Method for analyzing mRNA capping using an internal DNA motif

Abstract

A method for analyzing mRNA capping including hybridizing mRNA to a probe, the probe including a 3' ribonuclease segment, an interior deoxyribonuclease segment, and a 5' ribonuclease segment; cleaving the mRNA with a RNA:DNA specific ribonuclease; and analyzing the cleaved mRNA oligomer using liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS).

Description

METHOD FOR ANALYZING MRNA CAPPINGUSING AN INTERNAL DNA MOTIFCROSS-REFERENCE TO RELATED AFFLICTIONS

[0001] This application claims priority to the filing date of US Provisional Application S / N 63 / 504,674, entitled “Method for Analyzing MFNA Capping Using an Internal DNA Motiff,” filed on May 26, 2023, the disclosure of which is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.FIELD OF THE INVENTION

[0003] The present disclosure generally relates to the field of mass spectrometry including a method for analyzing mRNA capping using an internal DNA motif.BACKGROUND OF THE INVENTION

[0004] In Eukaryotic systems, non-mitochondrial messenger RNA (mRNA) are capped at the 5’ end by an inverted 7-methylguanosine (m7G) (Figure 1A) joined to the transcript by a 5’ to 5’ triphosphate linkage, m7G(5’)ppp(5’)N, referred to as Cap(0) (Figure 2A). Once added to the transcript the first nucleotide in the strand (N), is then methylated at the 2’ hydroxyl position to give the Cap(l) structure, m7G(5’)ppp(5’)Nm (Figure 2B). For example, Figure IB shows 2’-0-methyladenosine. Alternately, Nm can be 2’-0-methylguanosine (Gm), 2’ -O-methylcytosine Cm, or 2'-O-methyluridine (Um). Capping, as well as polyadenylation, help the transcript to resist degradation by the innate exonucleases in the cell. Besides blocking 5’ to 3’ exonucleases, the 5’ cap facilitates exportation from the nucleus to the cytoplasm and aids the translation initiation process by acting as a determinate for ribosomal docking complexes.

[0005] Further alteration of the transcript called splicing events also rely on the cap structure. Parts of the transcript termed intragenic regions, or introns, are removed and the cleaved sections rejoined, leaving the coding region to be expressed (exon). The coding region is flanked on both sides, 5’ and 3’, by an untranslated region, a stretch of RNA which is not translated into protein but does influence translation.A matured mRNA transcript then consists of 5 sections, the 5’ cap, the 5’ UTR, the coding region, the 3’ UTR and finally the polyadenylated tail (see Figure 3).

[0006] Due to recent events, particularly the COVID pandemic, mRNA has been widely adopted as a useful therapy, such as various mRNA based COVID vaccines. Production of non-natural transcripts by in vitro transcription, may require the addition of the modified cap structure post purification to achieve the desired therapeutic function. Modified 5' caps can include an anti-reverse cap analogs (ARCA) (U.S. Pat. No. 7,074,596), TriLink Biotechnology’s CleanCap analogs, or by a vaccinia capping enzyme followed by a methyltransferase reaction. Production of transcripts necessitate analytical examination to determine reaction outcome and yield, however, due to the size of the transcripts, a direct measurement, such as with mass spectrometry, is technologically challenging. As such, improved techniques for analyzing the capping and methylation efficiency are desirable.SUMMARY OF THE INVENTION

[0007] A method for analyzing mRNA capping can include hybridizing mRNA to a probe, the probe including a 3’ ribonuclease segment, an interior deoxyribonuclease segment, and a 5’ ribonuclease segment; cleaving the mRNA with a RNA:DNA specific ribonuclease; and analyze the cleaved mRNA oligomer using liquid chromatography-mass spectrometry or liquid chromatography-tandem mass spectrometry.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0009] Figures 1A and IB are diagrams illustrating the chemical structure of 7-methylguanosine and 2’- O-methyladenosine respectively.

[0010] Figures 2A and 2B are diagrams illustrating the chemical structure of Cap(0) and Cap(l ) respectively.

[0011] Figure 3 is a diagram illustrating the structure of mRNA.

