How to measure polyA tail length

Abstract

The present invention provides, inter alia, a method for measuring homopolymer nucleotide length in nucleic acids, including mRNA. In some aspects, provided herein is a method for measuring poly-A tail length in mRNA, comprising binding the mRNA with a minor groove binding dye, followed by ribonuclease digestion and capillary electrophoresis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 181,488, filed April 29, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the text file named "MRT-2220WOl_ST25.txt", created on April 15, 2022 and 688 bytes in size, are incorporated herein by reference in their entirety. [Background technology]

[0003] Messenger RNA therapy (MRT) is a promising approach to treat various diseases. MRT involves administering messenger RNA (mRNA) to a patient in need of treatment. The administered mRNA produces the protein or peptide encoded by the mRNA in the patient's body. The mRNA is typically synthesized using an in vitro transcription system (IVT), which involves an enzymatic reaction with RNA polymerase. The IVT synthesis process is usually followed by reaction(s) to add a 5'-cap (capping reaction) and a 3'-polyA tail (polyadenylation).

[0004] Effective mRNA therapy requires delivery of mRNA to patients and efficient production of the protein encoded by the mRNA in the patient's body. The 5' cap and 3' polyA tail play a role in optimizing mRNA delivery and protein production in vivo. The 5' cap prevents degradation and improves translation. The 3' polyA tail protects the mRNA from exonuclease degradation and improves the integrity and stability of the mRNA for mRNA therapy. Summary of the Invention [Problem to be solved by the invention]

[0005] Assessment of polyA tail length is performed as a quality control measurement for mRNA therapy. Accurate polyA tail length measurement is also used to establish dosage by accurately quantifying stable mRNA therapeutics that are intact, full-length, and translated into functional protein upon delivery. Currently available methods for determining polyA tail length have certain drawbacks, including, among others, low accuracy.

[0006] Current methods for determining polyA tail length include, for example, measurements using polyA binding protein assays, which require a polyA tail long enough to bind at least four monomers of polyA binding protein, with each monomer binding to a stretch of approximately 38 nucleotides. Other methods include ligation-mediated polyA measurement based on PCR assays, which require a reverse transcription step, and using cDNA synthesis from oligo-dT primers, which can be inaccurate for longer polyA tails. Another method, the RNase H-based method, involves removing the polyA tail from the mRNA of interest, and is not suitable for mRNA therapy, which requires an intact polyA tail.

[0007] Methods for measuring mRNA tail length for mRNA therapy include, for example, capillary electrophoresis (CE) and RNase A methods.

[0008] The CE method does not require enzymatic digestion and is achieved with a short run time of about 1 hour. It can be employed in a high-throughput manner with up to 48 samples being processed simultaneously. However, as the tail length increases, the measured tail length is often inaccurate because of the shift in retention time in capillary electrophoresis. CE methods generally employ intercalating dyes, such as Agilent™ intercalating dyes, which result in a weak signal with homopolymer stretches that contain poly-A tails.

[0009] The RNase A gel method requires an enzymatic digestion that allows for the subsequent specific degradation of C and U, leaving the polyA tail in a state to be measured. It is reproducible and provides a consistent measurement of tail length. However, it requires a 30 min enzymatic digestion step and 2 h 30 min gel running time. This method is performed in a low-throughput manner of approximately 10 samples / gel. [Means for solving the problem]

[0010] The present invention provides, inter alia, a method for accurately measuring poly-A tail length in an mRNA sample in a rapid and high-throughput manner. The present invention is based, in part, on the surprising and unexpected discovery that binding of a minor groove binding dye to mRNA, followed by ribonuclease (RNase) digestion and capillary electrophoresis (CE), provides an accurate method for determining the poly-A tail length of mRNA. In some embodiments, one or more steps of the method are automated. In some embodiments, the method is high-throughput.

[0011] Accurately measuring long polyA tail lengths of 50-200 nucleotides or more is difficult using current methods. Provided herein are methods that can reliably and accurately measure long polyA tail lengths of 50 or more, 100 or more, 150 or more, or 200 or more nucleotides in an efficient, high-throughput manner. In some embodiments, the polyA tail length measured using this method is equal to the theoretical tail length. In some embodiments, the measured tail length is 100% accurate. In some embodiments, the measured polyA tail length approaches the theoretical tail length. In some embodiments, the measured polyA tail length is greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% accurate. In some embodiments, the measured polyA tail length is more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% accurate.

[0012] Capillary electrophoresis (CE) is typically performed using an intercalating dye. In some embodiments, the intercalating dye is an Agilent™ intercalating dye. The present invention is based, in part, on the unexpected results obtained by using minor groove binding dyes in capillary electrophoresis. Thus, in some embodiments, the dye used in the methods described herein is a minor groove binding dye. In some embodiments, the minor groove binding dye is Sybr gold™.

[0013] The polyA tail is helical and composed of planarly stacked bases. Minor groove binding dyes selectively bind RNA non-covalently through hydrogen bonds and hydrophobic interactions. Without wishing to be bound by any particular theory, it is believed that minor groove binding dyes, including Sybr gold™, are able to bind to the planar structure formed by the polyA tail due to base stacking-independent interactions. Intercalating dyes, such as Agilent™ intercalating dyes, produce aqueous polyA signals, resulting in inaccurate polyA tail length measurements, because the intercalating dye cannot properly π-stack into the planar structure created by the polyA tail. In contrast, Sybr gold™ non-covalently binds to the helical structure of the polyA tail by binding to the minor groove formed by the single-stranded secondary structure, providing a way to accurately measure polyA tail length (Figure 1B).

[0014] In some aspects, provided herein are methods of measuring poly-A tail length in an mRNA sample, the methods comprising: (a) contacting the mRNA sample with a minor groove binding dye; (b) incubating the mRNA sample from (a) with one or more ribonucleases (RNases); and (c) assaying the sample from (b) by capillary electrophoresis (CE) to determine the poly-A tail length of the mRNA.

[0015] In some embodiments, provided herein is a method in which the minor groove binding dye is Sybr gold™, a Hoechst dye, or 4',6-diamidino-2-phenylindole (DAPI). In some embodiments, the minor groove binding dye is Sybr gold™. In some embodiments, the minor groove binding dye is a Hoechst dye. In some embodiments, the minor groove binding dye is 4',6-diamidino-2-phenylindole (DAPI).

[0016] In some embodiments, methods are provided herein, wherein an mRNA sample is incubated with one or more RNases selected from RNase A and RNase T1. In some embodiments, the one or more RNases are RNase A. In some embodiments, the one or more RNases are RNase T1. In some embodiments, the one or more RNases include RNase A1 and RNase T1. RNase A degrades RNA after C and U residues, and RNase T1 degrades after G residues. Digestion with RNase A and RNase T1 ensures that only the polyA tail remains.

[0017] In some embodiments, methods are provided herein in which capillary electrophoresis (CE) is coupled with fluorescence-based detection.

[0018] In some embodiments, methods are provided herein in which capillary electrophoresis (CE) is coupled with UV absorption spectroscopy detection.

[0019] In some embodiments, the mRNA sample is incubated with one or more ribonuclease (RNase) for about 15 minutes, 30 minutes, 45 minutes, or 60 minutes. In some embodiments, the mRNA sample is incubated with one or more ribonuclease (RNase) for about 15 minutes. In some embodiments, the mRNA sample is incubated with one or more ribonuclease (RNase) for about 30 minutes. In some embodiments, the mRNA sample is incubated with one or more ribonuclease (RNase) for about 45 minutes. In some embodiments, the mRNA sample is incubated with one or more ribonuclease (RNase) for about 60 minutes.

[0020] In some embodiments, the polyA tail length is 25 nucleotides or more.

[0021] In some embodiments, the polyA tail length is between 50 nucleotides and 5,000 nucleotides. In some embodiments, the polyA tail length is about 50 nucleotides. In some embodiments, the polyA tail length is about 100 nucleotides. In some embodiments, the polyA tail length is about 150 nucleotides. In some embodiments, the polyA tail length is about 200 nucleotides. In some embodiments, the polyA tail length is about 250 nucleotides. In some embodiments, the polyA tail length is about 300 nucleotides. In some embodiments, the polyA tail length is about 350 nucleotides. In some embodiments, the polyA tail length is about 400 nucleotides. In some embodiments, the polyA tail length is about 450 nucleotides. In some embodiments, the polyA tail length is about 500 nucleotides. In some embodiments, the polyA tail length is about 550 nucleotides. In some embodiments, the polyA tail length is about 600 nucleotides. In some embodiments, the polyA tail length is about 650 nucleotides. In some embodiments, the polyA tail length is about 700 nucleotides. In some embodiments, the polyA tail length is about 750 nucleotides. In some embodiments, the polyA tail length is about 800 nucleotides. In some embodiments, the polyA tail length is about 850 nucleotides. In some embodiments, the polyA tail length is about 900 nucleotides. In some embodiments, the polyA tail length is about 950 nucleotides. In some embodiments, the polyA tail length is about 1000 nucleotides. In some embodiments, the polyA tail length is about 1200 nucleotides. In some embodiments, the polyA tail length is about 1400 nucleotides. In some embodiments, the polyA tail length is about 1600 nucleotides. In some embodiments, the polyA tail length is about 1800 nucleotides. In some embodiments, the polyA tail length is about 2000 nucleotides. In some embodiments, the polyA tail length is about 2200 nucleotides. In some embodiments, the polyA tail length is about 2400 nucleotides. In some embodiments, the polyA tail length is about 2600 nucleotides. In some embodiments, the polyA tail length is about 2800 nucleotides.In some embodiments, the polyA tail length is about 3000 nucleotides. In some embodiments, the polyA tail length is about 3200 nucleotides. In some embodiments, the polyA tail length is about 3400 nucleotides. In some embodiments, the polyA tail length is about 3600 nucleotides. In some embodiments, the polyA tail length is about 3800 nucleotides. In some embodiments, the polyA tail length is about 4000 nucleotides. In some embodiments, the polyA tail length is about 4200 nucleotides. In some embodiments, the polyA tail length is about 4400 nucleotides. In some embodiments, the polyA tail length is about 4600 nucleotides. In some embodiments, the polyA tail length is about 4800 nucleotides. In some embodiments, the polyA tail length is about 5000 nucleotides.

[0022] In some embodiments, the polyA tail is 50 or more nucleotides in length. In some embodiments, the polyA tail is 100 or more nucleotides in length. In some embodiments, the polyA tail is 150 or more nucleotides in length. In some embodiments, the polyA tail is 200 or more nucleotides in length.

