Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
• • 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
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/33—Chemical structure of the base
  • C12N2310/334—Modified C
• • 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
  • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
• • 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
  • C12Q2521/00—Reaction characterised by the enzymatic activity
  • C12Q2521/10—Nucleotidyl transfering
  • C12Q2521/119—RNA polymerase
• • 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
  • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
  • C12Q2525/10—Modifications characterised by
  • C12Q2525/101—Modifications characterised by incorporating non-naturally occurring nucleotides, e.g. inosine
• • 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
  • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
  • C12Q2525/10—Modifications characterised by
  • C12Q2525/117—Modifications characterised by incorporating modified base
• • 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
  • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
  • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• RNA plays a fundamental role in human biology. It is the main player of the central dogma of biochemistry and a crucial regulator of gene expression via for instance micro and small interfering RNA, as well as through its intrinsic catalytic activity.
• RNA therapeutics have the potential to modify cellular function at the translational level, they may open up new opportunities to address previously undruggable targets.
• An increased molecular and mechanistic knowledge of the biological processes involving RNA is therefore vital to understanding diseases and treat them.
• RNA-based drugs lies in understanding the processes of cell uptake and endosomal release (Dowdy, S. F., Nat. Biotechnol.35, 222-229, [2017]).
• tC O TP modified nucleobase triphosphate
• mRNA functional messenger RNA
• this fluorophore constitutes a native-like alternative label, opening new possibilities not only to track the nucleic acid of interest but also to use fluorescent read-outs to obtain detailed information regarding nucleic acid structure and behaviour.
• the specification describes successful in vitro transcription and also effective in cell translation of a full-length mRNA internally labelled with this fluorescent nucleobase analogue.
• H2B:GFP Green Fluorescent Protein
• RNA molecules are not amenable to in cell “live” analysis of transcription, translation or delivery of long therapeutic RNAs (which are mRNA-based) and therefore do not allow the same level of mechanistic insight.
• WO2011/034895 concerns methods for labelling DNA and RNA. It mentions a structurally different fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (“tCTP”) which is used during in vitro transcription reactions to prepare labelled RNA.
• tCTP fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate
• RNA polymers accessible using the technology disclosed in the present specification also have advantageous properties over the labelled RNAs in WO2011/034895, for example 1) improved fluorescence levels and label photostability; 2) improved in vitro transcription fidelity; and 3) native-like levels of in cell translation of tC O -labelled mRNA resulting in the correct protein product and localization.
• this specification discloses a labelling technique that not only allows localisation and tracking of the tagged RNA, but also facilitates analysis of the biological functionality and delivery efficacy of mRNA, an important future drug modality.
• a primary objective of the present specification is to provide a modified nucleobase triphosphate that can be used to make labelled RNA especially suitable for in vitro and in vivo mechanistic investigations.
• This specification also describes, in part, a process for preparing a compound of formula (I) or a salt thereof as claimed in claim 5.
• This specification also describes, in part, a composition for preparing a tC O labelled RNA molecule comprising a compound of formula (I) as claimed in claim 16.
• This specification also describes, in part, the use of a compound of formula (I) or a salt thereof to enzymatically prepare a tC O labelled RNA molecule as claimed in claim 17.
• This specification also describes, in part, a process for preparing a tC O labelled RNA molecule as claimed in claim 19.
• a” or “an” may mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10000, 100000 or 1000000 (1 million).
• “Comprising” means that a given embodiment may contain other features.
• the given material may be formed of at least 10% w/w, at least 20% w/w, at least 30% w/w, or at least 40% w/w of the materials or elements (or combination of materials or elements).
• “comprising” may also mean “consisting of” (or “consists of”) or “consisting essentially of” (or “consists essentially of”).
• “consisting of” or “consists of” means the material or element is formed entirely of the material or element (or combination of materials or elements). In any embodiment where “consisting of” or “consists of” is mentioned the given material or element may be formed of 100% w/w of the material or element. [024] With respect to embodiments of a material, “consisting essentially of” or “consists essentially of” means that a given material or element consists almost entirely of that material or element (or combination of materials or elements).
• the given material or element may be formed of at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w or at least 99% w/w of the material or element.
• “is” or “may be” is used to define a material or element, “is” or “may be” may mean the material or element “consists of” or “consists essentially of” the material or element.