[0012] Figures 4A and 4B are diagram illustrating the capped mRNA hybridized to an RNA probe containing a section of deoxyribonucleotides to form a cleavage site for the ribonuclease, in accordance with various embodiments.

[0013] Figure 5 is a flow diagram a method of analyzing the capping efficacy and 2’-O- methyltransferase efficiency, in accordance with various embodiments.

[0014] Figure 6 is a block diagram of an exemplary chromatography system, in accordance with various embodiments.

[0015] Figure 7 is a block diagram of an exemplary mass spectrometry system, in accordance with various embodiments.

[0016] Figures 8 A and 8B are chromatograms showing the isolation of the probe and the cleaved product, in accordance with various embodiments..

[0017] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.DETAILED DESCRIPTION

[0018] Embodiments of a method for analyzing mRNA capping are described herein.

[0019] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

[0020] In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

[0021] All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless described otherwise, all technical and scientific terms used herein have a meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs.

[0022] It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0023] As used herein, "a" or "an" also may refer to "at least one" or "one or more." Also, the use of “or” is inclusive, such that the phrase “A or B” is true when “A” is true, “B” is true, or both “A” and “B” are true. Further, unless otherwise required by context, singular' terms shall include plur alities and plural terms shall include the singular.

[0024] A “system” sets forth a set of components, real or abstract, comprising a whole where each component interacts with or is related to at least one other component within the whole.

[0025] Production of transcripts can necessitate analytical examination to determine reaction outcome and yield. However, due to the size of the transcripts, a direct measurement, such as with mass spectrometry, is technically challenging. Published methods using enzymes to digest the transcripts into a more manageable size range have been developed. One method uses the digestive enzyme RNase H. RNase H will cleave the RNA strand in an RNA:DNA double stranded hybrid at the Watson-Crick RNA:DNA base pair. The method, published by Beverly (Beverly, M.; Dell, A.; Parmar, P.; Houghton, L. Label-Free Analysis of MRNA Capping Efficiency Using RNase H Probes and LC-MS. Anal. Bioanal. Chem. 2016, 408 (18), 5021-5030) involves designing a biotinylated molecular probe -25 nt in length, complementary to the 5’ end of the transcript. The probe is annealed to the 5’ end of the mRNA, followed by complexing to magnetic beads, then subjected to an RNase H digestion. Figure 4A shows a biotinylated molecular probe 402 hybridized to an mRNA 404 and bound to a magnetic bead 406. The biotinylated molecular probe 402 includes a segment of deoxyribonucleotides 408 at the 5’ end with a segment of ribonucleotides 410 at the 3' end. The cleaved products are separated by a magnet, washed, eluted from bead and probe with hot methanol water, dried, then analyzed by LC-MS. The results will show that capping has occurred. However, published data on RNase Hl, the commercially available enzyme referenced in the Beverly paper, will not cleave at a single RNA:DNA base pair, but will instead produce multiple cleavage products down strand from the chimeric base pairing. When replicating the digestion, it is possible to detect multiple different oligonucleotides, with 3’ cleaved ends 1 to 2 nucleotides down strand from the RNA:DNA base pair. Furthermore, due to sequence homology betweendifferent nucleotide sections of the 5’ end of the mRNA, off target RNase H cleavage products can also be detected at high abundance.

[0026] Described herein is a method using a molecular probe consisting of a DNA segment flanked on both sides by an RNA segment complementary to the 5’ end of the transcript. The probe can be annealed to the 5’ end of the mRNA. In various embodiments, the molecular probe can be biotinylated and can be bound to a bead, such as a magnetic bead, using a biotin-streptaviden interaction or similar'. The annealed probe:mRNA complex can subjected to an RNase digestion, preferably an RNase that cleaves the RNA strand of a RNA / DNA heteroduplex, such as RNase H. Figure 4B shows a biotinylated molecular probe 452 hybridized to an mRNA 454 and bound to a magnetic bead 456. The biotinylated molecular probe 452 includes a segment of deoxyribonucleotides 458 flanked by a segment of ribonucleotides 460 at the 3’ end and a segment of ribonucleotides 462 at the 5’ end. In various embodiments, the molecular probe can include between about 15 and 30 nucleotides. The segment of ribonucleotides 460 can include at least 3 ribonucleotides, generally less than about 8 ribonucleotides. The segment of deoxyribonucleotides 458 can include one or more deoxyribonucleotides, generally less than about 8 ribonucleotides. The segment of ribonucleotides 462 can include at least 10 ribonucleotides, generally less than about 20 ribonucleotides. In various embodiments, the molecular probe can include modified deoxyribonucleotides within the segment of deoxyribonucleotides 458. In various embodiments, the molecular probe can contain modified ribonucleotides within segment of ribonucleotides 460, segment of ribonucleotides 462, or any combination thereof.