[0023] In some embodiments, the polyA tail length is between 100 nucleotides and 1,500 nucleotides. In some embodiments, the polyA tail length is about 100 nucleotides. In some embodiments, the polyA tail length is about 200 nucleotides. In some embodiments, the polyA tail length is about 300 nucleotides. In some embodiments, the polyA tail length is about 400 nucleotides. In some embodiments, the polyA tail length is about 500 nucleotides. In some embodiments, the polyA tail length is about 600 nucleotides. In some embodiments, the polyA tail length is about 700 nucleotides. In some embodiments, the polyA tail length is about 800 nucleotides. In some embodiments, the polyA tail length is about 900 nucleotides. In some embodiments, the polyA tail length is about 1000 nucleotides.

[0024] In some embodiments, the polyA tail length is between 250 and 500 nucleotides. In some embodiments, the polyA tail length is about 250 nucleotides. In some embodiments, the polyA tail length is about 260 nucleotides. In some embodiments, the polyA tail length is about 270 nucleotides. In some embodiments, the polyA tail length is about 280 nucleotides. In some embodiments, the polyA tail length is about 290 nucleotides. In some embodiments, the polyA tail length is about 300 nucleotides. In some embodiments, the polyA tail length is about 310 nucleotides. In some embodiments, the polyA tail length is about 320 nucleotides. In some embodiments, the polyA tail length is about 330 nucleotides. In some embodiments, the polyA tail length is about 340 nucleotides. In some embodiments, the polyA tail length is about 350 nucleotides. In some embodiments, the polyA tail length is about 360 nucleotides. In some embodiments, the polyA tail length is about 370 nucleotides. In some embodiments, the polyA tail length is about 380 nucleotides. In some embodiments, the polyA tail length is about 390 nucleotides. In some embodiments, the polyA tail length is about 400 nucleotides. In some embodiments, the polyA tail length is about 410 nucleotides. In some embodiments, the polyA tail length is about 420 nucleotides. In some embodiments, the polyA tail length is about 430 nucleotides. In some embodiments, the polyA tail length is about 440 nucleotides. In some embodiments, the polyA tail length is about 450 nucleotides. In some embodiments, the polyA tail length is about 460 nucleotides. In some embodiments, the polyA tail length is about 470 nucleotides. In some embodiments, the polyA tail length is about 480 nucleotides. In some embodiments, the polyA tail length is about 490 nucleotides. In some embodiments, the polyA tail length is about 500 nucleotides.

[0025] In some embodiments, one or more steps of the method are automated.

[0026] In some embodiments, incubating the mRNA sample with one or more ribonucleases (RNases) is automated.

[0027] In some embodiments, the minor groove binding dye non-covalently binds to single-stranded RNA (ssRNA).

[0028] In some embodiments, the minor groove binding dye is not an intercalating dye.

[0029] In some aspects, provided herein are methods of measuring poly-A tail length in mRNA, the methods comprising: (a) contacting an mRNA sample with Sybr gold™ minor groove binding dye; (b) incubating the mRNA sample from (a) with RNase A and RNase T1; and (c) assaying the sample from (b) by capillary electrophoresis (CE) to determine the poly-A tail length of the mRNA.

[0030] In some embodiments, the method comprises capillary electrophoresis (CE) coupled with fluorescence-based detection.

[0031] In some embodiments, the method comprises capillary electrophoresis (CE) coupled with UV absorption spectroscopy detection.

[0032] In some embodiments, the method includes incubating the mRNA sample with RNase A and RNase T1 for about 15 minutes, 30 minutes, 45 minutes, or 60 minutes. In some embodiments, the mRNA sample is incubated with RNase A and RNase T1 for about 15 minutes. In some embodiments, the mRNA sample is incubated with RNase A and RNase T1 for about 30 minutes. In some embodiments, the mRNA sample is incubated with RNase A and RNase T1 for about 45 minutes. In some embodiments, the mRNA sample is incubated with RNase A and RNase T1 for about 60 minutes.

[0033] In some embodiments, the polyA tail length is 25 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 150 or more nucleotides, or 200 or more nucleotides. In some embodiments, the polyA tail length is 25 or more nucleotides. In some embodiments, the polyA tail length is 50 or more nucleotides. In some embodiments, the polyA tail length is 100 or more nucleotides. In some embodiments, the polyA tail length is 150 or more nucleotides. In some embodiments, the polyA is 200 or more nucleotides.

[0034] In some embodiments, one or more steps of the method are automated.

[0035] In some embodiments, the method is high throughput.

[0036] In some aspects, provided herein are methods for measuring homopolymeric nucleotide lengths in an mRNA sample, the methods comprising: (a) contacting the mRNA sample with a minor groove binding dye; (b) incubating the mRNA sample from (a) with one or more ribonucleases (RNases); and (c) assaying the sample from (b) by capillary electrophoresis (CE) to determine the homopolymeric nucleotide length of the mRNA.

[0037] In some embodiments, the homopolymer nucleotide length is 25 nucleotides or more.

[0038] In some embodiments, the homopolymer nucleotide length is between 50 nucleotides and 5,000 nucleotides. In some embodiments, the homopolymer nucleotide length is 50 nucleotides. In some embodiments, the homopolymer nucleotide length is 100 nucleotides. In some embodiments, the homopolymer nucleotide length is 150 nucleotides. In some embodiments, the homopolymer nucleotide length is 200 nucleotides. In some embodiments, the homopolymer nucleotide length is 250 nucleotides. In some embodiments, the homopolymer nucleotide length is 300 nucleotides. In some embodiments, the homopolymer nucleotide length is 350 nucleotides. In some embodiments, the homopolymer nucleotide length is 400 nucleotides. In some embodiments, the homopolymer nucleotide length is 450 nucleotides. In some embodiments, the homopolymer nucleotide length is 500 nucleotides. In some embodiments, the homopolymer nucleotide length is 550 nucleotides. In some embodiments, the homopolymer nucleotide length is 600 nucleotides. In some embodiments, the homopolymer nucleotide length is 650 nucleotides. In some embodiments, the homopolymer nucleotide length is 700 nucleotides. In some embodiments, the homopolymer nucleotide length is 750 nucleotides. In some embodiments, the homopolymer nucleotide length is 800 nucleotides. In some embodiments, the homopolymer nucleotide length is 850 nucleotides. In some embodiments, the homopolymer nucleotide length is 900 nucleotides. In some embodiments, the homopolymer nucleotide length is 950 nucleotides. In some embodiments, the homopolymer nucleotide length is 1000 nucleotides.

[0039] In some embodiments, the homopolymer nucleotide length is 1100 nucleotides. In some embodiments, the homopolymer nucleotide length is 1200 nucleotides. In some embodiments, the homopolymer nucleotide length is 1300 nucleotides. In some embodiments, the homopolymer nucleotide length is 1400 nucleotides. In some embodiments, the homopolymer nucleotide length is 1500 nucleotides. In some embodiments, the homopolymer nucleotide length is 1600 nucleotides. In some embodiments, the homopolymer nucleotide length is 1700 nucleotides. In some embodiments, the homopolymer nucleotide length is 1800 nucleotides. In some embodiments, the homopolymer nucleotide length is 1900 nucleotides. In some embodiments, the homopolymer nucleotide length is 2000 nucleotides. In some embodiments, the homopolymer nucleotide length is 2100 nucleotides. In some embodiments, the homopolymer nucleotide length is 2200 nucleotides. In some embodiments, the homopolymer nucleotide length is 2300 nucleotides. In some embodiments, the homopolymer nucleotide length is 2400 nucleotides. In some embodiments, the homopolymer nucleotide length is 2500 nucleotides. In some embodiments, the homopolymer nucleotide length is 2600 nucleotides. In some embodiments, the homopolymer nucleotide length is 2700 nucleotides. In some embodiments, the homopolymer nucleotide length is 2800 nucleotides. In some embodiments, the homopolymer nucleotide length is 2900 nucleotides. In some embodiments, the homopolymer nucleotide length is 3000 nucleotides. In some embodiments, the homopolymer nucleotide length is 3100 nucleotides. In some embodiments, the homopolymer nucleotide length is 3200 nucleotides. In some embodiments, the homopolymer nucleotide length is 3300 nucleotides. In some embodiments, the homopolymer nucleotide length is 3400 nucleotides. In some embodiments, the homopolymer nucleotide length is 3500 nucleotides. In some embodiments, the homopolymer nucleotide length is 3600 nucleotides.In some embodiments, the homopolymer nucleotide length is 3700 nucleotides. In some embodiments, the homopolymer nucleotide length is 3800 nucleotides. In some embodiments, the homopolymer nucleotide length is 3900 nucleotides. In some embodiments, the homopolymer nucleotide length is 4000 nucleotides. In some embodiments, the homopolymer nucleotide length is 4100 nucleotides. In some embodiments, the homopolymer nucleotide length is 4200 nucleotides. In some embodiments, the homopolymer nucleotide length is 4300 nucleotides. In some embodiments, the homopolymer nucleotide length is 4400 nucleotides. In some embodiments, the homopolymer nucleotide length is 4500 nucleotides. In some embodiments, the homopolymer nucleotide length is 4600 nucleotides. In some embodiments, the homopolymer nucleotide length is 4700 nucleotides. In some embodiments, the homopolymer nucleotide length is 4800 nucleotides. In some embodiments, the homopolymer nucleotide length is 4900 nucleotides. In some embodiments, the homopolymer nucleotide length is 5000 nucleotides.

[0040] In some embodiments, the homopolymer nucleotide length is 50 or more nucleotides, 100 or more nucleotides, 150 or more nucleotides, or 200 or more nucleotides. In some embodiments, the homopolymer nucleotide length is 50 or more nucleotides. In some embodiments, the homopolymer nucleotide length is 100 or more nucleotides. In some embodiments, the homopolymer nucleotide length is 150 or more nucleotides. In some embodiments, the homopolymer nucleotide length is 200 or more nucleotides.

[0041] In some embodiments, the nucleotides comprising the homopolymeric nucleotide tract are selected from A, U, G, or C. In some embodiments, the nucleotides comprising the homopolymeric nucleotide tract are adenine. In some embodiments, the nucleotides comprising the homopolymeric nucleotide are uracil. In some embodiments, the nucleotides comprising the homopolymeric nucleotide are guanine. In some embodiments, the nucleotides comprising the homopolymeric nucleotide are cytosine.

[0042] In this application, the use of "or" means "and / or" unless otherwise specified. As used in this disclosure, the term "comprise" and variations of terms such as "comprising" and "comprises" are not intended to exclude other additives, components, integers or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Both terms are meant to cover any normal fluctuations recognized by one of ordinary skill in the relevant art.

