analog during transcription (co-transcriptional capping).
  • Suitable cap analogues include 7-methyl guanosine (m7G) and 3 ' O-me 7-meGpppG cap analog (ARC A).
  • ARCA is methylated at the 3' position of the m7G, preventing RNA elongation by phosphodiester bond formation at this position.
  • transcripts synthesized using ARCA contain 5 ' -m7G cap structures in the correct orientation, with the 7-methylated G as the terminal residue.
  • Suitable for posttranscriptional capping is, for example, the Vaccinia Capping System.
  • a poly-A-tail may be added during transcription and/or after transcription.
  • a reverse PCR primer comprising a polyT-stretch can be used for amplifying the template sequence.
  • the polyA tail can be also added after transcription using e.g. an E. coli polyA polymerase. The length of the added tail can be adjusted by titrating the polyA polymerase.
  • a polyA tail refers to a sequence comprising, preferably 5 to 300, covalently linked adenines.
  • a 5’ cap is added at the 5’ end and a 3’ polyA tail is added at the 3’ end of the in vitro transcribed RNA.
  • the 5’ cap and/or the 3’ polyA tail is added while the RNA is in vitro transcribed.
  • the 5’ cap and/or the 3’ polyA tail is added after the RNA is in vitro transcribed.
  • the polyA tail is transcribed from an IVT template comprising a polyT- stretch at the 3 ’ end of the IVT template.
  • the RNA is purified before and/or after a 5’ cap and/or a 3’ polyA tail is added.
  • the nucleotide sequence encoding the RNA to be transcribed is an mRNA and the mRNA synthesized in vitro in step (b) is used to produce a recombinant protein in vitro or within a cell after providing an IVT mRNA.
  • the synthesis of the recombinant protein occurs in vitro.
  • the nucleotide sequence encoding the RNA to be transcribed is an mRNA and the mRNA synthesized in vitro in step (b) is then transfected into a cultured cell.
  • a cultured cell can be a mammalian cell line, an insect cell line, a yeast or a bacterium.
  • the cultured cell is a bacterium, even more preferably a cultured cell from E. coli.
  • Transfection of a cell with an mRNA is particularly advantageous to only temporarily express the protein encoded by the mRNA in the cell and/or expressing said protein without changing the genome of the cell.
  • Temporary expression is especially advantageous in case of a genome editing enzyme as the risk of unspecific genome-editing activity can be reduced.
  • DNA polynucleotide according to the present invention can thus be used for producing, e.g. a recombinant, protein.
  • IVT is advantageous for amplifying a polynucleotide, wherein the amplified polynucleotide is DNA or RNA.
  • the polynucleotide to be amplified is RNA which is reverse transcribed into cDNA which in turn is amplified by IVT.
  • the RNA is an mRNA which is amplified as antisense RNA (aRNA).
  • the present invention relates to a method for transcribing RNA within a cultured cell, said method comprising the steps of (a) providing a DNA polynucleotide according to the present invention comprising a T7 promoter sequence and downstream thereof a nucleotide sequence encoding the RNA to be transcribed and (b) introducing the DNA polynucleotide into a cultured cell expressing a T7 polymerase.
  • the RNA which is transcribed in a cultured cell is an mRNA which is further translated into a protein within the cultured cell.
  • the cultured cell wherein the RNA is transcribed is a bacterium.
  • the present invention relates to a cultured cell expressing a T7 polymerase comprising a DNA polynucleotide according to the present invention comprising a T7 promoter sequence and downstream thereof a nucleotide sequence encoding the RNA to be transcribed.
  • the cultured cell comprising the DNA polynucleotide of the invention is a bacterium.
  • DNA polynucleotide according to the present invention comprising an inventive T7 promoter provided herein may be used as a primer, i.e. for incorporating the sequence of the inventive DNA polynucleotide comprising the T7 promoter of the invention, into a copy of a target polynucleotide.
  • said DNA polynucleotide may be used for synthesizing the complementary strand of at least one target polynucleotide, wherein said synthesizing comprises annealing said DNA polynucleotide to said at least one target polynucleotide, in particular, thereby generating an IVT template according to the invention.
  • said IVT template may be used in an inventive IVT method provided herein.
  • the DNA polynucleotide according to the present invention is used as a reverse transcription primer.
  • the DNA polynucleotide is used for reverse transcribing RNA to cDNA, thereby incorporating the sequence of the DNA polynucleotide at least partially into the cDNA.
  • DNA polynucleotide of the invention as a primer or reverse transcription primer, the same applies as has been described above in connection with the T7 promoter, and in particular the downstream flanking region and/or the upstream flanking region of the T7 promoter, according to the invention.
  • RTs reverse transcriptases
  • RNA as template
  • a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which may be used directly as a template for PCR.
  • This combination referred to as RT-PCR, allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA.
  • the first strand cDNA can be made double-stranded using e.g. DNA Polymerase I and DNA Ligase. This is for example advantageous for cloning approaches without amplification.
  • Suitable RT polymerases are, for example, Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Moloney Murine Leukemia Virus (M-MuLV, MMLV), reverse Transcriptase or, preferably a Superscript RT polymerase.
  • AMV Avian Myeloblastosis Virus
  • M-MuLV Moloney Murine Leukemia Virus
  • MMLV Moloney Murine Leukemia Virus
  • reverse Transcriptase or, preferably a Superscript RT polymerase.