• a suitable salt of a compound of formula (I) is for example a base-addition salt.
• a base-addition salt is formed by bringing the compound of formula (I) into contact with a suitable organic or inorganic base.
• a base addition salt may be formed using a suitable organic base like a nitrogen base, for example ammonia or a trialkylamine like triethylamine.
• a base addition salt may also for example be formed using a suitable inorganic base like an alkali metal or rare earth hydroxide, for example potassium hydroxide, sodium hydroxide, magnesium hydroxide or manganese hydroxide.
• a compound of formula (I) which is a free acid.
• a compound of formula (I) which is a salt.
• a compound of formula (I) which is a sodium, potassium, magnesium, or ammonium salt.
• a compound of formula (I) which is a sodium, potassium, or ammonium salt.
• a compound of formula (I) which is a sodium or ammonium salt.
• a compound of formula (I) which is a monosodium, disodium, trisodium, tetrasodium, monoammonium, diammonium, triammonium or tetraammonium salt.
• a compound of formula (I) which is a monosodium, disodium, trisodium or tetrasodium salt.
• a compound of formula (I) which is a monosodium salt.
• a compound of formula (I) which is a disodium salt.
• a compound of formula (I) which is a trisodium salt.
• a compound of formula (I) which is a monoammonium, diammonium, triammonium or tetraammonium salt.
• a compound of formula (I) which is a monoammonium salt.
• a compound of formula (I) which is a diammonum salt.
• a compound of formula (I) which is a triammonium salt.
• a protecting group (“PG”, for example PG 1 and PG 2 ) is any group suitable for temporarily protecting a reactive centre, for example a hydroxyl group. Suitable protecting groups for the reactive centres disclosed herein may be found for example in “Greene's Protective Groups in Organic Synthesis, Fourth Edition”, Greene T. W., Wuts P. G. M.; John Wiley & Sons, Inc. 2007, doi: 10.1002/0470053488), the contents of all of which are herein incorporated by reference. [049] A “hydro” group is equivalent to a hydrogen atom.
• a “C 1-3 alkyl group” is a straight chain or branched saturated alkyl group with the indicated number of carbons.
• Example C 1-3 alkyl groups include methyl, ethyl, propyl and isopropyl.
• the secondary alcohols to be capped may be those on the ribose part of the molecule.
• R 1 may be a C 1-3 alkyl group. It has been observed that when R 1 is a C 1- 3alkyl group, the phosphoramidite reagent preparation is easier and higher yielding, but performs at least as well in step v above as when R 1 is a hydro group. [055] In some embodiments R 1 may be methyl. [056] In one embodiment there is provided a compound of formula (III): (III) Where R 1 is a C 1-3 alkyl group. [057] In one embodiment there is provided a compound of formula (IIIa): . (IIIa) [058] In some embodiments the support may be a solid polymer.
• the support may be a solid polymer selected from controlled-porosity glass and polystyrene. [060] In some embodiments the support may be polystyrene. [061] In some embodiments the support may be controlled-porosity glass. [062] In some embodiments the support may be functionalised with a primary amino group. This may form the reactive point of attachment to the support. [063] In some embodiments the support may be controlled-porosity glass functionalised with a primary amino group (for example Amino-SynBaseTM). [064] In some embodiments PG 1 may be selected from trityl, dimethoxytrityl and trimethoxytrityl.
• PG 2 may be selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, tert- butyldimethylsilyl and methoxyethyl.
• PG 2 may be acetyl. Where an immobilised molecule is base labile, this allows for an efficient synthesis in which removal of the PG 2 group and cleavage from the resin may be accomplished in a single step.
• PG 1 may be dimethoxytrityl and PG 2 may be acetyl.
• immobilisation of the compound of formula (II) in step i) may occur mainly at the 2’-hydroxy position.
• immobilisation occurs mainly at the 2’-hydroxy position, this may be >50%, >60%, >70%, >80%, >90% or 100% of the total immobilisation (i.e. the total covalent binding of both secondary hydroxyl groups to the support).
• the tetraalkylammonium pyrophosphate may be tetrabutylammonium pyrophosphate.
• a process for preparing a compound of formula (I) or a salt thereof comprising: i. Providing a compound of formula (II) or a salt thereof: (II) Where PG 1 is selected from trityl, dimethoxytrityl and trimethoxytrityl; ii. Immobilising the compound of formula (II) or a salt thereof by linking one of its secondary alcohol groups to a controlled-porosity glass support; iii.
