WO2025193768A1

WO2025193768A1 - Methods and kits for analyzing poly(a) tails

Info

Publication number: WO2025193768A1
Application number: PCT/US2025/019466
Authority: WO
Inventors: Mustafa FAKHRI; Samuel Crowley; Jonathan William Cooper
Original assignee: Aldevron LLC
Current assignee: Aldevron LLC
Priority date: 2024-03-12
Filing date: 2025-03-11
Publication date: 2025-09-18
Anticipated expiration: 2026-09-12

Abstract

The present disclosure provides methods, kits, and systems to efficiently identify the length of poly(A) tails, including exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP). Performing reverse-phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates. Determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample.

Description

METHODS AND KITS FOR ANALYZING POLY(A) TAILS

CROSS REFERENCE TO RELATED APPLICATIONS

[01] This application is being filed as a PCT International patent application and claims priority to U.S. Provisional Patent Application Serial Number 63/564,460 filed March 12, 2024, the subject matter of which is hereby incorporated by reference in its entirety.

INTRODUCTION

[02] Frequently, eukaryotic messenger ribonucleic acid (mRNA) has a long chain of adenine nucleotides on its 3’ end known as the poly(A) tail. In the nucleus, RNAs are co-transcriptionally cleaved at a poly(A) site and then polyadenylated before being exported to the cytoplasm. The poly(A) tail aids in the transportation of mRNA and protects it from degradation. In the cytoplasm, poly(A) tails play pivotal roles in the translation and stabi 11 ty of the mRNA. The presence and length of the poly(A) tail increases the stability and decreases immunogenicity of mRNA. As mRNA has diagnostic and therapeutic applications, researchers have an interest in determining poly(A) tail lengths.

[03] One challenge in studying poly(A) tails is that they are difficult to sequence and accurately measure. The long, repetitive nature of the poly(A) tail poses difficulties for analytical methodologies. In one known approach. Ribonuclease H is used with an RNA sequence specific probe to cleave the poly(A) tail that can then be measured via gel electrophoresis. This approach is limited in application due to its probe sequence specificity, which requires extensive work every’ new construct, and different sequence gel migration rates requiring different ladders. In another known approach, RNA is reverse transcribed into deoxyribonucleic acid (DNA) that is then sequenced through next-generation sequencing (NGS) and Sanger sequencing. Both NGS and Sanger sequencing of poly(A) tail lengths have low accuracy due to the long repeat of the adenine nucleotide. This approach is further limited in utility as its multi-step technique is time consuming and susceptible to error. In a third known approach, a Ribonuclease T1 (RNase Tl) endonuclease cleaves RNA to remove mRNA poly(A) tails that are measured through high-performance liquid chromatography (HPLC). mass spectrometry (MS), and capillary electrophoresis (CE). This approach is limited in utility as it results in a distribution of tail lengths which poses quality control issues. This approach also has limits to measurable tail length size that vary with whether HPLC. MS, or CE is used.

[04] Accordingly, the present disclosure addresses this problem via a simple, universal, and elegant method described in more detail herein. One advantage of the present disclosure is that is allows analyzation of both long and short poly(A) tails, e.g., allowing characterization of a practically infinite number of poly(A) tail lengths. Another advantage of the present disclosure is that is allows high resolution characterization of poly(A) tails, e.g., 0.7 nucleotides based on a 1 kilobase sequence. Another advantage of the present disclosure is that it allows analysis and characterization of modified nucleotides in addition to poly(A) tails.

[05] An embodiment of the present disclosure includes a method for determining a length of a poly(A) tail comprising exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, and determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample. In embodiments, comparing the peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and a GMP in the RNA sample is at a wavelength of about 260 nm. In embodiments, determining a length of the poly(A) tail of the RNA sample comprises comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and a GMP from the RNA sample.