  • the DNA polynucleotide according to the present invention may be used as a primer for synthesizing the complementary strand of a target DNA molecule, i.e. in a PCR reaction, thereby incorporating the sequence of the DNA polynucleotide of the invention at least partially into copies of the target polynucleotide.
  • the synthesis of the complementary strand may be accompanied and/or followed by synthesizing the complementary strand of the strand having incorporated the DNA polynucleotide of the invention, thereby generating a double stranded DNA molecule comprising at least part of the target DNA molecule, and at one end the T7 promoter of the invention.
  • This procedure may be considered as one cycle of a PCR reaction, wherein the DNA molecule of the invention is one PCR primer.
  • typical PCR reaction conditions may be applied for this step, i.e. employing a DNA polymerase, for example, inter alia , a Taq polymerase.
  • said PCR reaction comprises only few cycles, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles, preferably 1, 2, 3, 4 or 5 cycles, preferably 1 or 2 cycles, preferably 1 cycle.
  • the target polynucleotide which is amplified by using a primer comprising the T7 promoter of the invention may be obtained, in particular, from a few cells (i.e. less than 100 cells), a single cell, a single cell equivalent, a liquid biopsy, and/or a body fluid, as described herein.
  • the few cells may be 100 cells or more, for example 200, 400, 600, 800, 1000, 1500 or 2000 cells but less than 10000 cells.
  • the few cells, as described herein are preferably less than 100 cells, more preferably less than 10 cells.
  • the target polynucleotide i.e the target polynucleotide that is amplified, analyzed and/or sequenced, according to the invention is from a prokaryote or a eukaryote, preferably an animal, preferably a mammal, preferably a human.
  • said target polynucleotide may be associated with a disease and/or is suspected to be associated with a disease, i.e. as described herein.
  • said target polynucleotide may be not from a virus, i.e. not from a virus that exists for less than 1000, 100 or 10 years.
  • a liquid biopsy refers to the sampling of non-solid biological tissue, in particular, blood, and the analysis of at least one target polynucleotide comprised therein as described herein in the context of the invention.
  • a liquid biopsy is mainly used as a diagnostic and monitoring tool, e.g. for diseases such as cancer, and may be largely non-invasive.
  • a liquid biopsy may contain circulating tumor cells, e.g. inter alia , from metastatic breast, metastatic colon, and/or metastatic prostate cancer, and/or circulating tumor DNA.
  • a liquid biopsy may contain circulating endothelial cells which are an indicator of vascular dysfunction or damage, e.g. associated with a heart attack.
  • the presence or abundance of circulating tumor cells or circulating endothelial cells may be determined by a method, e.g. a sequencing method according to the invention, comprising amplifying target polynucleotides, e.g. mRNA molecules, from such single circulating cells by using a primer, e.g. a reverse transcription primer comprising an inventive T7 promoter, according to the invention.
  • a sequencing method comprising amplifying target polynucleotides, e.g. mRNA molecules, from such single circulating cells by using a primer, e.g. a reverse transcription primer comprising an inventive T7 promoter, according to the invention.
  • Circulating tumor DNA is tumor-derived fragmented DNA in the bloodstream that is not associated with cells.
  • ctDNA may reflect the entire tumor genome, it may be useful in clinics, i.e. for diagnosing and/or monitoring cancer.
  • the presence or abundance of circulating tumor DNA may be determined by a method, i.e. a sequencing method according to the invention, comprising amplifying target tumor DNA polynucleotides from a blood sample by using a primer comprising an inventive T7 promoter according to the invention.
  • a liquid biopsy may be from blood or amniotic fluid, and contain cell-free fetal DNA (cffDNA).
  • cffDNA originates from placental trophoblasts and is fragmented when placental microparticles are shed into the maternal blood circulation.
  • cffDNA fragments are approximately 200 base pairs (bp) in length which is significantly smaller than maternal DNA fragments. This difference in size allows cffDNA to be distinguished from maternal DNA fragments.
  • the presence or abundance of circulating tumor DNA may be determined by a method, e.g. a sequencing method according to the invention, comprising amplifying target cell-free fetal DNA polynucleotides from a blood sample (or an amniotic fluid sample) by using a primer comprising an inventive T7 promoter according to the invention.
  • the ctDNA or cffDNA is linearly amplified by in vitro transcription employing the T7 promoter of the invention, and the IVT template is generated by few or one cycle(s) of PCR employing the inventive primer comprising said T7 promoter.
  • the DNA polynucleotide and/or the T7 promoter i.e. the primer or reverse transcription primer of the invention may be used for diagnostic purposes, e.g. for diagnosing and/or monitoring cancer, a cardiovascular disease such as a heart attack, or a fetal gene defect or gene variant.
  • the sensitivity and accuracy of single cell sequencing methods can be increased by employing a primer comprising an inventive T7 promoter provided herein for capturing target polynucleotides and amplifying them by in vitro transcription. This increase in sensitivity and accuracy may be particularly useful for diagnostic approaches, e.g. to determine certain cell states, e.g. of circulating tumor cells or leukemic cells, more reliably.
  • cell state determinants such as transcription factors and chromatin binders, which are missed when using a conventional T7 promoter, are detected by the improved sequencing method of the invention employing a reverse transcription primer comprising an inventive T7 promoter provided herein.