• a protecting group PG 2 selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, tert- butyldimethylsilyl and methoxyethyl; iv. Removing the protecting group PG 1 ; v. Reacting the exposed primary alcohol group with a compound of formula (III): (III) Where R 1 is a C 1-3 alkyl group; vi.
• a process for preparing a compound of formula (I) or a salt thereof comprising: i. Providing a compound of formula (II) or a salt thereof: (II) Where PG 1 is dimethoxytrityl; ii.
• step ii) Removing the protecting group PG 2 ; and ix. Cleaving the resultant triphosphate from the support to generate a compound of formula (I) or salt thereof.
• Suitable conditions and reagents to effect each of steps i) to ix) above are known to the skilled person or can be found in the Detailed Description.
• immobilising the compound of formula (II) or salt thereof in step ii) may be accomplished by a coupling reagent (for example succinic anhydride catalysed by dimethylaminopyridine when the support is functionalised with a primary amino group).
• a coupling reagent for example succinic anhydride catalysed by dimethylaminopyridine when the support is functionalised with a primary amino group.
• reaction of the exposed primary alcohol group with a compound of formula (III) may be accomplished using an activator (for example BTT activator or Activator 42®).
• the phosphorus (III) compound in step vi) may be oxidised to a phosphorus (V) compound using aqueous pyridine and iodine.
• cleaving the triphosphate from the support may be accomplished using basic conditions (for example by treating with AMA). When there is a base-labile support and a base- labile protecting group is chosen for PG 2 , using these conditions allows simultaneous deprotection and cleavage.
• RNA Synthesis in one embodiment there is provided a composition for preparing a tC O labelled RNA molecule comprising a compound of formula (I) and a natural ribonucleotide triphosphate.
• a “natural ribonucleotide triphosphate” comprises the appropriate natural ribonucleoside with a triphosphate group bonded to the 5’ hydroxy position. It is equivalent to a natural ribonucleoside triphosphate.
• a natural ribonucleotide triphosphate may be selected from cytidine 5’-triphosphate, uridine 5’-triphosphate, adenosine 5'-triphosphate and guanidine 5’-triphosphate.
• a composition of natural ribonucleotide triphosphates i.e. one comprising a ribonucleotide triphosphate as defined herein
• a compound of formula (I) or a salt thereof to enzymatically prepare a tC O labelled RNA molecule.
• a tC O labelled RNA molecule comprises at least one tC O residue but is otherwise similar to the natural RNA molecule (i.e. one with an unmodified cytosine residue at the same location as the tC O residue).
• a tC O labelled RNA molecule may comprise >10%, >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90% or 100% of tC O residues in place of unmodified cytosine residues.
• a tC O labelled RNA molecule may comprise 10%-20%, 10%-30%, 10%- 40%, 20%-50%, 30%-60%, 40%-70%, 50%-80% or 50%-90% of tC O residues in place of unmodified cytosine residues.
• a process for preparing a tC O labelled RNA molecule comprising providing a DNA template to composition comprising a compound of formula (I) and a natural ribonucleotide triphosphate (for example a combination of varying amounts of cytidine 5’- triphosphate, uridine 5’-triphosphate, adenosine 5'-triphosphate and/or guanidine 5’-triphosphate in amounts sufficient to construct the target RNA molecule, for example as provided in NTP mix), then treating the resultant mixture with an RNA polymerase.
• the tC O labelled RNA molecule may be a tC O labelled mRNA.
• the tC O labelled RNA molecule may encode for a protein fused to a fluorescent protein.
• Example fusable fluorescent proteins include Green Fluorescent Protein (GFP) and mFruit family proteins.
• GFP Green Fluorescent Protein
• mFruit family proteins When the target protein is fluorescent (either inherently or due to a tag), it is possible to simultaneously visualise both the labelled RNA molecule and the protein it is being used to synthesise, giving a greater degree of mechanistic insight.
• the tC O labelled RNA molecule may encode for a protein selected from H2B, calmodulin, H2B:GFP and calmodulin-3:GFP.
• :GFP Green Fluorescent Protein
• the tC O labelled RNA molecule may encode for H2B:GFP.
• the tC O labelled RNA molecule may encode for calmodulin-3.