[0043] Other features, objects, and advantages of the present invention will be apparent in the following detailed description, drawings, and claims. It should be understood, however, that the detailed description, drawings, and claims, while indicating embodiments of the present invention, are given by way of illustration only, and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

[0044] The drawings are for illustrative purposes only, and not for limitation. [Brief description of the drawings]

[0045] [Figure 1A] 1 is a schematic diagram showing the binding of dyes and other ligands by different mechanisms to nucleic acids, in which an exemplary double-stranded DNA molecule is used to indicate the dye binding position, for example, by an intercalating dye, a major groove binder, a minor groove binder or a bisintercalator. [Figure 1B] FIG. 1B is a schematic diagram showing the RNA secondary structure formed such that a dye can be attached, as shown in FIG. 1A. [Figure 2A] 1 shows a gel showing RNase A analysis of EPO mRNA containing co-transcriptionally added polyA tails with polyA tail lengths of 25 nt, 50 nt and 114 nt for Sybr gold™ dye-treated CE samples and conventional RNase A digested samples. [Figure 2B] A graph is shown between observed tail length on the y-axis and theoretical tail length on the x-axis for EPO mRNA with poly-A tail lengths of 25 nt, 50 nt and 114 nt. Results are shown for Agilent™ dye treated CE samples, Sybr gold™ dye treated CE samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths. [Figure 3A] 1 shows a capillary electrophoresis gel showing an exemplary EPO mRNA with poly-A tail lengths of 100-600 nucleotides after Sybr Gold™ binding followed by RNase A digestion. [Figure 3B] 1 shows a capillary electrophoresis gel showing an exemplary EPO mRNA with a poly-A tail length between 100 and 600 nucleotides after Agilent™ intercalating dye binding followed by RNase A digestion. [Figure 4A] 1 shows a gel showing conventional RNase A analysis of EPO mRNA with polyA tails ranging from 100 to 600 nucleotides in length. [Figure 4B] Figure 1 shows a graph between observed tail length on the y-axis and theoretical tail length on the x-axis for EPO mRNA with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt. Results are shown for Agilent™ dye treated CE samples, Sybr gold™ dye treated CE samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths. [Figure 5A]Capillary electropherograms of 1.2 μg of Sybr gold™ dye-treated EPO mRNA samples with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt are shown after RNase A digestion. [Figure 5B] Capillary electropherograms of 1.2 μg Agilent™ dye-treated EPO mRNA samples with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt are shown after RNase A digestion. [Figure 6A] Figures 6A and 6B show a comparison of signal intensities of Sybr gold™ dye-treated EPO mRNA samples and Agilent™ dye-treated EPO mRNA samples: Figure 6A shows a capillary electropherogram of 1.2 μg of Sybr gold™ dye-treated EPO mRNA with a polyA tail length of 200 nt after RNase A digestion. [Figure 6B] Figures 6A and 6B show a comparison of signal intensity of Sybr gold™ dye-treated EPO mRNA samples and Agilent™ dye-treated EPO mRNA samples, and Figure 6B shows a capillary electropherogram of 1.2 μg of Agilent™ dye-treated EPO mRNA with a polyA tail length of 200 nt after RNase A digestion. [Figure 7] FIG. 1 shows a graph between observed tail length on the y-axis and theoretical tail length on the x-axis for EPO mRNA with polyA tail lengths of 100-1800 nucleotides comparing RNase A and RNase A / T1 digested products. [Figure 8A] A gel is shown following RNase A digestion of Sybr gold™ dye-treated CFTR mRNA with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 8B]A gel is shown following RNase A digestion of Agilent™ dye-treated CFTR mRNA with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 9A] Capillary electropherograms are shown following RNase A digestion of 1.2 μg Sybr gold™ dye-treated CFTR mRNA samples with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 9B] Capillary electropherograms are shown following RNase A digestion of 1.2 μg Agilent™ dye-treated CFTR mRNA samples with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 10] Figure 1 shows a graph between observed tail length on the y-axis and theoretical tail length on the x-axis for CFTR mRNA with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt. Results are shown for Agilent™ dye treated CE samples, Sybr gold™ dye treated CE samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths. [Figure 11A] Shown is a gel after RNase A digestion of Sybr gold™ dye treated OTC mRNA with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 11B] A gel is shown following RNase A digestion of Agilent™ dye-treated OTC mRNA with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 12A] Capillary electropherograms of 1.2 μg Sybr gold™ dye-treated OTC mRNA samples after RNase A digestion with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control are shown. [Figure 12B] Capillary electropherograms of 1.2 μg Agilent™ dye-treated OTC mRNA samples with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt are shown following RNase A digestion compared to an undigested control. [Figure 13] Figure 1 shows a graph between observed tail length on the y-axis and theoretical tail length on the x-axis for OTC mRNA with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt. Results are shown for Agilent™ dye treated CE samples, Sybr gold™ dye treated CE samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths. [Figure 14A] Shown is a gel following RNase A digestion of Sybr gold™ dye treated MMA mRNA with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 14B] A gel is shown following RNase A digestion of Agilent™ dye-treated MMA mRNA with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control. [Figure 15A] Capillary electropherograms of 1.2 μg Sybr gold™ dye-treated MMA mRNA samples with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt are shown following RNase A digestion compared to undigested controls. [Figure 15B] Capillary electropherograms of 1.2 μg Agilent™ dye-treated MMA mRNA samples after RNase A digestion with polyA tail lengths of 100, 200, 300, 400, 500 and 600 nt compared to an undigested control are shown. [Figure 16]Figure 1 shows a graph between observed tail length on the y-axis and theoretical tail length on the x-axis for MMA mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt. Results are shown for Agilent™ dye treated CE samples, Sybr gold™ dye treated CE samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are incorporated herein by reference.

[0047] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the reference value referred to. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater or smaller) of the reference value mentioned above, unless otherwise stated or clear from the context (unless such number exceeds 100% of possible values).

[0048] Batch: As used herein, the term "batch" refers to the content or amount of mRNA synthesized at one time, for example, produced according to a single production sequence during the same production cycle. A batch can refer to the amount of mRNA synthesized in one reaction that occurs through a single aliquot of enzyme and / or a single aliquot of DNA template to synthesize continuously under one set of conditions. In some embodiments, a batch includes mRNA produced from a reaction in which not all reagents and / or components are replenished and / or supplemented as the reaction proceeds. The term "batch" does not refer to mRNAs synthesized at different times that are combined to achieve a desired amount.

[0049] Biological activity: As used herein, the term "biologically active" refers to the characteristic of any agent that has activity in a biological system, particularly an organism. For example, an agent that, when administered to an organism, has a biological effect on the organism, is considered to be biologically active.

[0050] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, the delivery of mRNA encompasses the situation where mRNA is delivered to a target tissue, and the encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), and the situation where mRNA is delivered to a target tissue, and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), distributed throughout the body, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery"). In some embodiments, delivery is pulmonary delivery, including, for example, nebulization.

[0051] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides (e.g., heavy or light chains of an antibody) into an intact protein (e.g., an antibody), and / or the post-translational modification of a polypeptide or a fully assembled protein (e.g., an antibody). In this application, the terms "expression" and "production," and their grammatical equivalents, are used interchangeably.

[0052] Full-length mRNA: As used herein, "full-length mRNA" is characterized when using a specific assay, such as gel electrophoresis or UV detection with separation by capillary electrophoresis and UV absorption spectroscopy. The length of the mRNA molecule that encodes a full-length polypeptide and is obtained according to any of the purification methods described herein is at least 50% of the length of the full-length mRNA molecule transcribed from target DNA, such as at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.01%, 99.05%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the length of the full-length mRNA molecule transcribed from target DNA before being purified according to any of the methods described herein.

[0053] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0054] Homopolymer or homopolymeric nucleotide: As used herein, "homopolymer" or "homopolymeric nucleotide" and their grammatical equivalents refer to a sequence of contiguous identical bases. In some embodiments, the nucleotides comprising the homopolymeric nucleotide are selected from A, U, G, or C. In some embodiments, the term "homopolymer" or "homopolymeric nucleotide" refers to a sequence of substantially identical bases. For example, in some embodiments, the term includes a contiguous series of nucleotides having one or more non-identical nucleotides.

[0055] Intercalating dye: as used herein, an intercalating dye or a ligand or drug binds between base pairs of a DNA double helix. Intercalating dyes are hydrophobic heterocyclic molecules that resemble the ring structure of base pairs, such as ethidium bromide, acridine orange and actinomycin D. In some embodiments, the intercalating dye is an Agilent intercalating dye.

[0056] Improve, increase, or decrease: As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject of approximately the same age as the subject being treated, and suffering from the same form of the disease as the subject being treated.

[0057] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0058] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cellular systems, the term is used to refer to events that occur within living cells (e.g., in contrast to in vitro systems).

[0059] Isolated: As used herein, the term "isolated" refers to substances and / or entities that are (1) separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) produced, manufactured, and / or created by the hand of man. Isolated substances and / or entities are separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of the percent purity of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0060] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA contains one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' direction.

[0061] Minor groove or minor groove binding dye: As used herein, "minor groove" refers to the narrower of the two grooves in the DNA double helix to which the minor groove binding dye binds by hydrogen bonds or hydrophobic interactions. In RNA molecules, the minor groove binding dye binds non-covalently to the secondary structure formed by single-stranded nucleic acid. In some embodiments, the minor groove binding dye is Sybr gold™, Hoechst dye, or 4',6-diamidino-2-phenylindole (DAPI).

[0062] mRNA integrity: As used herein, the term "mRNA integrity" generally refers to the quality of mRNA. In some embodiments, mRNA integrity refers to the percentage of mRNA that is not degraded after purification process (e.g., the method described herein). The integrity of mRNA is determined by using the method specifically described herein, such as TAE agarose gel electrophoresis, or by SDS-PAGE with silver staining, or by methods well known in the art, such as RNA agarose gel electrophoresis (e.g., Ausubel et al., John Wiley & Sons, Inc., 1997, Current Protocols in Molecular Biology).

[0063] Nucleic acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain that includes individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered to be within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or that encode the same amino acid sequence. Protein-encoding nucleotide sequences and / or RNAs include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, the nucleic acid is selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, The nucleic acid may be or include C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). In some embodiments, the present invention specifically relates to "unmodified nucleic acids," meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified to facilitate or achieve delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[0064] π-stack: As used herein, π-stack refers to the attractive non-covalent interactions between aromatic rings because they involve π-bonds. These interactions are important in nucleobase stacking within DNA and RNA molecules.