  • the invention relates to a method for amplifying a target polynucleotide, wherein said method comprises the steps of
  • synthesizing the complementary strand of a target polynucleotide comprising annealing a DNA polynucleotide comprising the T7 promoter sequence according to the invention to said target polynucleotide, thereby obtaining an IVT template comprising said T7 promoter and a nucleotide sequence downstream thereof, i.e. wherein said nucleotide sequence reflects the target polynucleotide sequence, and
  • said amplification is a linear amplification as described herein.
  • reflecting the target polynucleotide sequence refers to the target polynucleotide sequence and the complementary sequence or antisense sequence thereof, and/or a well attributable fragment thereof.
  • a fragment can be attributed to the target polynucleotide sequence by methods well known in the art, as described herein or illustrated in the appended Examples, e.g. by a sequence alignment algorithm.
  • the target polynucleotide is an RNA which is reverse-transcribed into cDNA.
  • said RNA is thereby amplified as aRNA.
  • the IVT template is obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from less than 100 cells, preferably less than 10 cells, preferably a single cell.
  • the cell containing an RNA that is to be reverse transcribed to cDNA or a target polynucleotide that is to be amplified, and/or a polynucleotide that is to be analyzed and/or sequenced may be a prokaryotic cell or an eukaryotic cell, preferably an eukaryotic cell, preferably an animal cell, preferably a mammalian cell, preferably a human cell.
  • the human and/or animal cell may be associated with a disease and/or be suspected to be associated with a disease, e.g. inter alia cancer or a cardiovascular disease.
  • Said cancer may be, in particular, characterized by metastatic cells, circulating tumor cells and/or blood cancer cells / leukemic cells.
  • the IVT templated may be obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide, wherein said RNA and/or at least one target polynucleotide is present at a concentration of less than 1 ng/pl, preferably of less than 100 pg/m ⁇ , e.g. less than 50 pg/m ⁇ , and even more preferably of less than 10 pg/m ⁇ , in particular less than 1 pg/m ⁇ , or even less than 0.1 pg/m ⁇ , e.g. 0.05 pg/m ⁇ .
  • the IVT template may be obtained by reverse transcribing RNA to cDNA and/or synthesizing the complementary strand of at least one target polynucleotide from a liquid biopsy and/or a body fluid.
  • At least one target polynucleotide is amplified from less than 100 cells, preferably less than 10 cells, preferably a single cell, wherein the T7 promoter further comprises the sequence AATT directly upstream of the T7 core promoter sequence.
  • the T7 promoter further comprises a poly-T-stretch as described herein.
  • mRNA molecules from less than 100 cells, preferably less than 10 cells, preferably a single cell corresponding to at least 5000, preferably at least 7000, preferably at least 9000 different genes may be amplified.
  • mRNA molecules from a single cell corresponding to at least 9000 different genes may be amplified.
  • An mRNA molecule corresponding to a gene refers, in particular, to a transcript of said gene.
  • the maximum amount of different genes detected and/or amplified is limited by the genes expressed in a cell, and may thus vary between individual cells or samples.
  • a gene may refer to the genomic sequence of a gene comprising the exons of said gene. Moreover, said genomic sequence may comprise the intronic regions and/or, in some cases, the regulatory regions of said gene such as a promoter. Moreover, a gene may refer to a cDNA or a coding sequence. In particular, in the context of RNA sequencing, transcripts, reverse transcription, mRNA and/or cDNAs, a gene may preferably refer to the part of the gene that is transcribed (i.e. a cDNA).
  • the RNA which is reverse transcribed into cDNA is obtained from a single cell or a single cell equivalent.
  • a single cell equivalent as used herein, is a fraction of a sample comprising RNA, wherein the concentration of the RNA and/or the total amount of RNA is similar to what is obtained from a single cell by methods used in the art.
  • the amount of RNA obtained from a single cell is about 10 to 30 pg and the amount of mRNA obtained from a single cell is about 0.1 to 1.5 pg.
  • a single cell equivalent may be a sample of a liquid biopsy and/or a body fluid, such as blood, urine or cerebrospinal fluid, comprising a similar amount and/or concentration of RNA, mRNA and/or DNA as a single cell.
  • a single cell equivalent may comprise about 0.1 to 1.5 pg and/or at least one target polynucleotide, e.g. at least one target mRNA and/or at least one target DNA, at a very low concentration as described herein.
  • a sample of a body fluid and/or liquid biopsy may be concentrated or diluted and/or subsampled to comprise about 0.1 to 1.5 pg and/or at least one target polynucleotide at a very low concentration, as described herein.
  • a polyT-stretch is particularly useful when the DNA polynucleotide of the invention is used as a reverse transcription primer for reverse transcribing mRNA to cDNA.
  • the DNA polynucleotide comprising the T7 promoter of the invention and further comprising a polyT-stretch is used as a primer for reverse transcribing mRNA to cDNA, wherein the polyT-stretch binds to the polyA tail of the mRNA.
  • a polyT-stretch is useful for reverse-transcribing a non-coding RNA comprising a polyA tail to cDNA.
  • a cDNA comprising the T7 promoter of the invention and downstream thereof the antisense sequence of an mRNA is used as IVT template for in vitro transcribing antisense RNA (aRNA).