• the RNA polymerase may be selected from T7 polymerase and SP6 polymerase.
• a process for preparing a tC O labelled RNA molecule may be carried out in the presence of transcription buffer (e.g. 5X transcription buffer), magnesium salt (e.g.
• a process for preparing a tC O labelled RNA molecule may be carried out in the presence of transcription buffer (e.g. 5X transcription buffer), magnesium salt (e.g. magnesium(II) chloride) and an RNase inhibitor (e.g. Ribolock).
• a process for preparing a process for preparing a tC O labelled RNA molecule may be carried out substantially as described in the experimental section (e.g. as detailed in the section headed “H2B:GFP RNA transcription and purification”).
• kits for preparing a tC O labelled RNA molecule comprising: i. A compound of formula (I); ii. A composition of natural ribonucleotide triphosphates; iii. An RNA polymerase; optionally iv. A DNA template; and optionally v. Instructions for use.
• Labelled RNA Translation [095] In one embodiment there is provided the use of a tC O labelled mRNA molecule to prepare a protein encoded by the mRNA by translation.
• Translation refers to the central biological process whereby mRNA is decoded in a ribosome to produce a specific polypeptide, which may fold into an active protein before performing its functions in a cell.
• a tC O labelled mRNA molecule to prepare a protein encoded by the mRNA by in vitro translation (for example, substantially as described in the part of the experimental section (e.g. as detailed under the heading “cell-free translation”).
• in vitro translation may be performed using E.coli bacterial lysates and/or the Expressway® mini cell-free expression system.
• a tC O labelled mRNA molecule to prepare a protein encoded by the mRNA by in cell translation (for example, substantially as described in the parts of the experimental section (e.g. as detailed under the headings “cell culture” and “electroporation or chemical transfection”).
• in cell translation may be performed in human neuroblastoma cells (e.g. SH-SY5Y cells).
• a tC O labelled RNA molecule into a protein.
• in vitro translation of a tC O labelled RNA molecule into a protein there is provided.
• the in cell translation of a tC O labelled RNA molecule into a protein there is provided the in cell translation of a tC O labelled RNA molecule into a protein.
• the encoded protein may be fused to a fluorescent protein (for example a GFP or mFruit family protein). When this is the case, it is possible to simultaneously visualise both the labelled RNA molecule and the protein it is being used to synthesise, giving a greater degree of mechanistic insight.
• the tC O labelled mRNA and the encoded protein may be simultaneously analysed spatiotemporally using confocal microscopy (for example fluorescence confocal microscopy).
• FIGURES [106] Figure 1: Schematic showing minimally perturbing tC O labelled RNA compared to a common externally labelled RNA [107] Figure 2: Basic synthetic scheme for the preparation of compound (I). [108] Figure 3: Incorporation of tC O into full length mRNA by T7 RNA polymerase assisted in vitro transcription. Denaturing agarose bleach gels showing RNA transcripts formed at five different tC O TP/CTP ratios (0-100%). Direct visualization of tC O fluorescence (a) and after ethidium bromide staining (b).
• RNA samples were heat-denatured (65 °C for 5 min, 1.5 % bleach in the gel) prior to loading.
• Figure 4 Incorporation of tC O into full length mRNA by SP6 and T7 RNA polymerase assisted in vitro transcription.
• RNA samples were heat- denatured (65 °C for 5 min, 1.5 % bleach in the gel) prior to loading on the gel.
• Figure 5 Spectroscopic characterization of in vitro synthesized tC O -modified RNA transcripts. Four reactions charged with different molar fractions of tC O TP in the total cytosine triphosphate pool (tC O TP + CTP) were performed. The product transcripts were purified to wash out unreacted triphosphates prior to characterization.
• Representative images (3x zoomed-in, scale bars: 10 ⁇ m), scatter plots and histograms, show the signal distribution in single living cells at (a, b) 24 h post-electroporation or (c) 48 h post-chemical transfection.
• the boxplots display the GFP mean fluorescence intensities (MFI GFP) up to 72 h from 3 independent experiments performed in triplicate.
• Cells overexpressing mRFP-Rab5 were transfected with 75 % tC O mRNA and followed overtime to validate tC O as an intracellular tracking probe (white arrows) not altering the translation, scale bars: 10 ⁇ m.