[0065] Substantially: As used herein, the term "substantially" refers to a quantitative state that exhibits a degree or degree of all or nearly all of a desired feature or characteristic. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0066] Detailed Description The present invention provides, inter alia, a method for accurately measuring poly-A tail length in an mRNA sample in a rapid and high-throughput manner. The present invention is based, in part, on the surprising and unexpected discovery that binding of a minor groove binding dye to mRNA, followed by ribonuclease (RNase) digestion and capillary electrophoresis (CE) provides an accurate method for determining the poly-A tail length of mRNA. Capillary electrophoresis (CE) is typically performed using an intercalating dye. In some embodiments, the intercalating dye is an Agilent™ intercalating dye. The present invention is based, in part, on the use of a minor groove binding dye in capillary electrophoresis. In some embodiments, the minor groove binding dye is Sybr gold™.

[0067] PolyA tails are helical; they are composed of planarly stacked bases. Without wishing to be bound by any particular theory, it is believed that minor groove binding dyes, including Sybr gold™, are able to bind to the planar structure formed by the polyA tail due to interactions that are independent of base stacking. Intercalating dyes, such as Agilent™ intercalating dyes, generate aqueous polyA signals because the intercalating dyes cannot properly π-stack into the planar structure created by the polyA tail. In contrast, Sybr gold™ non-covalently binds to the helical structure of the polyA tail through hydrogen bonds and hydrophobic signals by binding to the minor groove formed by the single-stranded secondary structure (Figure 1B).

[0068] Various aspects of the invention are described further below.

[0069] Measurement of mRNA polyA tail length As described in more detail in the specification below, the mRNA is synthesized according to any of a variety of known methods, including in vitro transcription (IVT). In some embodiments, the mRNA is capped and a polyA tail is added. In some embodiments, the polyA tail is added post-transcriptionally or co-transcriptionally. The polyA tail confers stability to the mRNA therapeutic product.

[0070] In some aspects, provided herein is a method of measuring poly-A tail length in an mRNA sample, the method comprising: (a) contacting the mRNA sample with a minor groove binding dye; (b) incubating the mRNA sample from (a) with one or more ribonucleases (RNases); and (c) assaying the sample from (b) by capillary electrophoresis (CE) to determine the poly-A tail length of the mRNA. In some embodiments, one or more steps of the method are automated. In some embodiments, the method is high throughput.

[0071] Minor groove binding dye In some aspects, the methods of the present invention are based, in part, on the binding of nucleic acids, including mRNA, to minor groove binding dyes (FIGS. 1A and 1B). In some embodiments, the minor groove binding dye is Sybr gold™, Hoechst dye, or 4',6-diamidino-2-phenylindole (DAPI). In some embodiments, the minor groove binding dye is Sybr gold™. In some embodiments, the minor groove binding dye is Hoechst dye. In some embodiments, the minor groove binding dye is 4',6-diamidino-2-phenylindole (DAPI).

[0072] Ribonuclease In some embodiments, methods are provided herein, wherein an mRNA sample is incubated with one or more RNases selected from RNase A and RNase T1. In some embodiments, the one or more RNases are RNase A. In some embodiments, the one or more RNases are RNase T1. In some embodiments, the one or more RNases include RNase A1 and RNase T1. RNase A degrades RNA after C and U residues, while RNase T1 degrades after G residues. Digestion with RNase A and RNase T1 ensures that only the polyA tail remains.

[0073] In some embodiments, the mRNA sample is incubated with one or more RNases for about 15 minutes, 30 minutes, 45 minutes, or 60 minutes. In some embodiments, the mRNA sample is incubated with one or more RNases for about 15 minutes. In some embodiments, the mRNA sample is incubated with one or more RNases for about 30 minutes. In some embodiments, the mRNA sample is incubated with one or more RNases for about 45 minutes. In some embodiments, the mRNA sample is incubated with one or more RNases for about 60 minutes.

[0074] Capillary Gel Electrophoresis The method of the present invention employs capillary electrophoresis coupled with a detection system for the separation of mRNAs based on the equal mass-to-charge ratio of the mRNAs. Capillary gel electrophoresis separates digestion products of various lengths, from 25 nt to greater than about 5000 nucleotides. The lengths are precisely quantified based on standard size markers.

[0075] In some embodiments, capillary electrophoresis is coupled with fluorescence-based detection. For example, in some embodiments, the fluorescence-based detection method comprises laser-induced fluorescence detection.

[0076] In some embodiments, capillary electrophoresis is coupled with UV absorption spectroscopy detection.

[0077] Synthesis of mRNA mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized through in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template that contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that can include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions vary depending on the specific application.

[0078] In some embodiments, to prepare the mRNA according to the present invention, the DNA template is transcribed in vitro.Suitable DNA template typically has a promoter for in vitro transcription, such as T3, T7 or SP6 promoter, followed by the desired nucleotide sequence for the desired mRNA and termination signal.

[0079] Synthesis of mRNA using T3 RNA polymerase In some embodiments, mRNA is produced using T3 RNA polymerase. T3 RNA polymerase is a DNA-dependent RNA polymerase from the T3 bacteriophage that catalyzes the formation of RNA from DNA in the 5'→3' direction on either single-stranded or double-stranded DNA and can incorporate modified nucleotides. T3 polymerase is highly promoter specific and only transcribes DNA downstream of the T3 promoter. T3 binds to the consensus promoter sequence of 5'-AATTAACCCTCACTAAAGGGAGA-3' (SEQ ID NO: 1).

[0080] Synthesis of mRNA using T7 RNA polymerase In some embodiments, the mRNA is generated using T7 RNA polymerase. T7 RNA polymerase is a DNA-dependent RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in a 5' to 3' direction. T7 polymerase is highly promoter specific and only transcribes DNA downstream of the T7 promoter. T7 binds to the consensus promoter sequence of 5'-TAATACGACTCACTATAGGGAGA-3' (SEQ ID NO: 2). T7 polymerase also requires a double-stranded DNA template and Mg as cofactors for RNA synthesis. 2+ ions, which has a very low error rate.

[0081] Synthesis of mRNA using SP6 RNA polymerase In some embodiments, the mRNA is produced using SP6 RNA polymerase. SP6 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for the SP6 promoter sequence. SP6 polymerase catalyzes the 5'→3' in vitro synthesis of RNA on single-stranded or double-stranded DNA downstream from the promoter, incorporating natural and / or modified and / or labeled ribonucleotides into the polymerized transcript. SP6 binds to the consensus promoter sequence of 5'-ATTTACGACACACTATAGAAGAA-3' (SEQ ID NO: 3). Examples of such labeled ribonucleotides include biotin-, fluorescein-, digoxigenin-, aminoallyl-, and isotope-labeled nucleotides.

[0082] DNA template Typically, the DNA template is either fully double-stranded or mostly single-stranded with an appropriate promoter sequence (eg, T3, T7 or SP6 promoter).

[0083] Linearized plasmid DNA (linearized via one or more restriction enzymes), linearized genomic DNA fragments (via restriction enzymes and / or physical means), PCR products, and / or synthetic DNA oligonucleotides can be used as templates for in vitro transcription, provided that they contain a double-stranded promoter upstream (and in the forward orientation) of the DNA sequence to be transcribed.

[0084] In some embodiments, the linearized DNA template has blunt ends.

[0085] In some embodiments, the transcribed DNA sequence is optimized to promote more efficient transcription and / or translation. For example, the DNA sequence is optimized for cis-regulatory elements (e.g., TATA boxes, termination signals and protein binding sites), artificial recombination sites, Chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements related to transcription; the DNA sequence is optimized for potential splice sites, mRNA secondary structures, stable free energy of mRNA, repetitive sequences, RNA instability motifs, and / or other elements related to mRNA processing and stability; the DNA sequence is optimized for codon usage bias, codon compatibility, internal Chi sites, ribosome binding sites (e.g., IRES), premature polyA sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA sequence is optimized for codon context, codon-anticodon interactions, translation pause sites, and / or other elements related to protein folding. Optimization methods known in the art can be used in the present invention, such as GeneOptimizer by ThermoFisher and OptimumGene™, described in U.S. Patent Application Publication No. 2011 / 0081708, which is incorporated by reference in its entirety.

[0086] In some embodiments, the DNA template comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be between about 50 and 500 nucleotides in length.

[0087] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA in its location within a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region can be between 50-500 nucleotides in length or more.

[0088] Exemplary 3' and / or 5' UTR sequences can be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. For example, the 5' UTR sequence can include a subsequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the polynucleotide. It is also contemplated to include a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3' end or untranslated region of the polynucleotide (e.g., mRNA) to further stabilize the polynucleotide. In general, these modifications include modifications made to improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to its unmodified counterpart, e.g., to improve the resistance of such polynucleotides to in vivo nuclease digestion.

[0089] Large-scale mRNA synthesis In some embodiments, mRNA with poly-A tail is synthesized on a large scale. In some embodiments, mRNA is synthesized in a single batch of at least 100mg, 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1g, 5g, 10g, 25g, 50g, 75g, 100g, 250g, 500g, 750g, 1kg, 5kg, 10kg, 50kg, 100kg, 1000kg or more. As used herein, the term "batch" refers to the content or amount of mRNA synthesized at one time, for example, the content or amount of mRNA produced according to a single manufacturing setup. A batch can refer to the amount of mRNA synthesized in one reaction that occurs via a single aliquot of enzyme and / or a single aliquot of DNA template for continuous synthesis under one set of conditions. mRNA synthesized in a single batch does not include mRNA synthesized at different times that are combined to achieve a desired quantity.

[0090] According to the present invention, 1-100 mg of RNA polymerase is typically used per gram (g) of mRNA produced. In some embodiments, about 1-90 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 10-100 mg, 10-80 mg, 10-60 mg, 10-50 mg of RNA polymerase is used per gram of mRNA produced. In some embodiments, about 5-20 mg of RNA polymerase is used to produce about 1 gram of mRNA. In some embodiments, about 0.5-2 grams of RNA polymerase is used to produce about 100 grams of mRNA. In some embodiments, about 5-20 grams of RNA polymerase is used to produce about 1 kilogram of mRNA. In some embodiments, at least 5 mg of RNA polymerase is used to produce at least 1 gram of mRNA. In some embodiments, at least 500 mg of RNA polymerase is used to produce at least 100 grams of mRNA. In some embodiments, at least 5 grams of RNA polymerase are used to produce at least 1 kilogram of mRNA. In some embodiments, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg of plasmid DNA are used per gram of mRNA produced. In some embodiments, about 10-30 mg of plasmid DNA are used to produce about 1 gram of mRNA. In some embodiments, about 1-3 grams of plasmid DNA are used to produce about 100 grams of mRNA. In some embodiments, about 10-30 grams of plasmid DNA are used to produce about 1 kilogram of mRNA. In some embodiments, at least 10 mg of plasmid DNA is used to produce at least 1 gram of mRNA. In some embodiments, at least 1 gram of plasmid DNA is used to produce at least 100 grams of mRNA. In some embodiments, at least 10 grams of plasmid DNA is used to produce at least 1 kilogram of mRNA.