[06] In embodiments, a method further comprises identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP in a control RNA sample. In further embodiments, identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP. and GMP further comprises performing HPLC on a control RNA sample comprising a known number of C nucleobases, a known number of U nucleobases, a known number of G nucleobases, and a number of A nucleobases. In embodiments, identifying a coefficient for a ratio of absorbance comprises identifying a coefficient for a ratio of absorbance for each of AMP to CMP, UMP, and GMP in a control RNA sample. In particular embodiments, determining a length of the poly(A) tail of the RNA sample by comparing a peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP. and GMP from the RNA sample comprises using formula 1: wherein N is the amount of a nucleobase in the RNA sample other than A nucleobase, A is the amount of A nucleobases in the non-tail portion of the RNA sample (excludes any enzymatic tail that has been added (e.g., via a tailing reaction) and any tail that is encoded already in the DNA template), NA is the coefficient for a ratio of absorbance for AMP with a corresponding nucleobase N from the control RNA sample, AMP is the AMP peak area in the RNA sample, and NMP is the corresponding nucleobase peak area in the RNA sample.

[07] Embodiments of the present disclosure include a method for determining a length of a poly(A) tail of an RNA sample, the method comprising hydrolyzing an RNA sample with a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike at a wavelength of about 260 nm, and determining a length of a poly(A) tail of the RNA sample by comparing the concentration of AMP in the RNA sample with a reference curve. In embodiments, a method further comprises performing HPLC on an external standard to create the reference spike, wherein the performing HPLC on an external standard to create the reference spike is simultaneous with performing HPLC on the mixture of nucleotide monophosphates from the RNA sample, and wherein the external standard is cap-0.

[08] In embodiments, a method further comprising creating a reference curve, wherein the reference curve is generated by identifying an expected concentration of AMP from a number of nucleotides in a sequence for at least three different tail lengths using formula 4:

Expected concentration \iM) (C, U, G, or ) = wherein N is the number of C. U, G. or A nucleobases in the known sequence.

[09] Embodiments of the present disclosure include kits for practicing any of the disclosed methods. For example, an embodiment includes a kit for performing any method of the present disclosure, the kit comprising a cleaving enzyme capable of hydrolyzing the RNA sample to a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), and either (a) a first RNA sample comprising known amounts of C nucleobases, U nucleobases, G nucleobases, and A nucleobase that is capable of generating a coefficient indicative of a ratio of absorbance of AMP to at least one of CMP. UMP, and GMP or is capable of acting as an external standard by generating a 3 to 7 point calibration curve for molarity of CMP, UMP, GMP, and AMP or (b) an external standard capable of eluting at a time different than at least one of CMP, UMP, GMP, and AMP, and instructions for performing high-performance liquid chromatography (HPLC) comprising one or more of a mobile phase, a column, a mobile phase gradient, a flow rate, a run length, a column temperature, a detector, or any combination thereof.

BRIEF SUMMARY OF THE INVENTION

[010] The methods and kits of the present disclosure offer significant advantages over the currently known and used methods for determining the length of a poly(A) tail, including but not limited to the ability to analyze both long and short poly(A) tails thereby allowing characterization of a practically infinite number of poly(A) tail lengths, the ability to complete high resolution characterization of poly(A) tails, e g., 0.7 nucleotides based on a 1 kilobase sequence, and the ability to analyze and characterize modified nucleotides incorporated within or adjacent to poly(A) tails.

Methods for characterization of poly(A) tails using internal standards. [OH] An embodiment of the present disclosure includes a method for determining a length of a poly(A) tail comprising exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, and determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample. In embodiments, comparing the peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and a GMP in the RNA sample is at a wavelength of about 260 nm. In embodiments, determining a length of the poly(A) tail of the RNA sample comprises comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and a GMP from the RNA sample.

[012] In embodiments, a method further comprises identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP in a control RNA sample. In further embodiments, identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP further comprises performing HPLC on a control RNA sample comprising a known number of C nucleobases, a known number of U nucleobases, a known number of G nucleobases, and a number of A nucleobases. In embodiments, identifying a coefficient for a ratio of absorbance comprises identifying a coefficient for a ratio of absorbance for each of AMP to CMP, UMP, and GMP in a control RNA sample. In particular embodiments, determining a length of the poly(A) tail of the RNA sample by comparing a peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP. and GMP from the RNA sample comprises using formula 1:

[013] wherein N is the amount of a nucleobase in the RNA sample other than A nucleobase, A is the amount of A nucleobases in the non-tail portion of the RNA sample, NA is the coefficient for a ratio of absorbance for AMP with a corresponding nucleobase N from the control RNA sample, AMP is the AMP peak area in the RNA sample, and NMP is the corresponding nucleobase peak area in the RNA sample. [014] In embodiments, determining a length of the poly(A) tail of the RNA sample includes comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and GMP from the RNA sample using formula 1 to generate a length of poly(A) tail based upon the comparison of a peak area of AMP to CMP, a peak area of AMP to UMP, and a peak area of AMP to GMP, and averaging each of the three tail lengths determined by formula 1 to generate the length of a poly(A) tail of the RNA sample. In certain embodiments, NA for AMP to CMP is about 2.6 to about 1.8, or about 2.5 to about 2.0, or about 2. 1 to about 2.0, or about 2.029, NA for AMP to UMP is about 1.8 to about 1.3, or about 1.7 to about 1.4, or about 1.6 to about 1.5, or about 1.514, and/or NA for AMP to GMP is about 1.5 to about 1.0, or about 1.4 to about 1.1, or about 1.3 to about 1.2, or about 1.217.

[015] In embodiments, a cleaving enzyme comprises nuclease Pl, nuclease SI, benzonase, phosphodiesterase, or any combination thereof. In embodiments, exposing an RNA sample to the cleaving enzy me is for an incubation time ranging from about 3 minutes to about 24 hours, from about 30 minutes to about 4 hours, or about 3 hours. In embodiments, exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C, from about 35 °C to about 39 °C, or about 37 °C.

[016] The methods of the present disclosure may be used to analyze and characterize a poly(A) tail of an RNA sample ranging having a length from about 5 nucleotides to about 2000 nucleotides, from about 20 nucleotides to about 2000 nucleotides, from about 30 nucleotides to about 2000 nucleotides, from about 50 nucleotides to about 2000 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 200 nucleotides to about 2000 nucleotides, or from about 300 nucleotides to about 2000 nucleotides. The methods of the present disclosure may be used to analyze and characterize an RNA sample having a length from about 200 nucleotides to about 10,000 nucleotides, from about 1,000 nucleotides to about 10,000 nucleotides, from about 2,000 nucleotides to about 10,000 nucleotides, from about 3,000 nucleotides to about 10,000 nucleotides, from about 5,000 nucleotides to about 10,000 nucleotides.

Methods for characterization of poly(A) tails using external standards.

[017] Embodiments of the present disclosure include a method for determining a length of a poly(A) tail of an RNA sample, the method comprising hydrolyzing an RNA sample with a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike at a wavelength of about 260 nm, and determining a length of a poly(A) tail of the RNA sample by comparing the concentration of AMP in the RNA sample with a reference curve. In embodiments, a method further comprises performing HPLC on an external standard to create the reference spike, wherein the performing HPLC on an external standard to create the reference spike is simultaneous with performing HPLC on the mixture of nucleotide monophosphates from the RNA sample, and wherein the external standard is cap-0.

[018] In embodiments, a method further comprising creating a reference curve, wherein the reference curve is generated by identifying an expected concentration of AMP from a number of nucleotides in a sequence for at least three different tail lengths using formula 4: wherein N is the number of C, U, G. or A nucleobases in the known sequence.

[019] In embodiments, a cleaving enzyme comprises nuclease Pl, nuclease SI, benzonase, phosphodiesterase, or any combination thereof. In embodiments, exposing an RNA sample to the cleaving enzy me is for an incubation time ranging from about 3 minutes to about 24 hours, from about 30 minutes to about 4 hours, or about 3 hours. In embodiments, exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C, from about 35 °C to about 39 °C, or about 37 °C.