• RNA-tC O constructs Representative confocal images (large view, scale bar: 10 ⁇ m) of RNA-tC O constructs and mRNAs from TriLink® transfected by (a, e) electroporation or (b, f) chemical transfection.
• Example 1 Synthesis of Modified Nucleobase Triphosphates
• Compound (I) may be prepared according to the scheme shown in Fig. 2. Unless otherwise noted reagents were commercially available and used without further purification. The following reagents used for the triphosphorylation were bought from Sigma-Aldrich: DCA deblock for ⁇ KTA, CAP A for ⁇ KTA, CAP B1 and B2 for ⁇ KTA, BTT Activator. 1 H (500 MHz) and 13 C (126 MHz) NMR spectra were recorded at 300 K on a Bruker 500 MHz system equipped with a CryoProbe.
• CPG solid support 3 [117] Amino-SynBaseTM CPG 500/110 (LCAA) from LinkTech (Nu. 1397-C025, 1 g, 0.08 mmol) was activated by shaking in trichloroacetic acid 3% in DCE (8 mL, 0.08 mmol) for 18 h. The activated support was then filtered off and washed with 9:1 triethylamine:diisopropylethylamine (20 mL), dichloromethane (20 mL) and diethyl ether (20 mL). The activated support was dried under vacuum for 2 days before use.
• 5-chlorosalicylic acid was reduced with LAH (0.5 equiv.) at -20 °C and the resulting 5-chlorosalicylic alcohol was cyclized into 2,6-dichloro-4H-benzo[d][1,3,2]dioxaphosphinine using PCl 3 (1.2 equiv.) and triethylamine (2.3 equiv.) at -20 °C under argon.
• PCl 3 1.2 equiv.
• triethylamine 2.3 equiv.
• Oxidation Pyridine/Water/Iodine (9/1/12.7 v/v/w, 5mL) for 45 s, followed by ACN wash (3x5 mL) and drying of the support in an argon flow.
• Triphosphorylation Two injections of bis(tetrabutylammonium) dihydrogen diphosphate 6 (0.5 M, 5 ml) for 15 min and 18 hours, respectively. The support was subsequently rinsed with DMF (5 mL), water (3x5 mL), ACN (5 mL) and then dried in an argon flow.
• DMF diMF
• ACN ACN
• Compound (I) can also be made by a slightly modified route wherein the coupling step (b above) is carried out with a modified phosphoramidite such as 6-chloro-N,N-diisopropyl-4-methyl-4H- benzo[d][1,3,2]dioxaphosphinin-2-amine 8 (compound (IIIa) above).
• a modified phosphoramidite such as 6-chloro-N,N-diisopropyl-4-methyl-4H- benzo[d][1,3,2]dioxaphosphinin-2-amine 8 (compound (IIIa) above).
• RNA transcripts run as one single band on the gels, with a size corresponding to the expected 1247 nt mRNA product (H2B:GFP), demonstrating that the full-length mRNA is formed.
• H2B:GFP 1247 nt mRNA product
• tC O can be successfully incorporated into full-length RNA transcripts even under conditions where all canonical CTP is replaced with tC O TP (0% CTP; i.e. 100% C-labelling efficiency).
• Example 3 Spectroscopic characterization of in vitro synthesized tC O -modified RNA transcripts [134] A spectroscopic approach was used to quantify the incorporation efficiency of tC O TP, compared to the canonical CTP. To enable this, all RNA transcripts were purified using a Monarch RNA Cleanup kit, ensuring complete removal of unreacted tC O TP.
• Example 4 Translation of tC O -labelled mRNA in bacterial lysates [138]
• the labelled mRNAs encoding for the 17 kDa protein were transcribed from a commercial Calmodulin-3 DNA template plasmid using the same tC O TP/CTP ratios as for the H2B:GFP encoding mRNA (0 to 100% of tC O TP).
• the presence of Calmodulin-3 was confirmed by Coomassie staining (Fig.
• the mRNAs were capped with a 5 ⁇ -Cap 0 analogue and 3’-protected by poly-adenylation (by ca.300 nt).
• Live-cell confocal microscopy and flow cytometry showed that GFP fluorescence in the cell nuclei could be detected in 32, 25, 18, and 12% of the cells 24 hours post-electroporation for mRNA’s containing 25, 50, 75 and 100% of tC O , respectively (Fig.7a and Fig.8a). In comparison, the transfection efficiency with unmodified mRNA was 46%.