[0027] The cleaved products can be separated, such as by a magnet, washed, eluted from bead and probe with hot methanol water, dried, then analyzed by LC-MS or LC-MS / MS. The probe with RNA on both sides of the interior DNA segment allows for controlled cleavage, substantially reducing the off-target RNase H cleavage products. Additionally, the cleavage site is closer to the 5’ end of the transcript resulting in a shorter cleavage product. The shorter cleavage product is easier to dissolve for LC separation and easier to fragment in the mass spectrometer for MS / MS analysis.

[0028] Figure 5 illustrates method 500 of measuring 5’ capping efficiency and / or 2’-O-methyltransferase efficiency. At 502, the mRNA sample can be hybridized to a probe. In various embodiments, the mRNA can include modified ribonucleotides. In various embodiments, the probe can contain 3’ modifications, such as an attached biotin or other modification capable of coupling to a solid support such as a bead, including magnetic beads. In various embodiments, the probe may be immobilized on a surface. Alternatively, the probe may not be immobilized. The probe can include a 5’ RNA segment, an internal DNA segment, and 3’ RNA segment. In various embodiments, the molecular probe can include between about 10 and about 50 nucleotides, such as between about 15 and about 30 nucleotides. The segment ofribonucleotides 460 can include at least 3 ribonucleotides, generally less than about 10 ribonucleotides, such as less than about 8 ribonucleotides. The segment of deoxyribonucleotides 458 can include one or more deoxyribonucleotides, generally less than about 10 deoxyribonucleotides, such as about 3 to about 6 deoxyribonucleotides. The segment of ribonucleotides 462 can include at least about 5 ribonucleotides, such as at least about 10 ribonucleotides, generally less than about 40 ribonucleotides, such as less than about 20 ribonucleotides. In various embodiments, the probe can include modified deoxyribonucleotides within the internal DNA segment. In various embodiments, the probe can contain modified ribonucleotides within segment of ribonucleotides 460, segment of ribonucleotides 462, or any combination thereof.

[0029] Optionally, at 504, the hybridized probe: mRNA can be bound to a magnetic bead or other solid support. In various embodiments, the solid support can include binding sites for the probe. For example, a biotinylated probe can be captured by streptavidin bound on the surface of the solid support. In various embodiments, the biotinylated hybridized probe:mRNA can be incubated with the streptavidin coated magnetic beads for 5 to 60 minutes at 20°C to 40°C. Significantly, the incubation temperature should be below the annealing temperature of the probe and mRNA, which can be calculated by those of skill in the art based on the sequence. In other embodiments, the hybridized probemRNA may not be immobilized on a support and can be free floating in solution.

[0030] At 506, the hybridized probe and mRNA sample can be cleaved with an RNase that cleaves the RNA strand in a DNA:RNA duplex, such as RNase H. In various embodiments, the hybridized probe can be incubated with RNase H at a temperature of between about 30°C and about 70°C, such as between about 30°C and about 50°C, even between about 35°C and about 45°C for a period of time sufficient to cleave the DNA:RNA duplex. It is known that increasing the amount of enzyme can shorten the time to completion and one of skill in the art can identify a suitable amount of enzyme for a desired incubation period. In various embodiments, the enzymatic digestion can be carried out for at least about 30 minutes, such as at least about 45 minutes, such as at least about 1 hour, such as at least about 4 hours, such as at least about overnight (-12-16 hours). Generally, the enzymatic digestion can be less than about a day.