[020] The methods of the present disclosure may be used to analyze and characterize a poly(A) tail of an RNA sample ranging having a length from about 5 nucleotides to about 2000 nucleotides, from about 20 nucleotides to about 2000 nucleotides, from about 30 nucleotides to about 2000 nucleotides, from about 50 nucleotides to about 2000 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 200 nucleotides to about 2000 nucleotides, or from about 300 nucleotides to about 2000 nucleotides. The methods of the present disclosure may be used to analyze and characterize an RNA sample having a length from about 200 nucleotides to about 10,000 nucleotides, from about 1,000 nucleotides to about 10,000 nucleotides, from about 2,000 nucleotides to about 10,000 nucleotides, from about 3,000 nucleotides to about 10,000 nucleotides, from about 5,000 nucleotides to about 10.000 nucleotides. Kits for characterization of poly(A) tails.

[021] Embodiments of the present disclosure include kits for practicing any of the disclosed methods. For example, an embodiment includes a kit for performing any method of the present disclosure, the kit comprising a cleaving enzyme capable of hydrolyzing the RNA sample to a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP). and guanosine monophosphate (GMP), and either (a) a first RNA sample comprising known amounts of C nucleobases, U nucleobases, G nucleobases, and A nucleobase that is capable of generating a coefficient indicative of a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP, or is capable of acting as an external standard by generating a 3 to 7 point calibration curve for molarity of CMP, UMP, GMP, and AMP or (b) an external standard capable of eluting at a time different than at least one of CMP, UMP, GMP, and AMP, and instructions for performing high-performance liquid chromatography (HPLC) comprising one or more of a mobile phase, a column, a mobile phase gradient, a flow rate, a run length, a column temperature, a detector, or any combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

[022] Figure 1 (FIG. 1) shows an embodiment of the present disclosure for analyzing a poly(A) tail, comprising exposing an RNA sample to a cleaving enzyme to generate a mixture of components comprising nucleotide monophosphates, performing high-performance-liquid chromatography (HPLC) on the mixture of components comprising nucleotide monophosphates, and determining a length of the poly(A) tail of the RNA sample based on the peak area of AMP.

[023] Figure 2 (FIG. 2) shows an embodiment of the present disclosure for analyzing a poly(A) tail using an internal intrinsic standard, comprising hydrolyzing an RNA sample to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing high- performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, optionally (if not previously performed or performed after), performing HPLC on a sequence having a know n number of A nucleobases, a known number of C nucleobases, a known number of G nucleobases, and a known number of U nucleobases to create a universal multiplier (coefficient) for at least one of AMP:CMP, AMP:GMP. and AMP:UMP, and determining the length of the poly(A) tail of the sample by comparing a peak area of adenosine monophosphate to a peak area of at least one of a cytidine monophosphate, a uridine monophosphate, and a guanosine monophosphate.

[024] Figure 3 (FIG. 3) shows a representative chromatograph from reverse phase HPLC of an internal intrinsic standard comprising a known number of C nucleobases. a known number of U nucleobases, a known number of G nucleobases. and a known number of A nucleobases where the HPLC profile is shown as absorbance (mAU) at 260 nm as a function of time (minutes). 1=CMP, 2=UMP, 3=GMP, and 4= AMP.

[025] Figure 4 (FIG. 4) shows an embodiment of the present disclosure for analyzing a poly(A) tail using an external spike, comprising simulating a tail length curve based on multiple tail lengths of a given construct, determining a concentration (pM) of AMP in a sample by comparing an AMP signal to a known reference spike, and comparing the observed concentration (pM) of AMP with an expected concentration (pM) of AMP on the simulated tail length curve.

[026] Figure 5 (FIG. 5) shows a representative simulated tail length curve for multiple tail lengths of a given construct as a function of tail length (# of A) and concentration of AMP (pM).

[027] Figure 6 (FIG. 6) shows an embodiment of the present disclosure where the concentration of AMP was determined in a sample by comparing the AMP signal with a known reference spike.

[028] Figure 7 (FIG. 7) shows an embodiment of the present disclosure where the observed concentration of AMP from FIG. 6 was then compared with the expected concentration of AMP on a simulated curve from FIG. 5. to determine the length of a poly(A) tail from a sample.

DETAILED DESCRIPTION

[029] While the concepts of the present disclosure are illustrated and described in detail in the figures and descriptions herein, results in the figures and their description are to be considered as examples and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. [030] Unless defined otherwise, the scientific and technology nomenclatures have the same meaning as commonly understood by a person in the ordinary skill in the art pertaining to this disclosure.