• RNA transcript can be accurately and efficiently translated by human ribosomal machineries, resulting in a correctly localized and folded protein product.
• Fig.7b the levels of H2B:GFP fluorescence in the cells was quantified (Fig.7b), showing a decrease in mean cellular H2B:GFP fluorescence intensity upon increasing the percentage of tC O in the transcript (approximately one order of magnitude difference between 0% and 100% of tC O (Fig.7a and 7f). This suggests that under these conditions, translation, as opposed to transcription, is somewhat impeded by the tC O modification, especially at the highest incorporation fraction.
• H2B:GFP fluorescence was found to increase gradually with time between 24 h and 72 h (Fig. 7c), which is a contrasting behaviour compared to electroporated cells (Fig.7a), suggesting that the lipofectamine-mRNA complexes are continuously internalized and, potentially, that endocytosed complexes progressively release more transcripts with time, counteracting the degradative effect in the cytosol.
• the tC O -labelled mRNAs were found to promote very similar H2B:GFP translation compared to the corresponding non-labelled mRNA, as indicated by the fluorescence levels in Fig. 7f.
• Cy5-tagged mRNA results in an average fluorescence level that is 80 % lower than that of its corresponding non-labelled transcript. This suggests that tC O does not impair the ability of mRNA to be processed by ribosomes upon chemical transfection, possibly because of an absence of charge and reduced steric hindrance, whereas Cy5, currently the most common commercial fluorophore for mRNA labelling, quite considerably impacts the translation process and/or interferes with a native-like uptake process of mRNA since it introduces significant amphiphilicity to the mRNA and, hence, possibly non-native interactions between the CY5- mRNA and the lipophilic membrane constituents.
• the complexation of the tC O -labelled mRNA with lipofectamine enabled its direct visualization inside cells using live cell confocal microscopy (Fig. 7c). This represents the first observation of fluorescent base analogue-labelled nucleic acids inside live cells. It was also found to be possible to simultaneously visualize, in real time, the uptake and subsequent translation of a fluorescent base analogue-modified mRNA by time-lapse recordings. We observed co-localization of the tC O signal with an mRFP-labelled Rab5 protein, thus highlighting that the mRNA transits through the early endosome (Fig. 7d).
• this technology allows tracking of both the intrinsically labelled mRNA transcripts and their translation products live, to gather spatiotemporal information on the translation product, even with as low as 50 % tC O content.
• Example 6 Biochemical Methods Generation of H2B:GFP DNA template
• the original coding sequence for H2B:GFP was taken from pCS2-H2B:GFP plasmid (Addgene, Plasmid #53744, manually codon-optimized to minimize the occurrence of poly-Cn stretches (n ⁇ 3), in silico-assembled with an additional T7 promoter and other desired features (Shine-Dalgarno/Kozak consensus sequences for enhancement of translation and a 3xStop, respectively at the 5 ⁇ and 3 ⁇ of the coding sequence itself, plus the needed HindIII/SnaBI restriction sites, to generate the ligation-prone sticky ends) and ordered from Twist Bioscience as a synthetic gene block.
• the obtained sequence was then PCR-amplified, using a Phusion Hot Start High-Fidelity Taq (Thermo Scientific), and subcloned into a HindIII/SnaBI-digested (Fast Digest enzymes, Thermo Scientific) empty pCS2 backbone. After ligation with T4 ligase for 1 h at room temperature (Roche), DH5 ⁇ E. coli competent cells (Invitrogen) were transformed following the recommended protocol, and obtained colonies were screened by colony-PCR.
• RNA was then enzymatically added with a polyA tail (with a Poly(A) Polymerase, NEB protocol #M0276 with incubation extended to 1 hour) and a Cap 0 analogue (using a Vaccinia capping system, NEB protocol #M2080), following the recommended procedures.
• Denaturing bleach-agarose gels [151] For a qualitative check of all in vitro synthesized RNAs, a denaturing agarose gel was run, in presence of 1,5 % bleach (Sigma Aldrich), as recommended in Aranda, P. S., LaJoie, D. M. & Jorcyk, C. L., Electrophoresis 33, 366-369 [2012].