[0091] In some embodiments, the concentration of RNA polymerase in the reaction mixture can be about 1-100 nM, 1-90 nM, 1-80 nM, 1-70 nM, 1-60 nM, 1-50 nM, 1-40 nM, 1-30 nM, 1-20 nM, or about 1-10 nM. In certain embodiments, the concentration of RNA polymerase is about 10-50 nM, 20-50 nM, or 30-50 nM. Concentrations of RNA polymerase in the range of 100 to 10,000 units / ml, for example, 100 to 9,000 units / ml, 100 to 8,000 units / ml, 100 to 7,000 units / ml, 100 to 6,000 units / ml, 100 to 5,000 units / ml, 100 to 1,000 units / ml, 200 to 2,000 units / ml, 500 to 1,000 units / ml, 500 to 2,000 units / ml, 500 to 3,000 units / ml, 500 to 4,000 units / ml, 500 to 5,000 units / ml, 500 to 6,000 units / ml, 1,000 to 7,500 units / ml, and 2,500 to 5,000 units / ml can be used.

[0092] The concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture is about 0.1 mM to about 10 mM, e.g., between about 1 mM to about 10 mM, between about 2 mM to about 10 mM, between about 3 mM to about 10 mM, between about 1 mM to about 8 mM, between about 1 mM to about 6 mM, between about 3 mM to about 10 mM, between about 3 mM to about 8 mM, between about 3 mM to about 6 mM, between about 4 mM to about 5 mM. In some embodiments, each ribonucleotide is about 5 mM in the reaction mixture. In some embodiments, the total concentration of rNTPs (e.g., a combination of ATP, GTP, CTP, and UTP) used in the reaction ranges between 1 mM and 40 mM. In some embodiments, the total concentration of rNTPs (e.g., a combination of ATP, GTP, CTP, and UTP) used in the reaction ranges between 1 mM and 30 mM, or between 1 mM and 28 mM, or between 1 mM and 25 mM, or between 1 mM and 20 mM. In some embodiments, the total rNTP concentration is less than 30 mM. In some embodiments, the total rNTP concentration is less than 25 mM. In some embodiments, the total rNTP concentration is less than 20 mM. In some embodiments, the total rNTP concentration is less than 15 mM. In some embodiments, the total rNTP concentration is less than 10 mM.

[0093] RNA polymerase reaction buffers typically contain salts / buffers such as Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, sodium chloride, and magnesium chloride.

[0094] The pH of the reaction mixture can be about 6-8.5, 6.5-8.0, 7.0-7.5, and in some embodiments, the pH is 7.5.

[0095] Combine a linearized or straightened DNA template (e.g., as described above, in an amount / concentration sufficient to provide the desired amount of RNA), RNA polymerase reaction buffer, and RNA polymerase to form a reaction mixture. Incubate the reaction mixture at about 37°C to about 42°C for 30 minutes to 6 hours, e.g., about 60 to about 90 minutes.

[0096] In some embodiments, about 5 mM NTPs, about 0.05 mg / mL RNA polymerase, and about 0.1 mg / ml DNA template in a suitable RNA polymerase reaction buffer (pH of the final reaction mixture is about 7.5) are incubated at about 37° C. to about 42° C. for 60 to 90 minutes.

[0097] In some embodiments, the reaction mixture contains a linearized double-stranded DNA template with an RNA polymerase-specific promoter, RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTPs, 10 mM DTT, and reaction buffer (800 mM HEPES, 20 mM spermidine, 250 mM MgCl2, pH 7.7 for 10x) and sufficient content (QS) for the desired reaction volume with RNase-free water. The reaction mixture is then incubated at 37°C for 60 minutes. The polymerase reaction is then quenched by the addition of DNase I and DNase I buffer (100 mM Tris-HCl, 5 mM MgCl2 and 25 mM CaCl2, pH 7.6 for 10x) to facilitate digestion of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to generate 100 grams of mRNA.

[0098] In some embodiments, the reaction mixture comprises NTPs at a concentration ranging from 1 to 10 mM, DNA template at a concentration ranging from 0.01 to 0.5 mg / ml, and RNA polymerase at a concentration ranging from 0.01 to 0.1 mg / ml, for example, the reaction mixture comprises NTPs at a concentration of 5 mM, DNA template at a concentration of 0.1 mg / ml, and RNA polymerase at a concentration of 0.05 mg / ml.

[0099] nucleotide A variety of naturally occurring or modified nucleosides can be used to generate the mRNA according to the present invention. In some embodiments, the mRNA can be modified with any of the following nucleosides: natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanidine ... The base may be or contain: anosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0100] In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. The non-standard nucleotide residues may include, for example, 5-methyl-cytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thio-uridine ("2sU"). For a discussion of such residues and their incorporation into mRNA, see, for example, U.S. Pat. No. 8,278,036 or WO 2011 / 012316. The mRNA may be RNA, defined as RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings for the use of RNA are disclosed in U.S. Pat. App. Pub. No. 2012 / 0195936 and WO 2011 / 012316, both of which are incorporated herein by reference in their entireties. The presence of non-standard nucleotide residues can make an mRNA more stable and / or less immunogenic than a control mRNA having the same sequence but containing only standard residues. In further embodiments, the mRNA can contain one or more non-standard nucleotide residues selected from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine and 2-chloro-6-aminopurine cytosine, and combinations of these and other nucleobase modifications. Some embodiments can further include additional modifications to the furanose ring or nucleobase. Additional modifications include, for example, sugar modifications or substitutions (e.g., one or more of 2'-O-alkyl modifications, locked nucleic acids (LNAs)). In some embodiments, the RNA is complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications include, but are not limited to, a 2'-deoxy-2'-fluoro modification, a 2'-O-methyl modification, a 2'-O-methoxyethyl modification, and a 2'-deoxy modification.In some embodiments, any of these modifications can be present in 0-100% of the nucleotides - for example, individually or in combination, 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, greater than 95% or 100% of the constituent nucleotides.

[0101] Post-synthesis processing Typically, a 5' cap and / or a 3' tail can be added post-synthetically. The presence of a cap is important to provide resistance to nucleases found in most eukaryotic cells. The presence of a "tail" helps protect the mRNA from exonuclease degradation.

[0102] 5' Cap A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via guanylate transferase, generating a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in U.S. Patent Application Publication No. 2016 / 0032356, filed February 27, 2017, and U.S. Provisional Patent Application No. 62 / 464,327, both of which are incorporated herein by reference.

[0103] 3'-Poly A tail The presence of a "tail" at the 3' end helps protect the mRNA from exonuclease degradation. The 3' tail can be added before, after, or at the same time as the 5' cap.

[0104] In some embodiments, a polyA tail is added co-transcriptionally. In some embodiments, a polyA tail is added post-transcriptionally. In some embodiments, a polyC tail is added co-transcriptionally. In some embodiments, a polyC tail is added post-transcriptionally.

[0105] In some embodiments, the polyA tail is between 25 and 5,000 nucleotides in length. In some embodiments, the polyA tail is 25 nucleotides in length. In some embodiments, the polyA tail is 50 nucleotides in length. In some embodiments, the polyA tail is 75 nucleotides in length. In some embodiments, the polyA tail is 100 nucleotides in length. In some embodiments, the polyA tail is 150 nucleotides in length. In some embodiments, the polyA tail is 200 nucleotides in length. In some embodiments, the polyA tail is 250 nucleotides in length. In some embodiments, the polyA tail is 300 nucleotides in length. In some embodiments, the polyA tail is 350 nucleotides in length. In some embodiments, the polyA tail is 400 nucleotides in length. In some embodiments, the polyA tail is 450 nucleotides in length. In some embodiments, the polyA tail is 500 nucleotides in length. In some embodiments, the polyA tail is 550 nucleotides in length. In some embodiments, the polyA tail is 300 nucleotides in length. In some embodiments, the polyA tail is 600 nucleotides in length. In some embodiments, the polyA tail is 650 nucleotides in length. In some embodiments, the polyA tail is 700 nucleotides in length. In some embodiments, the polyA tail is 750 nucleotides in length. In some embodiments, the polyA tail is 800 nucleotides in length. In some embodiments, the polyA tail is 850 nucleotides in length. In some embodiments, the polyA tail is 900 nucleotides in length. In some embodiments, the polyA tail is 950 nucleotides in length. In some embodiments, the polyA tail is 1000 nucleotides in length.

[0106] In some embodiments, the polyA tail is 1500 nucleotides in length. In some embodiments, the polyA tail is 2000 nucleotides in length. In some embodiments, the polyA tail is 2500 nucleotides in length. In some embodiments, the polyA tail is 3000 nucleotides in length. In some embodiments, the polyA tail is 3500 nucleotides in length. In some embodiments, the polyA tail is 4000 nucleotides in length. In some embodiments, the polyA tail is 4500 nucleotides in length. In some embodiments, the polyA tail is 5000 nucleotides in length.

[0107] Typically, the tail structure comprises a polyA and / or polyC tail. (A, adenosine; C, cytosine). In some embodiments, the polyA or polyC tail at the 3' end of the mRNA comprises at least 25 adenine or cytosine nucleotides, at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 1000 adenosine or cytosine nucleotides, at least 1500 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotides, at least 2 kb of adenosine or cytosine nucleotides, at least 3 kb of adenosine or cytosine nucleotides, at least 4 kb of adenosine or cytosine nucleotides, at least 5 kb of adenosine or cytosine nucleotides.In some embodiments, the polyA or polyC tail comprises about 10-800 adenosine or cytosine nucleotides, respectively (e.g., about 10-200 adenosine or cytosine nucleotides, about 10-300 adenosine or cytosine nucleotides, about 10-400 adenosine or cytosine nucleotides, about 10-500 adenosine or cytosine nucleotides, about 10-550 adenosine or cytosine nucleotides, about 10-600 adenosine or cytosine nucleotides, about 50-600 adenosine or cytosine nucleotides, about 100-600 adenosine or cytosine nucleotides, about 150-600 adenosine or cytosine nucleotides, about 200-60 0 adenosine or cytosine nucleotides, about 250-600 adenosine or cytosine nucleotides, about 300-600 adenosine or cytosine nucleotides, about 350-600 adenosine or cytosine nucleotides, about 400-600 adenosine or cytosine nucleotides, about 450-600 adenosine or cytosine nucleotides, about 500-600 adenosine or cytosine nucleotides, about 10-150 adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides). In some embodiments, the tail structure is a combination of poly-A tails and poly-C tails of various lengths as described herein. In some embodiments, the poly-A tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, the poly-A tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0108] As described herein, the addition of a 5' cap and / or a 3' tail facilitates the detection of aborted transcripts produced during in vitro synthesis, since in the absence of capping and / or tailing, the size of premature aborted mRNA transcripts is too small to be detected. Thus, in some embodiments, a 5' cap and / or a 3' tail is added to the synthesized mRNA before the mRNA is tested for purity (the level of aborted transcript present in the mRNA). In some embodiments, a 5' cap and / or a 3' tail is added to the synthesized mRNA before the mRNA is purified. In other embodiments, a 5' cap and / or a 3' tail is added to the synthesized mRNA after the mRNA is purified.