[0031] At 508, the cleavage product can be isolated, such as by using the magnetic beads to separate the cleavage product from the solution. For example, the beads can be magnetically held in place while the supernatant is removed. For example, magnetic beads can be precipitated using a magnet to pull the beads, along with the attached probe and cleavage product, to the bottom and the supernatant can be removed from the top. Alternatively, a centrifuge can be used to precipitate beads or other solid support particles with the attached probe and cleavage product. In other embodiments, the probe and cleavage products can be precipitated from solution by changing solvent conditions and centrifugation.

[0032] At 510, the oligomer can be separated from the probe, such as by using a solution of 75% methanol or incubating at an elevated temperature.

[0033] At 512, the digested sample can be chromatographically separated, and the chromatographically separated sample can be analyzed by mass spectrometry. This can include obtaining m / z and intensity data using the mass spectrometer, identifying and integrating the peaks. Further, the oligo can be fragmented in the mass spectrometer and the fragment ions can be analyzed to further elucidate the structure of the oligo.

[0034] Figure 6 depicts a liquid chromatography system 600 according to one aspect of the invention. The liquid chromatography system 600 comprises an analytical pump 602 to pump a solvent through the system 600. The system 600 comprises a sample reservoir 604 comprising a sample to be analyzed. The system 600 further comprises a separation column 606 and a detector 608. System 600 also comprises a controller 610.

[0035] The liquid chromatography system 600 is adapted to retrieve a sample from the sample reservoir 604. The sample can then be introduced into the system.

[0036] The system 600 is also adapted to inject the sample into the separation column 606 by means of the analytical flow. This can be done by guiding the sample by means of the analytical pump 602. The separation column 606 can separate the sample into component species based on retention time within the separation column 606. After separation of the sample by the separation column 606, the separated components can be detected by a detector 608. In some embodiments, the detector 608 can be an optical detector, such as an absorption detector, refractive index detector, a fluorescence detector, or the like. In other embodiments, the detector 608 can be a conductivity detector or an electrochemical detector. In yet other embodiments, the detector 608 can be a mass spectrometer.

[0037] In various embodiments, the separation column 606 generally consists of a tube packed with a stationary phase medium. The stationary phase medium can affect the time it takes for a compound to travel through the column (retention time). The effect can be different for different compounds, such that individual components of a sample can be separated based on their respective retention times. There are a variety of stationary phase mediums, including porous materials, ionic materials, polar- materials, nonpolar materials, and the like. Porous materials can affect retention time based on the size of a molecule and the ability of the molecule to enter the porous material. Ionic materials can affect retention time based on charge attraction or repulsion between the ionic material and the compounds. Polar and nonpolar materials can affect retention time based on the hydrophobicity or hydrophilicity of the compounds.

[0038] In various embodiments, nucleotides and nucleosides can be separated using a reverse phase separation in which a hydrophobic non-polar stationary phase material is used along with mobile phase with varying hydrophobicity depending on the ratio of polar to organic solvents. For example, a Cl 8 column can be used with an ammonium acetate or ammonium formate buffer system to separate the nucleosides. In particular embodiments, an aqueous mobile phase of 5 mM ammonium acetate at a pH of about 5 can be used and a gradient of increasing concentrations of acetonitrile (up to about 40%) or methanol (up to about 50%) can be used to separate the nucleosides. Suitable columns and buffer systems would be apparent to one of skill in the art and are within the scope of this disclosure.

[0039] In other embodiments, nucleotides and nucleosides can be separated using hydrophilic interaction liquid chromatography (HILIC) in which a hydrophilic stationary phase material is used along with hydrophobic mobile phase, such as acetonitrile. Suitable columns and buffer systems would be apparent to one of skill in the art and are within the scope of this disclosure.

[0040] Various embodiments of mass spectrometry platform 700 can include components as displayed in the block diagram of Figure 7. In various embodiments, mass spectrometry platform 700 can operate as a detector 608 of system 600. In various embodiments, elements of Figure 7 can be incorporated into mass spectrometry platform 700. According to various embodiments, mass speedometer 700 can include an ion source 702, a mass analyzer 704, an ion detector 706, and a controller 708.