• RNAs were first mixed with a 6x DNA loading dye (Invitrogen) and then heat-denatured at 70°C for 10 min in a heating block, then immediately transferred and kept on ice.
• the RiboRuler High Range RNA Ladder (Thermo Scientific) underwent the same treatment; 2 ⁇ l of RNA ladder were loaded along the samples and the gel was run at constant voltage (70 V) for 1 h and then imaged, under UV transillumination (302 nm) using a ChemiDoc Touch (BioRad).
• an in-house generated model of human hepatic Huh-7 cells stably overexpressing mRFP- Rab5 were cultured in DMEM/GlutaMax/High glucose (Gibco) supplemented with 10 % FBS. The cells are detached with trypsin-EDTA 0.05 % (Gibco) and passaged twice a week.
• Electroporation or chemical transfection Cells were electroporated either with 9.7 ⁇ g of tC O TP (for in vitro incorporation experiments) or 100 ng of tC O -labelled mRNA per 105 cells (for in vitro translation, cytotoxicity assessment, flow cytometry analysis and confocal microscopy), using a Neon Transfection System (Invitrogen, Carlsbad, CA, US) and following the protocol for 10 ⁇ L Neon Tip provided by the manufacturer, with a triple pulse of 1200 V and a pulse width of 20 ms.
• Neon Transfection System Invitrogen, Carlsbad, CA, US
• SH-SY5Y cells were seeded one day prior transfection at a density of 0.8106 cells/mL, in 48-well plate or glass-bottomed culture dishes for flow cytometry or confocal microscopy analysis, respectively.
• Lipofectamine MessengerMAX was used as chemical reagent for transfection according to the manufacturer’s instructions. Briefly, the reagent was diluted and incubated for 10 min at room temperature in Opti- MEM medium. The tC O -mRNA constructs were added to the reagent to reach a 1:1 final ratio reagent- mRNA (v/w), followed by a 5 min incubation at room temperature allowing the complex mRNA-lipid to form.
• LDH released in the supernatants of cells 24 h post-electroporated or post-transfected with tC O -labelled mRNA, or Cy5-mRNA was measured with a coupled enzymatic assay which results in the conversion of a tetrazolium salt into a red formazan product.
• the absorbance was recorded at 490 nm and 680 nm.
• the toxicity was expressed as the percentage of LDH release in supernatant compared to maximum LDH release (supernatant + cell lysate). Data are means ⁇ SD from three experiments performed in triplicate.
• Flow cytometry Following electroporation of tC O -labelled mRNA, cells were seeded in 48-well plate (2.105 cells/well) and the expression of H2B:GFP in cells was quantified by flow cytometry. Briefly, 24 h, 48 h or 72 h post-electroporation or post-transfection with tC O -mRNA, non-labelled mRNA or Cy5-mRNA, cells were harvested and analysed on a Guava EasyCyte 8HT flow cytometer (Millipore). Data are mean fluorescence intensities ⁇ SD of gated single living cells from three experiments performed in triplicate.
• the Huh-7 cells stably overexpressing mRFP-Rab5 were incubated with lipofectamine/tC O -mRNA complex and time-lapse was recorded up to 20 h post-chemical transfection.
• Confocal images were acquired on a Nikon C2+ confocal microscope equipped with a C2-DUVB GaAsP Detector Unit and using an oil-immersion 60 ⁇ 1.4 Nikon APO objective (Nikon Instruments, Amsterdam, Netherlands). Data were processed with the Fiji software.
• RNAs once purified, were used as templates for the cell-free translation reaction according to the manufacturer’s recommendations: E. coli slyD- Extract - 20 ⁇ l; 2.5X IVPS E. coli Reaction Buffer (-A.A.) - 20 ⁇ l; 50 mM Amino Acids (-Met) - 1.25 ⁇ l; 75 mM Methionine* - 1 ⁇ l; T7 Enzyme Mix - 1 ⁇ l (omitted when using tC O -labelled RNAs); DNA Template - 1 ⁇ g (when testing the tC O -labelled RNAs, added the same amount of RNA instead); DNase/RNase-free distilled water qsp 50 ⁇ l.