  • aRNA antisense RNA
  • the aRNA is thereby linearly amplified.
  • the DNA polynucleotide comprising the T7 promoter of the invention is used for linearly amplifying RNA from cDNA by in vitro transcription.
  • the DNA polynucleotide comprising the T7 promoter comprising the downstream flanking region of the invention and the upstream flanking region of the invention is used for linearly amplifying RNA from cDNA by in vitro transcription.
  • cDNA is generated from RNA by using the DNA polynucleotide comprising the T7 promoter comprising the downstream flanking region of the invention and the upstream flanking region of the invention as reverse transcription primer.
  • the RNA is obtained from less than 100 cells, preferably less than 10 cells, very preferably from a single cell.
  • the T7 promoter comprising the downstream flanking region of the invention and the upstream flanking region of the invention is particularly suitable for in vitro transcribing RNA, when the IVT template concentration is low, i.e. very low as described herein.
  • the IVT template concentration is low, for example, when the IVT template is a cDNA which is reverse transcribed from an RNA obtained from a single cell.
  • the DNA polynucleotide of the invention comprising a T7 promoter comprising a downstream flanking region of the invention and an upstream flanking region of the invention is particularly useful for in vitro transcribing RNA from cDNA, wherein the cDNA has been reverse transcribed from RNA present in a very low concentration and/or derived or obtained from less than 100 cells, preferably a single cell.
  • said DNA polynucleotide comprising a T7 promoter comprising a downstream flanking region of the invention and an upstream flanking region of the invention is very useful for in vitro transcribing RNA from a DNA polynucleotide, i.e.
  • DNA polynucleotide has been generated from a target DNA polynucleotide that is present in a very low concentration and/or derived or obtained from less than 100 cells, preferably a single cell.
  • a polynucleotide that is derived or obtained from a cell has been contained in said cell, and/or is contained in such a cell.
  • aRNA is linearly amplified by in vitro transcribing aRNA from a cDNA IVT template, wherein the cDNA is obtained by reverse transcription from an mRNA, wherein the DNA polynucleotide of the invention is comprised in the reverse transcription primer, and wherein the mRNA is obtained from a single cell.
  • the method for in vitro transcribing RNA further comprising the steps of reverse transcribing an RNA to cDNA and subsequently transcribing the cDNA to RNA, wherein the DNA polynucleotide of the invention is used as reverse transcription primer and the cDNA is the IVT template.
  • the reverse transcribed RNA is an mRNA
  • the DNA polynucleotide comprises a poly-T-stretch, preferably downstream of the T7 core promoter
  • the in vitro transcribed RNA is an antisense RNA (aRNA).
  • a second strand of the cDNA comprising the T7 promoter of the invention is synthesized upon first strand cDNA synthesis.
  • the linearly amplified RNA is further reverse transcribed to cDNA, preferably by using random primers for first strand synthesis and the DNA polynucleotide of the invention comprising a polyT-stretch as primer for second strand synthesis.
  • the cDNA derived from linearly amplified RNA can again be used as an IVT template to again in vitro transcribe RNA.
  • the cDNA comprising the T7 promoter of the invention and downstream thereof a nucleotide sequence encoding preferably an RNA is used as IVT template for in vitro transcribing RNA.
  • the RNA is thereby linearly amplified.
  • the DNA polynucleotide of the invention can be used for, preferably linearly, amplifying a nucleotide sequence which is complementary to a nucleotide sequence comprised in said DNA polynucleotide.
  • the presence and/or abundance of a target polynucleotide in sample such as a liquid biopsy, a sample from a body fluid, a few cells and/or a single cells, as described herein, may be determined by employing the inventive IVT and T7 promoter based amplification methods provided herein.
  • the amount of the amplified target polynucleotide(s) e.g. aRNA
  • the amplified target polynucleotide(s) may be determined by methods known in the art and/or as described herein, e.g. by quantitative PCR, digital PCR and/or next-generation sequencing.
  • Further methods to detect the amplified target polynucleotide(s) may be also used, e.g. fluorescence in-situ hybridization (FISH) and/or dCas9-based imaging.
  • FISH fluorescence in-situ hybridization
  • dCas9-based imaging e.g. fluorescence in-s
  • the invention relates to a method for amplifying a target polynucleotide, wherein said target polynucleotide is a genomic DNA sequence, and wherein said method comprises the steps of
  • step b As regards the in vitro transcription (step b), the amplification of the target polynucleotide, and “reflecting the target polynucleotide sequence”, the same applies as is described herein in the context of further methods for amplifying a target polynucleotide.
  • a primer or reverse transcription primer comprising a T7 promoter with an inventive and defined downstream flanking region, as provided herein, allows to compare the abundances of target polynucleotides (e.g. non-poly-A RNA or DNA) with improved sensitivity and/or accuracy.
  • target polynucleotides e.g. non-poly-A RNA or DNA
  • T7-promoter based transposase-mediated amplification of genomic DNA target polynucleotides e.g. which are in proximity to a certain epigenetic modification or DNA-binding molecule.
  • the improved accuracy is due to the improved efficiency of amplifying the target polynucleotide, and furthermore, due to the lack of an amplification bias.
  • an amplification bias may occur when only a core T7 promoter or a T7 core promoter with less than 8 defined directly adjacent downstream bases is used because in such a case the efficiency of the T7 promoter may vary between different target polynucleotides since they may constitute a (random) part of the downstream flanking region.