[0041] In various embodiments, the ion source 702 generates a plurality of ions from a sample. The ion source can include, but is not limited to, a matrix assisted laser desorption / ionization (MALDI) source, electrospray ionization (ESI) source, atmospheric pressure chemical ionization (APCI) source, atmospheric pressure photoionization source (APPI), inductively coupled plasma (ICP) source, electron ionization source, chemical ionization source, photoionization source, glow discharge ionization source, thermospray ionization source, and the like.

[0042] In various embodiments, the mass analyzer 704 can separate ions based on a mass-to-charge ratio of the ions. For example, the mass analyzer 704 can include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., Orbitrap) mass analyzer, Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, and the like. In various embodiments, the mass analyzer 704 can also be configured to fragment the ions using collision induced dissociation (CID) electron transfer dissociation (ETD), electron capture dissociation (ECD), photo induced dissociation (PID), surface induced dissociation (SID), and the like, and further separate the fragmented ions based on the mass-to-charge ratio.

[0043] In various embodiments, the ion detector 706 can detect ions. For example, the ion detector 706 can include an electron multiplier, a Faraday cup, and the like. Ions leaving the mass analyzer can be detected by the ion detector. In various embodiments, the ion detector can be quantitative, such that an accurate count of the ions can be determined. In various embodiments, the functions of the mass analyzer 704 and the ion detector 706 can be combined, such as in an Orbitrap mass analyzer.

[0044] In various embodiments, the controller 708 can communicate with the ion source 702, the mass analyzer 704, and the ion detector 706. For example, the controller 708 can configure the ion source or enable / disable the ion source. Additionally, the controller 708 can configure the mass analyzer 704 to select a particular mass range to detect. Further, the controller 708 can adjust the sensitivity of the ion detector 706, such as by adjusting the gain. Additionally, the controller 708 can adjust the polarity of the ion detector 706 based on the polarity of the ions being detected. For example, the ion detector 706 can be configured to detect positive ions or be configured to detected negative ions.

[0045] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0046] Further, in describing various embodiments, the specification may have presented a method and / or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the ai t would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and / or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for analyzing mRNA capping comprising: hybridizing mRNA to a probe, the probe including a 3’ ribonuclease segment, an interior deoxyribonuclease segment, and a 5’ ribonuclease segment; cleaving the mRNA with a RNA:DNA specific ribonuclease; and analyzing the cleaved mRNA oligomer using liquid chromatography-mass spectrometry or liquid chromatography-tandem mass spectrometry.

2. The method of claim 1, wherein the probe contains a 3’ modification.

3. The method of claim 2, wherein the probe is immobilized on a solid support with the 3’ modification.

4. The method of claim 3, wherein the solid support is a bead.

5. The method of claim 4, wherein the bead is a magnetic bead.

6. The method of claim 1, wherein the probe contains modified deoxyribonucleotides.

7. The method of claim 1, wherein the probe contains modified ribonucleotides.

8. The method of claim 1, wherein the mRNA contains modified ribonucleotides.

9. The method of claim 1, wherein the deoxyribonuclease segment includes one or more deoxyribonucleotides.

10. The method of claim 9, wherein the deoxyribonuclease segment includes less than about 10 dcoxyribonuclcotidcs .

11. The method of claim 10, wherein the deoxyribonuclease segment includes about 3 to about 6 deoxyribonucleotides .

12. The method of claim 1, wherein the 3’ ribonuclease segment includes at least 3 ribonucleotides.

13. The method of claim 12, wherein the 3’ ribonuclease segment includes less than about 10 ribonucleotides.

14. The method of claim 13, wherein the 3' ribonuclease segment includes less than about 8 ribonucleotides.

15. The method of claim 1, wherein the 5’ ribonuclease segment includes at least about 5 ribonucleotides.

16. The method of claim 15, wherein the 5’ ribonuclease segment includes at least about 10 ribonucleotides.

17. The method of claim 15, wherein the 5’ ribonuclease segment includes less than about 40 ribonucleotides.

18. The method of claim 17, wherein the 5’ ribonuclease segment includes less than about 20 ribonucleotides.

19. The method of claim 1, wherein probe has a length of between about 10 and 50 nucleotides.

20. The method of claim 19, wherein probe has a length of between about 15 and 30 nucleotides.

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