• PVDF membranes were then washed 5 min in TBS-T (TBS and 0,1 % Tween-20, Sigma Aldrich), blocked in 5 % milk in TBS for 1 h at room temperature and incubated with the appropriate primary antibody dilutions. [160] After 3x5 min washes in TBST and an incubation of 1 h with the corresponding HRP-conjugated secondary antibodies, the membrane was washed again three times in TBS-T, once in TBS and once more in distilled water. Finally, membranes were incubated with a minimal volume of SuperSignal West Pico PLUS (Thermo Scientific) and imaged with a ChemiDoc Touch.
• TBS-T TBS and 0,1 % Tween-20, Sigma Aldrich
• mice monoclonal anti-6xHistidine tag Invitrogen
• mouse monoclonal anti-GAPDH Ref. 437000, Invitrogen
• Secondary antibodies HRP-conjugated polyclonal goat anti-Ms and anti-Rb Cross-Adsorbed IgG (H+L) (ref. A16072 and A16104, Invitrogen), used at 1:10000 dilution in TBS-T.
• H+L HRP-conjugated polyclonal goat anti-Ms and anti-Rb Cross-Adsorbed IgG (H+L) (ref. A16072 and A16104, Invitrogen), used at 1:10000 dilution in TBS-T.
• Example 8 Spectroscopic Methods [161] The tC O -RNA products from the cell-free transcription reactions (prior to polyadenylation and capping, see Methods: Bio for details) were measured as received, i.e.
• Photon counts were recorded on a R3809U 50 microchannel plate PMT (Hamamatsu) and fed into a LifeSpec multichannel analyser (Edinburgh Analytical Instruments) with 2048 active channels (24.4 ps/channel), until the stop condition of 104 counts in the top channel was met.
• Equation S5 and S6 follows upon assuming first order reaction kinetics with respect to the triphosphate species [CTP] and [tC O TP]. [170] Solving S5 and S6 for the respective rate constants renders equation S7, in which [C] and [tC O ] denote the concentration of incorporated C and tC O , respectively.
• RNA yield ( ) was calculated according to equation S16.
• This specification demonstrates that an artificial, size-expanded analogue of cytosine takes the role of natural cytosine and is correctly recognized by several enzymatic machineries, including the ribosome.
• This fluorescent base analogue, tC O is demonstrated to be a suitable intrinsic imaging label of different size RNAs which minimally perturbs native properties and is compatible with enzymatic labelling processes.
• Modified transcripts are non-toxic and translationally active both in bacterial lysate and in eukaryotic systems, regardless of their degree of tC O incorporation. This conveniently allows for simultaneous monitoring of mRNA uptake and translation into H2B:GFP in live-cell confocal microscopy using selective excitation, an approach that should be applicable to the translation of any protein similarly tagged with a GFP family protein.
• the intrinsic fluorescence RNA-labelling methodologies disclosed herein are therefore excellent non-invasive ways to, in real time, elucidate cellular trafficking mechanisms such as endosomal escape or exosomes formation, both of which are of fundamental importance for pharmaceutical applications. As such the technology for live cell imaging should enable new and improved delivery strategies for next-generation nucleic acid-based drugs.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Biomedical Technology (AREA)
• Immunology (AREA)
• Urology & Nephrology (AREA)
• Hematology (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• General Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• General Physics & Mathematics (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Biophysics (AREA)
• Crystallography & Structural Chemistry (AREA)
• Plant Pathology (AREA)
• Saccharide Compounds (AREA)
• Electroluminescent Light Sources (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (1)

Family

ID=71949604

Family Applications (1)

Country Status (8)

Families Citing this family (1)

Citations (2)

Family Cites Families (4)

• 2020
  • 2020-06-25 GB GB2009705.1A patent/GB2600372A/en not_active Withdrawn
• 2021
  • 2021-06-24 EP EP21734353.2A patent/EP4172170B1/en active Active
  • 2021-06-24 WO PCT/EP2021/067342 patent/WO2021260107A1/en not_active Ceased
  • 2021-06-24 US US18/001,805 patent/US20230219995A1/en active Pending
  • 2021-06-24 ES ES21734353T patent/ES2995470T3/es active Active
  • 2021-06-24 JP JP2022580419A patent/JP2023533484A/ja active Pending
  • 2021-06-24 IL IL299302A patent/IL299302A/en unknown
  • 2021-06-24 DK DK21734353.2T patent/DK4172170T3/da active

Patent Citations (2)

Non-Patent Citations (6)

Also Published As

Similar Documents

Legal Events