[0109] Purification of mRNA The mRNA synthesized according to the present invention can be used without a step of removing shortmers. In some embodiments, the mRNA synthesized according to the present invention can be further purified. Various methods can be used to purify the mRNA synthesized according to the present invention. For example, purification of the mRNA can be performed using centrifugation, filtration and / or chromatography methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution, well known to those skilled in the art. In some embodiments, the mRNA is purified using tangential flow filtration. Suitable purification methods include those described in U.S. Patent Application Publication No. 2016 / 0040154, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA," filed February 27, 2018, U.S. Patent Application Publication No. 2015 / 0376220, International Patent Application No. PCT / US18 / 19954, and International Patent Application No. PCT / US18 / 19978, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA," filed February 27, 2018, all of which are incorporated by reference herein and can be used to practice the present invention.

[0110] In some embodiments, the mRNA is purified before capping and tailing. In some embodiments, the mRNA is purified after capping and tailing. In some embodiments, the mRNA is purified both before and after capping and tailing.

[0111] In some embodiments, the mRNA is purified by centrifugation either before or after capping and tailing, or both before and after capping and tailing.

[0112] In some embodiments, the mRNA is purified by filtration either before or after capping and tailing, or both before and after capping and tailing.

[0113] In some embodiments, the mRNA is purified by tangential flow filtration (TFF) either before or after capping and tailing, or both.

[0114] In some embodiments, the mRNA is purified by chromatography either before or after, or both before and after capping and tailing.

[0115] mRNA characterization The full-length or interrupted transcripts of mRNA can be detected and quantified using any method available in the art. In some embodiments, the synthesized mRNA molecules are detected using blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV), or UPLC, or a combination thereof. Other detection methods known in the art are included in the present invention. In some embodiments, the synthesized mRNA molecules are detected using UV absorption spectroscopy with separation by capillary electrophoresis. In some embodiments, the mRNA is first denatured by glyoxal dye before gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, the synthesized mRNA is characterized before capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing.

[0116] In some embodiments, the mRNA produced by the methods disclosed herein contains less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% of impurities other than full-length mRNA. Impurities include IVT contaminants, such as proteins, enzymes, free nucleotides, and / or shortmers.

[0117] In some embodiments, the mRNA produced according to the present invention is substantially free of shortmers and interrupted transcripts. In particular, the mRNA produced according to the present invention contains undetectable levels of shortmers or interrupted transcripts by capillary electrophoresis or glyoxal gel electrophoresis. As used herein, the term "shortmer" or "interrupted transcript" refers to any transcript that is less than full length. In some embodiments, a "shortmer" or "interrupted transcript" is less than 100 nucleotides, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides in length. In some embodiments, the shortmers are detected or quantified after adding a 5'-cap and / or a 3'-polyA tail.

[0118] Homopolymeric Nucleic Acid Tracts In some aspects, the present invention provides a method for measuring homopolymeric nucleotide length in a nucleic acid, including mRNA, comprising contacting the mRNA with a minor groove binding dye, followed by treating the mRNA with one or more ribonucleases, performing capillary electrophoresis, and determining the homopolymeric nucleotide length. In some embodiments, one or more steps of the method are automated. In some embodiments, the method is high throughput.

[0119] In some embodiments, the nucleotides comprising homopolymeric nucleotides are selected from A, U, G, or C. In some embodiments, the nucleotides comprising homopolymeric nucleotides are adenine. In some embodiments, the nucleotides comprising homopolymeric nucleotides are uracil. In some embodiments, the nucleotides comprising homopolymeric nucleotides are guanine. In some embodiments, the nucleotides comprising homopolymeric nucleotides are cytosine.

[0120] The homopolymer nucleotide length measured according to the method of the present invention ranges from about 25 nucleotides to more than about 5000 nucleotides. In some embodiments, the homopolymer nucleotide length is about 25 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, or more than about 200 nucleotides. In some embodiments, the homopolymer nucleotide length is about 25 nucleotides, about 50 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, or more than about 200 nucleotides.

[0121] In some embodiments, the homopolymer nucleotide length is between 50 nucleotides and 5,000 nucleotides. In some embodiments, the homopolymer nucleotide length is 50 nucleotides. In some embodiments, the homopolymer nucleotide length is 100 nucleotides. In some embodiments, the homopolymer nucleotide length is 150 nucleotides. In some embodiments, the homopolymer nucleotide length is 200 nucleotides. In some embodiments, the homopolymer nucleotide length is 250 nucleotides. In some embodiments, the homopolymer nucleotide length is 300 nucleotides. In some embodiments, the homopolymer nucleotide length is 350 nucleotides. In some embodiments, the homopolymer nucleotide length is 400 nucleotides. In some embodiments, the homopolymer nucleotide length is 450 nucleotides. In some embodiments, the homopolymer nucleotide length is 500 nucleotides. In some embodiments, the homopolymer nucleotide length is 550 nucleotides. In some embodiments, the homopolymer nucleotide length is 600 nucleotides. In some embodiments, the homopolymer nucleotide length is 650 nucleotides. In some embodiments, the homopolymer nucleotide length is 700 nucleotides. In some embodiments, the homopolymer nucleotide length is 750 nucleotides. In some embodiments, the homopolymer nucleotide length is 800 nucleotides. In some embodiments, the homopolymer nucleotide length is 850 nucleotides. In some embodiments, the homopolymer nucleotide length is 900 nucleotides. In some embodiments, the homopolymer nucleotide length is 950 nucleotides. In some embodiments, the homopolymer nucleotide length is 1000 nucleotides. In some embodiments, the homopolymer nucleotide length is between about 1100 and 5000 nucleotides.

[0122] Homopolymeric nucleotides serve several functions, such as determining DNA position in protein binding regions, upstream promoter elements, and nucleosome structures. The methods of the present invention provide, inter alia, a method for measuring the length of homopolymeric sequences in nucleic acids, including DNA and RNA.

[0123] In some embodiments, the present invention further provides a method for measuring the length of homopolymer containing repeat units (SSRs), microsatellites, minisatellites and macrosatellites. SSRs are composed of tandem repeat units of 1-5 bp. For example, the most abundant SSRs are poly dA-poly dT and poly dG-poly dC, which are generally found in non-coding regions and are often greater than 9 bp in length. Poly dA-poly dT tracts are common in AT-rich sequences. SSRs play a role in sequence-specific DNA binding.

[0124] Microsatellites are found in regions including telomeres, consisting of approximately 10 bp repeats, often containing 6-8 bp repeats. In coding regions, frameshift errors resulting from homopolymers result in cancer. In some cancers, measuring the length of DNA microsatellites in tumor samples reveals a measure of microsatellite instability (whether they are shorter or longer) and provides an indication of cancer progression.

[0125] In some embodiments, the method of the present invention is used to measure the length of minisatellites, which are composed of repeat units of 10 to 100 bp. Minisatellites are often found in centromeres and heterochromatin regions. In some embodiments, the method of the present invention is used to measure the length of macrosatellites, which contain repeat units of more than 100 bp.

[0126] In some embodiments, the methods of the invention are used to measure the length of RNA homopolymers such as polyA and polyU used to make virus-like particles, as these offer advantages over RNA with a typical composition that includes a mixture of bases.

[0127] In some embodiments, the method of the present invention is used to measure homopolymer length as a quality control in next generation sequencing reads that contain homopolymer nucleotides. Repetitive DNA sequence assembly from short reads cannot determine the length of repetitive sequences such as microsatellites, which are often omitted from reported sequences.

[0128] In some embodiments, the methods of the invention are used to measure the length of homopolymeric nucleotides in tandem repeats, interspersed repeats, transposable elements, DNA transposons, retrotransposons, SINEs (short interspersed nuclear elements), LINEs (long interspersed nuclear elements), and CRISPR sequences. EXAMPLES

[0129] While certain compounds, compositions and methods of the invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the invention and are not intended to limit the compounds of the invention.

[0130] Synthesis of mRNA In each of the examples below, synthesis of mRNA was performed under completely RNase-free conditions. In the following examples, mRNA was synthesized via in vitro transcription from a linear DNA template. To generate the desired precursor-mRNA (IVT) construct, a mixture of approximately 8 mg of linearized DNA, rNTPs (7.25 mM), DTT (10 mM), T7 RNA polymerase, RNase inhibitor, pyrophosphatase and reaction buffer (10x, 800 mM HEPES (pH 8.0), 20 mM spermidine, 250 mM MgCl2, pH 7.7) was prepared to a final volume of 180 mL with RNase-free water. The reaction mixture is incubated at 37 °C for a time range between 20 min to 60 min. After completion, the mixture is treated with DNase I for an additional 15 min and quenched appropriately.

[0131] Addition of 5' cap and 3' tail The purified mRNA product from the IVT step described above was denatured at 65°C for 10 min. Separately, a portion of GTP (1.0 mM), S-adenosylmethionine, RNase inhibitor, 2'-O-methyltransferase and guanylyltransferase were mixed with reaction buffer (10x, 500 mM Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl2) to a final concentration of 1.6 L. Upon denaturation, the mRNA was chilled on ice and then added to the reaction mixture. The combined solution was incubated at 37°C for a time range of 25-90 min. After completion, an aliquot of ATP (2.0 mM), polyA polymerase and tailing reaction buffer (10x, 500 mM Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl2) was added and the entire reaction mixture was further incubated at 37°C for a time range of 20-45 min. Upon completion, the final reaction mixture was quenched and appropriately purified. PolyA tail length was measured in some embodiments according to the methods of the present invention, as described in the Examples below. EXAMPLES

[0132] Measurement of polyA tail length of EPO mRNA with short tail lengths of less than 150 nucleotides This example demonstrates how to measure the polyA tail length in exemplary EPO mRNAs having exemplary tail lengths of approximately 25 nt, 50 nt, and 114 nt.

[0133] Briefly, exemplary EPO mRNA samples were digested with one or more ribonucleases (RNases). In this example, in some embodiments, RNase A was used. The mRNA samples were then assayed by capillary electrophoresis to determine the polyA tail length of the mRNA.