  • the T7 promoter of the invention comprising an optimized and defined downstream flanking region is thus particularly useful for detecting and/or quantifying target polynucleotides in a sample, i.e.
  • an inventive sequencing method employing an inventive primer provided herein may be used.
  • the IVT method using a DNA polynucleotide according to the present invention is part of a sequencing method.
  • the term “sequencing” refers to approaches aiming at determining the identity of at least one nucleotide, preferably most nucleotides, even more preferably all nucleotides in a given nucleotide sequence.
  • the term “sequencing method” refers to a method for determining the partial or full sequence(s) of a polynucleotide and/or to determine the relative abundance of identical molecules of said polynucleotide.
  • a sequencing method may be a first, next (second) or third generation sequencing method. Preferred are next or third generation sequencing methods.
  • a preferred next-generation sequencing technique is Illumina sequencing.
  • the sequencing method is suitable for single cell sequencing. Suitable single cell sequencing techniques are for example based on SMART-seq2, CEL-seq2, inDrop, MARS-seq, Drop-seq, STRT, Quartz-seq, LIANTI, CHIL-Seq, Dam-seq (e.g.
  • scDam&T-seq and the sci-L3 method.
  • a preferred third generation sequencing method is Nanopore sequencing which allows direct sequencing of RNA and/or DNA in the absence of a library preparation step comprising PCR based amplification of reverse-transcribed aRNA.
  • the DNA polynucleotide of the present invention is used to reverse transcribe a target polynucleotide, e.g. an RNA, amplify it, i.e. by IVT, and then subject the amplified RNA directly and/or after reverse transcription to sequencing.
  • a target polynucleotide e.g. an RNA
  • IVT i.e. by IVT
  • the DNA polynucleotide used for the sequencing method comprises a polyT-stretch to capture mRNAs which are then amplified as antisense RNAs (aRNAs).
  • aRNAs antisense RNAs
  • the entire sequence of RNAs, including the sequence of the respective poly(A) tails, can be determined by sequencing the synthesized cDNA library.
  • the DNA polynucleotide of the invention used for the sequencing method further comprises a barcode and/or a unique molecular identifier (UMI) and preferably a polyT-stretch.
  • UMI unique molecular identifier
  • the sequencing method is suitable for scarce material, in particular for less than 10.000 cells, preferably less than 100 cells, preferably less than 10 cells. In a very preferred embodiment, the sequencing method is a single cell sequencing method.
  • the invention relates to a method for determining the partial or full sequence(s) of a polynucleotide, i.e. a target polynucleotide, and/or the relative abundance of identical molecules of said polynucleotide comprising the steps of (a) synthesizing a DNA strand which is complementary to the polynucleotide that is to be sequenced (target polynucleotide) or the complementary strand thereof comprising annealing the DNA polynucleotide of the invention (to said target polynucleotide), and (b) transcribing the polynucleotide that is to be sequenced (target polynucleotide) or the complementary strand thereof into RNA by using a T7 polymerase.
  • the present invention relates to a method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene comprising the steps of (a) reverse transcribing an RNA into a first strand of a cDNA comprising annealing the DNA polynucleotide according to the present invention; and (b) transcribing the cDNA into aRNA by using a T7 polymerase.
  • step (a)) of the method the same applies as it has been described above in connection with reverse transcribing RNA to cDNA, synthesizing the complementary strand of a target polynucleotide and/or the use of the DNA polynucleotide of the invention as a primer or reverse transcription primer.
  • annealing the DNA polynucleotide refers herein to the use of the DNA polynucleotide of the invention as a primer for reverse transcribing or synthesizing the complementary strand of the polynucleotide the sequence of which is to be determined (target polynucleotide).
  • the method further comprises a step (a’) of synthesizing a second strand of the first strand of the cDNA obtained from step (a) or during the method for amplifying a target polynucleotide according to the invention.
  • the invention relates to a method for determining the partial or full nucleotide sequence(s) and/or abundance of at least one target polynucleotide comprising the steps of
  • determining the abundance of at least one target polynucleotide may comprise counting the nucleotide sequences corresponding to said target polynucleotide. Methods for determining and normalizing the counts are well known in the art, and described herein and in the appended Examples. Moreover, the abundance of at least one mRNA may further refer to the transcript level of the corresponding gene.
  • the invention also relates to a method for determining the transcript level of at least one gene comprising the steps of
  • the partial or full nucleotide sequence(s), the abundance of at least one target polynucleotide and/or the transcript level of at least one gene is determined in a sample comprising less than 100 cells, preferably less than 10 cells, preferably a single cell.
  • the mRNA and/or at least one target polynucleotide may be present at a concentration of less than 1 ng/m ⁇ , preferably of less than 100 pg/m ⁇ , e.g. less than 50 pg/m ⁇ , and even more preferably of less than 10 pg/m ⁇ , in particular less than 1 pg/m ⁇ , or even less than 0.1 pg/m ⁇ , e.g. 0.05 pg/m ⁇ .
  • the expression of at least about 9750 genes can be detected in a single cell (e.g. a K562 cell) with an exemplary sequencing method according to the invention employing an inventive reverse transcription primer provided herein (e.g. SEQ ID NO: 15) comprising an T7 promoter of the invention (e.g. SEQ ID NO:20).