[031] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods, kits, and systems described herein are readily apparent from the description of the disclosure contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the disclosure or any embodiment thereof

[032] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are now described.

Definitions.

[033] As used herein, “g” represents gram; “L” represents liter; “mg” represents “milligram (10-3 gram);” “mL” or “cc” represents milliliter (10-3 liter). One “pL” equals to one microliter (10-6 liter). The unit of temperature used herein is degree Celsius (°C).

[034] The term “about” is used in conjunction with numeric values to include normal variations in measurements as expected by persons skilled in the art, and is understood to have the same meaning as “approximately” and to cover a typical margin of error, such as ±15%, ±10%. ±5%, ±1%, ±0.5%, or even ±0. 1% of the stated value. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

[035] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a method” includes having two or more methods that are either the same or different from each other. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[036] In the interest of brevity and conciseness, any ranges of values set forth in this specification contemplate all values within the range and are to be construed as support for claims reciting any sub-ranges having endpoints which are real number values within the specified range in question. By way of a hypothetical illustrative example, a disclosure in this specification of a range of from 1 to 5 shall be considered to support claims to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[037] The term “substantially” or “about” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” or “about” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[038] The term “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

General Description.

[039] Referring to FIG. 1. generally, the present disclosure relates to methods and kits for determining a length of a poly(A) tail. Broadly, the methods 100 comprise exposing an RNA sample to a cleaving enzy me to generate a mixture of components comprising nucleotide monophosphates 102, performing high-performance liquid chromatography (HPLC) on the mixture of components comprising nucleotide monophosphates 104, and determining a length of the poly(A) tail of the RNA sample based on the peak area of AMP 106. Reference to a peak area throughout this disclosure is a reference to the area underneath an absorbance curve resulting from high-performance liquid chromatograph as a function of time. For example, the peak area of AMP is a reference to the area underneath the peak for AMP on an absorbance curve as shown in FIG. 3 as peak 4. The methods of the present disclosure contemplate using an internal standard to calculate the length of a poly(A) tail and/or an external standard to calculate the length of a poly(A) tail as discussed more completely herein.

Methods Using an Internal Standard.

[040] Referring to FIG. 2, an embodiment of the present disclosure is shown 200 that relies on the use of an internal standard to determine the length of a poly(A) tail. In FIG. 2, a method for determining the length of a poly(A) tail 200 using an internal intrinsic standard is disclosed comprising hydrolyzing an RNA sample to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP) 202. performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates 204, optionally (if not previously performed or performed after), performing HPLC on a sequence having a known number of A nucleobases, a known number of C nucleobases, a known number of G nucleobases, and a known number of U nucleobases to create a universal multiplier (coefficient) for at least one of AMP:CMP. AMP:GMP, and AMP:UMP 206, and determining the length of the poly(A) tail of the sample by comparing a peak area of adenosine monophosphate to a peak area of at least one of a cytidine monophosphate, a uridine monophosphate, and a guanosine monophosphate 208.

[041] In certain embodiments, step 202 includes only AMP, only CMP, only UMP, only GMP, or any combination thereof. In certain embodiments, step 202 includes non-naturally occurring and/or modified nucleotides in combination with any one or all of AMP, CMP, UMP, and GMP. For example, in certain embodiments the RNA sample is an RNA sample like DasherGFP® mRNA available from Aldevron®.

[042] In embodiments, step 204 is performed at a temperature betw een 0 °C and 40 °C. In embodiments, step 204 is performed at roughly 4 °C, at roughly room temperature, or at roughly 37 °C. Typically, step 204 is performed on a reverse phase column (e.g.. C18 column) but can be performed on any HPLC column suitable for separation of AMP, CMP, UMP, and/or GMP. The HPLC column does not necessarily need to be a reverse phase column.