[0134] The mRNA samples were incubated with the organic solvent methanamide (formamide) at 75°C for approximately 10 minutes. The mRNA samples were then run using a fragment analyzer instrument to perform CE. During this process, Sybr Gold dye was added to the RNA separation gel at a 1:10,000 dilution of the stock before adding the samples and starting the run. The dye then non-covalently bound to the mRNA samples via hydrogen bonding and hydrophobic interactions at room temperature.

[0135] RNase A gel analysis of EPO mRNA containing a co-transcriptionally added polyA tail with polyA tail lengths of 25 nt, 50 nt and 114 nt for Sybr gold™ dye-treated CE samples and conventional RNase A digested samples is shown in Figure 2A. The results of the analysis are shown in a graph of observed tail length on the y-axis and theoretical tail length on the x-axis for EPO mRNA with polyA tail lengths of 25 nt, 50 nt and 114 nt (Figure 2B). Results are shown for Agilent™ dye-treated samples (plotted from very low raw signal), Sybr gold™ dye-treated samples, RNase A digested samples, followed by Biorad MW analysis and theoretical tail lengths.

[0136] As can be seen from the results shown in Figure 2B, the observed tail lengths are overestimated and highly inaccurate in the Agilent™ dye-treated samples at all three tail lengths assayed, i.e., 25 nt, 50 nt, and 114 nt. Sybr gold™ dye-treated CE samples and RNase A digested samples, followed by Biorad MW analysis, showed good correlation between observed and theoretical tail lengths.

[0137] Overall, the results of this example demonstrated that the method of conjugating mRNA with a minor groove binding dye followed by RNase digestion and capillary electrophoresis accurately measured polyA tail length. The method was highly accurate, and the observed polyA tail lengths were comparable to the theoretical tail lengths. The method was comparable in accuracy to traditional RNase A digestion followed by Biorad MW analysis. EXAMPLES

[0138] Measurement of polyA tail length of EPO mRNA with tail length longer than 100 nucleotides This example demonstrates how to measure the polyA tail length in exemplary EPO mRNAs having exemplary tail lengths of approximately 100 nt, 200 nt, 300 nt, 400 nt, 500 nt and 600 nt.

[0139] Briefly, exemplary EPO mRNA samples were digested with one or more ribonucleases (RNases). In this example, in some embodiments, RNase A was used. The mRNA samples were then assayed by capillary electrophoresis to determine the polyA tail length of the mRNA.

[0140] The mRNA samples were incubated with the organic solvent methanamide (formamide) at 75°C for approximately 10 minutes. The mRNA samples were then run using a fragment analyzer instrument to perform CE. During this process, Sybr Gold dye was added to the RNA separation gel at a 1:10,000 dilution of the stock before adding the samples and starting the run. The dye then non-covalently bound to the mRNA samples via hydrogen bonding and hydrophobic interactions at room temperature.

[0141] RNase A gel analysis of poly-A-tailed EPO mRNA with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Sybr gold™ dye-treated CE is shown in Figure 3A. Gel analysis of poly-A-tailed EPO mRNA with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Agilent™ intercalating dye-treated CE is shown in Figure 3B.

[0142] In some embodiments, polyA tail length was also assessed by conventional RNase A method. Briefly, mRNA samples were digested with RNase A for 30 minutes and run on a 2% agarose gel for 2 hours and 30 minutes. The products are shown in Figure 4A. PolyA tail length was then analyzed by Biorad MW analysis.

[0143] The results of the analysis are shown in a graph of observed tail length on the y-axis and theoretical tail length on the x-axis for EPO mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt (Figure 4B). Results are shown for Agilent™ dye treated samples (plotted from very low raw signal), Sybr gold™ dye treated samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths.

[0144] As can be seen from the results shown in Figure 4B, the observed tail lengths are highly inaccurate in the Agilent™ dye-treated samples at all six tail lengths assayed, i.e., 100 nt, 200 nt, 300 nt, 400 nt, 500 nt and 600 nt. The observed tail lengths are highly underestimated at 100 nt and overestimated at 200-600 nt tail lengths. The Sybr gold™ dye-treated CE samples showed good correlation between observed and theoretical tail lengths. The Sybr gold™ treated CE samples showed improved correlation between observed and theoretical tail lengths over the traditional RNase A method followed by Biorad MW analysis.

[0145] Capillary electrophoresis patterns of 1.2 μg of Sybr gold™ dye-treated EPO mRNA samples with poly-A tail lengths of 100, 200, 300, 400, 500 and 600 nt after RNase A digestion are shown in Figure 5A. Capillary electrophoresis patterns of 1.2 μg of Agilent™ dye-treated EPO mRNA samples with poly-A tail lengths of 100, 200, 300, 400, 500 and 600 nt after RNase A digestion are shown in Figure 5B.

[0146] A comparison of the signal intensities of the Sybr gold™ dye-treated EPO mRNA samples and the Agilent™ dye-treated EPO mRNA samples is shown in Figures 6A and 6B. A capillary electropherogram of 1.2 μg of Sybr gold™ dye-treated EPO mRNA with a polyA tail length of 200 nt after RNase A digestion is shown in Figure 6A. A capillary electropherogram of 1.2 μg of Agilent™ dye-treated EPO mRNA with a polyA tail length of 200 nt after RNase A digestion is shown in Figure 6B.

[0147] Overall, the results of this example demonstrated that the method of conjugating mRNA with a minor groove binding dye followed by RNase digestion and capillary electrophoresis accurately measured polyA tail length. The method was highly accurate, and the observed polyA tail lengths were comparable to the theoretical tail lengths. The method was comparable in accuracy to conventional RNase A digestion followed by Biorad MW analysis. EXAMPLES

[0148] Comparison of the accuracy of poly(A) tail length measurements by methods involving mRNA digestion with either RNase A alone or both RNase A and RNase T1 enzymes This example shows a comparison of the accuracy of polyA tail length measurements using methods involving mRNA digestion with either RNase A alone or both RNase A and RNase T1 enzymes.

[0149] RNase A degrades RNA after C and U residues, while RNase T1 degrades after G residues. Digestion with RNase A and RNase T1 ensures that only the polyA tail remains. In this example, the accuracy of polyA tail length measurements was compared for exemplary EPO mRNA samples with polyA tail lengths ranging from 50 to 1800 nucleotides that were digested with RNase A or RNase A and RNase T1.

[0150] In this example, samples of EPO mRNA with theoretical polyA tail lengths of 50-1800 nucleotides (e.g., 50nt, 100nt, 200nt, 500nt, 1000nt, 1500nt and 1800nt) were digested with RNase A for 30 minutes. The digestion products were run on a 2% agarose gel and the molecular weights were assessed on a BIORAD to determine the polyA tail length. The graph was plotted between the observed tail length on the y-axis and the theoretical tail length on the x-axis.

[0151] In parallel, samples of EPO mRNA with polyA tail lengths between 50 and 1800 nucleotides (e.g., 50nt, 100nt, 200nt, 500nt, 1000nt, 1500nt and 1800nt) were digested with RNase A and RNase T1 for 30 min. The digestion products were run on a 2% agarose gel and the molecular weights were assessed on a BIORAD to determine the polyA tail lengths. The graph was plotted between the observed tail length on the y-axis and the theoretical tail length on the x-axis. The results are shown in Figure 7 and Table 1. The graph showed a correlation between the observed tail length and the theoretical tail length. The observed tail length approached the theoretical tail length.

[0152] [Table 1]

[0153] This example also showed that the observed tail lengths were comparable in RNase A and RNase A / T1 digested EPO mRNA samples with tail lengths between 50 and 1800 nucleotides. EXAMPLES

[0154] Methods for measuring polyA tail length of CFTR mRNA with tail lengths greater than 100 nucleotides This example demonstrates how to measure polyA tail length in exemplary CFTR mRNAs having exemplary tail lengths of approximately 100 nt, 200 nt, 300 nt, 400 nt, 500 nt and 600 nt.

[0155] Briefly, exemplary CFTR mRNA samples were digested with one or more ribonucleases (RNases). In this example, in some embodiments, RNase A was used. The mRNA samples were then assayed by capillary electrophoresis to determine the polyA tail length of the mRNA.

[0156] The mRNA samples were incubated with the organic solvent methanamide (formamide) at 75°C for approximately 10 minutes. The mRNA samples were then run using a fragment analyzer instrument to perform CE. During this process, Sybr Gold dye was added to the RNA separation gel at a 1:10,000 dilution of the stock before adding the samples and starting the run. The dye then non-covalently bound to the mRNA samples via hydrogen bonds and hydrophobic interactions at room temperature. RNase A gel analysis of CFTR mRNA containing poly-A tails with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Sybr gold™ dye-treated samples versus undigested controls with poly-A tail lengths of 400nt, 500nt and 600nt is shown in Figure 8A. RNase A gel analysis of CFTR mRNA containing polyA tails with 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Agilent™ intercalating dye-treated CE versus undigested controls with polyA tail lengths of 400nt, 500nt and 600nt is shown in FIG. 8B.

[0157] Capillary electrophoresis diagrams showing peaks of Sybr gold™ dye-treated CFTR mRNA samples with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt, and 600nt, versus undigested controls with poly-A tail lengths of 400nt, 500nt, and 600nt, are shown in FIG. 9A. Capillary electrophoresis diagrams showing peaks of Agilent™ intercalating dye-treated CFTR mRNA samples with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt, and 600nt, versus undigested controls with poly-A tail lengths of 400nt, 500nt, and 600nt, are shown in FIG. 9B. The results show that the signal intensity of the Agilent™ dye-treated sample (FIG. 9B) is much lower than that of the Sybr gold™-treated sample (FIG. 9A).

[0158] The results of the analysis are shown in a graph of observed tail length on the y-axis and theoretical tail length on the x-axis for CFTR mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt (Figure 10). Results are shown for Agilent™ dye treated samples (plotted from very low raw signal), Sybr gold™ dye treated samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail length.

[0159] As can be seen in Figure 10, the observed tail lengths are underestimated at lengths of 100 nt and 200 nt, but overestimated at tail lengths between 300 and 600 nt in the Agilent™ dye-treated samples, resulting in an overall inaccurate measurement of polyA tail length.

[0160] RNase A digested samples followed by Biorad MW analysis showed accurate tail length measurements comparable to the theoretical tail length for short tail lengths such as 100 nt. However, for lengths between 200 and 600 nt, the observed tail lengths were underestimated compared to the theoretical tail lengths.

[0161] Sybr gold™ treated samples showed accurate short tail length measurements at 100 nt. Longer tail lengths were measured more accurately than observed with the RNase A method followed by Biorad MW measurements for tail lengths between 200 and 600 nt. EXAMPLES

[0162] A method for measuring the polyA tail length of OTC mRNA with a tail length greater than 100 nucleotides This example shows how to measure the polyA tail length in exemplary OTC mRNAs having exemplary tail lengths of approximately 100 nt, 200 nt, 300 nt, 400 nt, 500 nt and 600 nt.