  • an inventive reverse transcription primer provided herein (e.g. SEQ ID NO: 15) comprising an T7 promoter of the invention (e.g. SEQ ID NO:20).
  • the partial or full nucleotide sequence(s) and/or the abundance of at least 5000, preferably at least 7000, preferably at least 9000 different target polynucleotides and/or the transcript level of at least 5000, preferably at least 7000, preferably at least 9000 genes may be determined according to the sequencing method of the invention.
  • at least 5000, preferably at least 7000, preferably at least 9000 transcripts of unique genes may be detected in a single cell.
  • at least 9100, 9300 or 9500 transcripts of unique genes may be detected in a single cell.
  • the sequencing method of the invention e.g. the method for determining the partial or full nucleotide sequence(s) of an RNA and/or the transcript level of at least one gene comprises a step (c) of generating a double-stranded cDNA from the aRNA of step (b), and/or a step of sequencing the aRNA of step (b) and/or the cDNA of step (c).
  • the sequencing method of the invention further comprises a step (c’) of synthesizing the second strand of the cDNA of step (c) upon synthesis of the first strand of the cDNA.
  • the method further comprises a step (d) of amplifying the cDNA of step (c) by PCR, wherein a library preparation primer binds to the sequencing adapter.
  • the sequencing method further comprises a step of sequencing the amplified cDNA of step (d).
  • the partial or full nucleotide sequence(s) of an mRNA and/or the transcript level of at least one gene comprising a coding sequence is determined, wherein the DNA polynucleotide of the invention comprises a polyT-stretch and a sequencing adapter, wherein the polyT-stretch binds to the poly-A-tail of the mRNA.
  • the polynucleotide that is to be sequenced is derived or obtained from less than 10000 cells, preferably, less than 100 cells, preferably less than 10 cells.
  • the polynucleotide to be sequenced is derived or obtained from a single cell and/or a single cell equivalent.
  • the polynucleotide that is to be sequenced is derived from and/or obtained from a single cell.
  • the polynucleotide to be sequenced may be derived or obtained from a body fluid, i.e. a single cell equivalent obtained from a body fluid, as described herein.
  • the sequencing method of the invention further comprises a step of analyzing the sequencing data, wherein the analysis comprises a sequence alignment, counting reads, and/or normalizing read counts.
  • the present invention relates to the use of the DNA polynucleotide according to the present invention in a sequencing method, e.g. for determining the partial or full nucleotide sequence(s) of an RNA contained in a single cell or single cell equivalent and/or the transcript level of at least one gene of a single cell or single cell equivalent.
  • DNA polynucleotide and the determination of the partial or full nucleotide sequence(s) of an RNA contained in a single cell or single cell equivalent and/or the transcript level of at least one gene of a single cell or single cell equivalent the same applies as described above.
  • the present invention further relates to a kit comprising the DNA polynucleotide of the invention, one or more modified and/or unmodified ribonucleotide triphosphate(s), library preparation primer(s), sequencing primer(s), and/or a microfluidic chip.
  • the kit can further comprise an enzyme for 5’ capping of RNA and/or an enzyme for poly-A- tailing of RNA, and/or a manual describing an in vitro transcription method and/or a sequencing method comprising an IVT step using said kit.
  • an enzyme for 5’ capping of RNA and/or an enzyme for poly-A- tailing of RNA and/or a manual describing an in vitro transcription method and/or a sequencing method comprising an IVT step using said kit.
  • the polynucleotide of the invention is an RNA polynucleotide.
  • the RNA polynucleotide of the invention the same applies as it has been described in the context of a DNA polynucleotide.
  • RNA polynucleotide of the invention is reverse-transcribed to DNA, in particular before its use as a reverse transcription primer, for IVT and/or a sequencing method according to the invention.
  • FIG. 1 Scheme of 5’RACE-Seq for investigating the efficiency of different T7 promoter sequences.
  • a double stranded DNA (dsDNA) library (SEQ ID NO:2), wherein each DNA polynucleotide comprised a T7 core promoter sequence, followed by a guanine at position +1 and a randomized nucleotide sequence at positions +2 to +16, was transcribed in vitro using a T7 RNA polymerase. The resulting 210 nucleotides long RNAs were reverse transcribed, and the 5’ end of the respective cDNA converted into a library for deep sequencing. The activity of the T7 promoter sequences was determined by counting the reads comprising a respective flanking region directly adjacent downstream the T7 core promoter sequence. The DNA IVT template was amplified by PCR and sequenced for normalization of the read counts.
  • FIG. 4 Activity of T7 promoter variants carrying nucleotide sequences from positions +4 to +8 determined by 5’RACE-Seq shown as log2 fold change (FC) (relative abundances of individual sequence motifs). All promoters contained a G at positions +1 to +3. A high correlation (R 2 ) of 0.98 was observed between two independent experiments denoted as RepA and RepB. Highlighted are exemplary T7 promoter sequences with high, mid, or low activity.
  • Figure 5. Linear amplification of RNA by in vitro transcription.
  • FIG. 6 Comparison of the IVT activity of T7 promoter variants with different 5’RACE-Seq ranks as shown in Table 1.
  • All T7 promoter variants comprised a G at positions +1 to +3 and different nucleotide sequences at positions +4 to +8 and were used to in vitro transcribe a 410 nucleotides long RNA for 2h. Shown is the fold amplification relative to the template DNA.
  • Indicated below is the +4 to +8 RACE-Seq rank as shown in Table 1. Results are shown from left to right for SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:6, SEQ ID NO:3, and SEQ ID NO:4. Error bars represent standard deviation for triplicate experiments
  • FIG. 7 Comparison of the IVT activity of two T7 promoter variants using different IVT DNA template concentrations (SEQ ID NO:4 and SEQ ID NO: 8). IVT was performed for 2h.
  • FIG. 8 Comparison of the IVT activity of a T7 promoter variant with a 5’RACE-Seq rank as shown in Table 1 with or without an AT -rich 4 nucleotides long upstream flanking region. IVTs were performed for 2h using T7 promoter variants of the indicated 5'RACE-Seq rank. Shown is the resulting fold amplification of template DNA for SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO: 8 (from left to right). The upstream flanking region was GAATT located directly adjacent upstream the T7 core promoter (comprised in SEQ ID NO:5).
  • FIG. 9 An AT -rich upstream flanking region boosts the IVT activity of the T7 promoter in an IVT template that mimics the structure of CEL-seq2 derived cDNA, in particular at very low IVT template concentrations.
  • a 410 nucleotides long RNA was in vitro transcribed for 2h from 1 nanogram of standard IVT templates (SEQ ID NO:4 and SEQ ID NO:5).
  • the RNA was in vitro transcribed for 2h from 1 nanogram (50 pg/pl) of 305 nucleotides long CEL-seq cDNA mimics (SEQ ID NO: 10 and SEQ ID NO: 11).
  • RNA was in vitro transcribed for 15h from 1 picogram (0.05 pg/m ⁇ ) of those CEL-seq cDNA mimics.
  • the T7 promoter in all IVT templates further comprised a directly adjacent downstream flanking region with the sequence “GGGATAAT”.
  • Figure 10 A T7 promoter of the present invention, in particular with a respective upstream flanking region improves IVT from a dsDNA that mimics the structure of CEL-seq2 derived cDNA.
  • the relative activity of the T7 promoter as found in the CEL-seq2 adaptor is compared to the T7 promoter with rank #4 (see SEQ ID NO: 10) in the 5’RACE-Seq data as shown in Table 1, and the latter in combination with the directly adjacent upstream flanking region with the sequence GAATT (see SEQ ID NO: 11).
  • the template was pre-synthesized as double stranded DNA to monitor the specific effect of the promoter sequence, independent of the mRNA capturing step during CEL-seq2. 1 picogram template was in vitro transcribed for 15h.
  • the data for “CEL-seq + #4” and “CEL-seq up + #4” are the same as shown in the right panel of Figure 9.
  • T7 promoter in combination with specific directly adjacent upstream flanking regions improves aRNA yield during CEL-seq2 from 10 single cells.
  • CEL-seq2 experiments were performed until RNA amplification using the reverse transcription primers with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 14 or SEQ ID NO: 16 (from left to right).
  • the 5’RACE-Seq rank of the T7 promoter sequence is indicated as shown in Table 1. “upl” refers to the upstream flanking region GAATT (comprised in SEQ ID NO: 13 and SEQ ID NO: 15) and “up4” refers to the upstream flanking region AATTG (comprised in SEQ ID NO: 14 and SEQ ID NO: 16).
  • CEL-seq indicates the original CEL-seq2 reverse transcription primer (SEQ ID NO: 12). Error bars represent standard deviation for triplicate experiments.
  • FIG. 12 The combination of a directly adjacent up- and downstream region of the T7 improves IVT in a single cell RNA sequencing experiment based on CEL-seq2.
  • CEL-seq2 was performed from single K562 cells using the indicated primers (SEQ ID NO: 12 and SEQ ID NO: 15). After reverse transcription and second strand synthesis cDNA from 10 cells was pooled and in vitro transcribed for 15h. Purified RNA was fragmented and quantified on a Tapestation (Agilent). Error bars represent the standard deviation from triplicate experiments.
  • T7 T7 promoter
  • Illumina A partial sequencing adapter A
  • UMI unique molecular identifier
  • BC cellular barcode
  • a modified CEL-Seq2 reverse transcription primer (SEQ ID NO: 15; CEL-Seq+) harboring a modified T7 promoter (SEQ ID NO:20) significantly improved CEL-Seq2-based RNA-Sequencing of single cells, i.e. by enhancing the efficacy of the linear amplification of cDNA. Specifically, a significantly increased number of genes was detected with CEL-Seq+.
  • FIG. 16 Genes from deep sequenced bulk K562 RNA-Seq (Prost (2015), Nature 525) were sorted into quartiles by expression level with the lowest expressed genes in the first quartile and the highest expressed genes in the fourth quartile. Shown are the numbers of genes from the indicated bulk quartiles that were detected in individual cells by CEL-Seq2 (with the reverse transcription primer shown in SEQ ID NO: 12) or CEL-Seq+ (with the reverse transcription primer shown in SEQ ID NO: 15). Within each of the four quartile plots, CEL-Seq2 is shown on the left, and CEL-Seq+ on the right.