[0163] Briefly, exemplary OTC mRNA samples were digested with one or more ribonucleases (RNases), in this example, RNase A was used in part, and the mRNA samples were then assayed by capillary electrophoresis to determine the polyA tail length of the mRNA.

[0164] The mRNA samples were incubated with the organic solvent methanamide (formamide) at 75°C for approximately 10 minutes. The mRNA samples were then run using a fragment analyzer instrument to perform CE. During this process, Sybr Gold dye was added to the RNA separation gel at a 1:10,000 dilution of the stock before adding the samples and starting the run. The dye then non-covalently bound to the mRNA samples via hydrogen bonding and hydrophobic interactions at room temperature.

[0165] RNase A gel analysis of polyA-tailed OTC mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Sybr gold™ dye treated samples versus undigested controls with polyA tail lengths of 400nt, 500nt and 600nt is shown in Figure 11 A. RNase A gel analysis of polyA-tailed OTC mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Agilent™ intercalating dye treated CE versus undigested controls with polyA tail lengths of 400nt, 500nt and 600nt is shown in Figure 11 B.

[0166] Capillary electrophoresis diagrams showing peaks of Sybr gold™ dye-treated OTC mRNA samples with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt, and 600nt, versus undigested controls with poly-A tail lengths of 400nt, 500nt, and 600nt, are shown in FIG. 12A. Capillary electrophoresis diagrams showing peaks of Agilent™ intercalating dye-treated OTC mRNA samples with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt, and 600nt, versus undigested controls with poly-A tail lengths of 400nt, 500nt, and 600nt, are shown in FIG. 12B. The results show that the signal intensity of the Agilent™ dye-treated sample (FIG. 12B) is much lower than that of the Sybr gold™-treated sample (FIG. 12A).

[0167] The results of the analysis are shown in a graph of observed tail length on the y-axis and theoretical tail length on the x-axis for OTC mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt (Figure 13). Results are shown for Agilent™ dye treated samples (plotted from very low raw signal), Sybr gold™ dye treated samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths.

[0168] As can be seen in Figure 13, the observed tail lengths are underestimated at lengths of 100 nt, but overestimated at tail lengths between 200 and 600 nt in the Agilent™ dye-treated samples, resulting in an overall inaccurate measurement of polyA tail length.

[0169] RNase A digested samples followed by Biorad MW analysis showed accurate tail length measurements comparable to the theoretical tail length for short tail lengths such as 100 nt. However, for tail lengths between 200 and 600 nt, the observed tail lengths were underestimated compared to the theoretical tail lengths.

[0170] Sybr gold™ treated samples showed accurate tail length measurements between 100 nt and 400 nt. Longer tail lengths, such as 500 nt to 600 nt, corresponded more closely to theoretical tail lengths than those observed with the RNase A method followed by Biorad MW measurements for tail lengths between 100 nt and 600 nt. EXAMPLES

[0171] A method for measuring the polyA tail length of MMA mRNA with a tail length greater than 100 nucleotides This example shows how to measure the polyA tail length in exemplary MMA mRNAs having exemplary tail lengths of approximately 100 nt, 200 nt, 300 nt, 400 nt, 500 nt and 600 nt.

[0172] Briefly, exemplary MMA mRNA samples were digested with one or more ribonucleases (RNases). In this example, in some embodiments, RNase A was used. The mRNA samples were then assayed by capillary electrophoresis to determine the polyA tail length of the mRNA.

[0173] The mRNA samples were incubated with the organic solvent methanamide (formamide) at 75°C for approximately 10 minutes. The mRNA samples were then run using a fragment analyzer instrument to perform CE. During this process, Sybr Gold dye was added to the RNA separation gel at a 1:10,000 dilution of the stock before adding the samples and starting the run. The dye then non-covalently bound to the mRNA samples via hydrogen bonding and hydrophobic interactions at room temperature.

[0174] RNase A gel analysis of polyA-tailed MMA mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Sybr gold™ dye treated samples versus undigested controls with polyA tail lengths of 400nt, 500nt and 600nt is shown in Figure 14A. RNase A gel analysis of polyA-tailed MMA mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt for Agilent™ intercalating dye treated CE versus undigested controls with polyA tail lengths of 400nt, 500nt and 600nt is shown in Figure 14B.

[0175] Capillary electrophoresis diagrams showing peaks of Sybr gold™ dye-treated MMA mRNA samples with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt, and 600nt, versus undigested controls with poly-A tail lengths of 400nt, 500nt, and 600nt, are shown in FIG. 15A. Capillary electrophoresis diagrams showing peaks of Agilent™ intercalating dye-treated MMA mRNA samples with poly-A tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt, and 600nt, versus undigested controls with poly-A tail lengths of 400nt, 500nt, and 600nt, are shown in FIG. 15B. The results show that the signal intensity of the Agilent™ dye-treated sample (FIG. 15B) is much lower than that of the Sybr gold™-treated sample (FIG. 15A).

[0176] The results of the analysis are shown in a graph of observed tail length on the y-axis and theoretical tail length on the x-axis for MMA mRNA with polyA tail lengths of 100nt, 200nt, 300nt, 400nt, 500nt and 600nt (Figure 16). Results are shown for Agilent™ dye treated samples (plotted from very low raw signal), Sybr gold™ dye treated samples, RNase A digested samples followed by Biorad MW analysis and theoretical tail lengths.

[0177] As can be seen in Figure 16, the observed tail lengths are underestimated at lengths of 100 nt, but overestimated at tail lengths between 200 and 600 nt in the Agilent™ dye-treated samples, resulting in an overall inaccurate measurement of polyA tail length.

[0178] RNase A digested samples followed by Biorad MW analysis showed accurate tail length measurements comparable to the theoretical tail length for short tail lengths such as 100 nt. However, for tail lengths between 200 and 600 nt, the observed tail lengths were underestimated compared to the theoretical tail lengths.

[0179] Sybr gold™ treated samples showed accurate tail length measurements between 100 nt and 300 nt. Longer tail lengths such as 400 nt to 600 nt corresponded more closely to theoretical tail lengths than those observed with the RNase A method followed by Biorad MW measurements for tail lengths between 400 nt and 600 nt.

[0180] All publications, patent applications, patents and other references described herein are incorporated herein by reference in their entirety.In addition, materials, methods and examples are merely illustrative and are not intended to be limiting.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although similar or equivalent methods and materials to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0181] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above specification, but rather is as set forth in the following claims.

Claims

1. 1. A method for measuring poly A tail length in an mRNA sample, comprising: (a) contacting an mRNA sample with a minor groove binding dye; (b) incubating the minor groove binding dye-bound mRNA sample from (a) with one or more ribonucleases (RNases); (c) assaying the RNase digested samples from (b) by capillary electrophoresis (CE) to determine the poly A tail length of the mRNA; A method comprising:

2. 2. The method of claim 1, wherein the minor groove binding dye comprises SYBR GOLD™, a Hoechst dye, or 4',6-diamidino-2-phenylindole (DAPI).

3. The method of claim 2, wherein the minor groove binding dye is SYBR GOLD™.

4. The method of any one of claims 1 to 3, wherein the one or more RNases include RNase A and / or RNase T1.

5. The method of claim 1 , wherein CE is coupled with fluorescence-based detection or UV absorption spectroscopy detection.

6. The method of any one of claims 1 to 3 and 5, wherein the step of incubating the mRNA sample from (a) with one or more RNases is for about 15 minutes, 30 minutes, 45 minutes, or 60 minutes, preferably 30 minutes.

7. 6. The method of any one of claims 1 to 3 and 5, wherein the poly-A tail length is 25 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 150 nucleotides or more, or 200 nucleotides or more.

8. The polyA tail length is between 50 nucleotides and 5,000 nucleotides, between 100 nucleotides and 1,500 nucleotides, or between 250 nucleotides and 500 nucleotides. The method according to any one of claims 1 to 3 and 5,

9. The method according to any one of claims 1 to 3 and 5, wherein one or more steps of the method are automated, preferably step (b) is automated.

10. The method of any one of claims 1 to 3 and 5, wherein the minor groove binding dye non-covalently binds to single-stranded RNA (ssRNA).

11. The method of any one of claims 1 to 3 and 5, wherein the minor groove binding dye is not an intercalating dye.

12. The method of any one of claims 1 to 3 and 5, wherein the mRNA sample comprises in vitro synthesized mRNA.

13. A method for measuring poly A tail length in mRNA, comprising: (a) contacting an mRNA sample with a SYBR GOLD™ minor groove binding dye; (b) incubating the SYBR GOLD™ minor groove binding dye-bound mRNA sample from (a) with RNase A and RNase T1; (c) assaying the RNase A and RNase T1 digested mRNA samples from (b) by capillary electrophoresis (CE) to determine the poly A tail length of the mRNA; A method comprising:

14. The method of claim 13, wherein CE is coupled with fluorescence-based detection or UV absorption spectroscopy detection.

15. 15. The method of claim 13 or 14, wherein the step of incubating the SYBR GOLD™ minor groove binding dye-bound mRNA sample from (a) with RNase A and RNase T1 is for about 15 minutes, 30 minutes, 45 minutes, or 60 minutes, preferably about 30 minutes.

16. 15. The method of claim 13 or 14, wherein the polyA tail length is 25 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 150 or more nucleotides, or 200 or more nucleotides.

17. 15. The method of claim 13 or 14, wherein one or more steps of the method are automated and / or the method is high throughput.

18. The method of claim 13 or 14, wherein the mRNA comprises in vitro synthesized mRNA.

19. 1. A method for measuring homopolymeric nucleotide lengths in an mRNA sample, comprising: (a) contacting an mRNA sample with a minor groove binding dye; (b) incubating the minor groove binding dye-bound mRNA sample from (a) with one or more RNases; (c) assaying the RNase digested mRNA sample from (b) by capillary electrophoresis (CE) to determine the homopolymeric nucleotide length of the mRNA.

20. Homopolymer nucleotide length of 25 nucleotides or more, 50 nucleotides 20. The method of claim 19, wherein the nucleic acid sequence is 100 nucleotides or more, 100 nucleotides or more, 150 nucleotides or more, or 200 nucleotides or more.

21. The method of claim 19 or 20, wherein the homopolymer nucleotide length is between 50 nucleotides and 5,000 nucleotides.

22. 21. The method of claim 19 or 20, wherein the nucleotides comprising the homopolymeric nucleotides are selected from A, U, G, or C.

23. 21. The method of claim 19 or 20, wherein the mRNA comprises in vitro synthesized mRNA.

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