  • FIG. 17 Shown are the numbers of genes of different categories detected with CEL-Seq2 (with the reverse transcription primer shown in SEQ ID NO: 12) or CEL-Seq+ (with the reverse transcription primer shown in SEQ ID NO: 15) in individual cells.
  • the first category encompasses genes that are differentially expressed throughout chronic myeloid leukemia (CML) disease progression (Prost (2015), Nature 525; left plot), the second category encompasses genes with the GO association “DNA binding transcription factor (TF)” (middle plot), and the third category encompasses genes with the GO association “chromatin binding” (right plot).
  • CEL-Seq2 is shown on the left, and CEL-Seq+ on the right. Whiskers reach to 1.5x IQR away from the lst/3rd quartile.
  • a saturated randomized 5' RACE-Seq screen was performed to determine the self-transcription activity of T7 promoter sequences comprising the T7 core promoter, a guanine at position +1 (TSS) and different combinations of nucleotides at positions +2 to +16.
  • T7 promoter sequences comprising the T7 core promoter, a guanine at position +1 (TSS) and different combinations of nucleotides at positions +2 to +16.
  • TSS guanine at position +1
  • the 5’ end sequence of over 100 million 210 nucleotides long RNAs transcribed from said randomized promoter sequences comprised in a 500 nucleotides long dsDNA polynucleotide library (lOng; scale of 10 10 (10 billion) molecules of the +2 to +16 randomized T7 promoter template) was determined to interrogate the influence of the directly adjacent downstream flanking region of the core promoter on the transcriptional activity of the T7 promoter.
  • a scheme of the applied experimental procedure of 5' RACE-Seq is shown in Figure 1.
  • 5' “RACE-Seq” refers to a rapid amplification of cDNA ends and subsequent sequencing using a next-generation-sequencing method (deep sequencing).
  • a “saturated” screen refers to a screen of virtually any possible combination of nucleotides at positions +2 to +16 contained in a 5’RACE-Seq library. Any possible sequence at positions +2 to +8 is covered at least 100 times, but typically thousands of times during sequencing. The promoter strength of a given promoter sequence was assessed based on the relative production of transcripts from said promoter.
  • cDNA was purified using 1.6 volumes Ampure XP beads and eluted in 10 pi water.
  • a poly (A)-tailing reaction was carried out using 3 U/mI terminal transferase (Invitrogen 10533).
  • Second-strand cDNA synthesis was performed in 50 pi with 0.5 pM Oligo i5_5N_dT20VN, 2000 U Q5® High- Fidelity DNA Polymerase, and 0.2 mM dNTPs, using the temperature cycle 98 °C for 30 sec; 55 °C for 30 sec; 72 °C for 5 min.
  • 2 pL Exonuclease 1 and 48 pi water were added and the reaction was incubated for 1 h at 37 °C.
  • dsDNA was purified with 1.6 volumes AMPure XP beads, and eluted in 22 pi water. Sequencing adapters were introduced during 8 cycles of PCR with KAPA HiFi HotStart.
  • dsDNA template T7 15N was amplified by PCR using KAPA HiFi HotStart Polymerase, P5 and BG T7 sequencing adapters. Here, simply 7 cycles of PCR had to be performed.
  • Libraries were purified twice with 0.9 volumes AMPure XP beads and sequenced in single end mode using a NextSeq 500 instrument from Illumina. The first 15 nucleotides were used for data analysis.
  • the dsDNA template for RACE-Seq sequence was as follows (SEQ ID NO:2):
  • GCATCGAG wherein bold and italics refer to the T7 core sequence, bold and not in italics to the sequencing adapter binding site, and bold and underlined to the reverse transcription binding site, and wherein N denotes any nucleotide selected from the group consisting of A, C, G, and T.
  • T7 promoter variants comprising three guanines at positions +1 to +3 and a certain set of nucleotide sequences at positions +4 to +8 is shown in Table 1.
  • the downstream flanking regions (positions +1 to +8) of T7 promoter variants are ranked based on the transcriptional activity measured by 5’RACE-Seq.
  • the normalized activity measurements of two replicate experiments (A and B) and the mean of the normalized activity obtained in the two experiments is shown.
  • GGGAACCC 1.593360743 1.994660137 1.79401044

Abstract

L'invention concerne un polynucléotide d'ADN, un kit comprenant ledit polynucléotide d'ADN, et un procédé de transcription in vitro d'ARN À l'aide dudit polynucléotide d'ADN, ledit polynucléotide d'ADN comprenant (i) une séquence de promoteur T7 comprenant (1) une séquence de promoteur principal T7 et (2) une région de flanquement en aval directement adjacente, la région de flanquement en aval comprenant huit bases (+1 à +8) comprenant trois guanines à des positions +1 à +3, une adénine ou une thymine en position +4, au moins deux adénosines à des positions +4 à +8 et/ou une thymine en position +4, et au plus une cytosine à des positions +4 à +8, et en aval de celle-ci (ii) une séquence nucléotidique codant pour l'ARN à transcrire ; et le procédé comprenant (a) la fourniture dudit polynucléotide d'ADN en tant que matrice IVT, et (b) à transcrire in vitro ledit modèle IVT en présence d'une polymérase T7 et de ribonucléotides triphosphates.
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Publication number Priority date Publication date Assignee Title
US11898186B1 (en) 2022-08-10 2024-02-13 Genscript Usa Inc. Compositions and methods for preparing capped mRNA

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