[043] In embodiments, step 206 is performed prior to method 200, after method 200, or at any time during method 200. Step 206 may be performed more than once per method 200. It may only be necessary to only perform step 206 once to generate a universal multiplier (coefficient) for at least one of AMP:CMP, AMP:GMP, and AMP: UMP, which can be subsequently used for method 200. In embodiments, a universal multiplier (coefficient) for at least one of AMP:CMP. AMP:GMP, and AMP: UMP (or all off AMP UMP. AM PUMP, and AMP: UMP) is supplied with a kit thereby removing the need to perform step 206. In embodiments, step 206 may be run monthly, weekly, daily, and/or with each method 200 to provide the most accurate universal multiplier (coefficient) based on current column and HPLC conditions, which may include but is not limited to buffer, temperature, flow rate, column medium, and/or column size. [044] In embodiments, the determination of a length of poly(A) tail of the sample in step 208 is based on a comparison of the peak area of AMP to the peak area of just one of CMP, UMP, and GMP. In certain embodiments the determination of a length of poly(A) tail of the sample in step 208 is based on a comparison of the peak area of AMP to the peak area of at least two of CMP, UMP, and GMP. In certain embodiments the determination of a length of poly(A) tail of the sample in step 208 is based on a comparison of the peak area of AMP to the peak area of all three of CMP, UMP. and GMP.

[045] FIG. 3 shows a representative chromatograph from HPLC of an internal intrinsic standard comprising a known number of C nucleobases, a known number of U nucleobases. a known number of G nucleobases, and a known number of A nucleobases where the HPLC profile is shown as absorbance (mAU) at 260 nm as a function of time (minutes). 1=CMP, 2=UMP, 3=GMP, and 4= AMP. The retention time for CMP is 10.026, the retention time for UMP is 11. 166, the retention time for GMP is 17.032, and the retention time for AMP is 21.352. In embodiments, the absorbance is measured at about 260 nm, however, additional wavelengths my be used so long as the wavelength is suitable for absorbance of the internal intrinsic standard and RNA sample.

[046] In embodiments of the present disclosure, a method for determining a length of a poly(A) tail is disclosed comprising exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high- performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, and determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample. The cleaving enzyme may comprise any singular or combination of enzymes suitable to cleave the RNA sample into individual nucleotide monophosphates. Some examples of suitable cleaving enzymes include nuclease Pl, nuclease SI, benzonase, phosphodiesterase, or any combination thereof.

[047] Exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates will generally occur for the time period recommended by the commercial company manufacturing the cleaving enzyme taking into account the concentration of the cleaving enzyme, the activity of the cleaving enzyme, and the concentration and length of the RNA sample that is subjected to the cleaving enzyme. In an embodiment, the length of exposure (incubation) will be for a time sufficient to substantially digest the entirety' of the RNA sample. In embodiments, the length of exposure (incubation) will result in greater than about 90% digestion, greater than about 93% digestion, greater than about 95% digestion, greater than about 97% digestion, greater than about 98% digestion, greater than about 99% digestion, or about 100% digestion of the RNA sample. In embodiments, the length of exposure (incubation) time will be greater than about 3 minutes, greater than about 30 minutes, greater than about 1 hour, or greater than about 2 hours. In embodiments, the length of exposure (incubation) time will be less than about 48 hours, less than about 24 hours, less than about 12 hours, less than about 6 hours, or less than about 4 hours. In embodiments, the length of exposure (incubation) time will be from about 3 minutes to about 24 hours, from about 30 minutes to about 4 hours, from about 2 hours to about 4 hours, or about 3 hours. In embodiments, exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C, from about 35 °C to about 39 °C, or about 37 °C. Generally, the incubation temperature will be the temperature recommended by the supplier of the cleaving enzyme. In embodiments, exposing an RNA sample to a cleaving enzyme generates a mixture of nucleotide monophosphates, not a mixture of nucleosides or a mixture of nucleosides and nucleotide monophosphates. In embodiments, an RNA sample is only exposed to a single cleaving enzyme. In embodiments, an RNA sample is exposed to one or more cleaving enzymes. In embodiments, a single cleaving enzyme is used where the cleaving enzyme generates nucleotide monophosphates as opposed to nucleosides.

[048] In embodiments, comparing the peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and a GMP in the RNA sample is at a wavelength of about 260 nm. However, the present disclosure contemplates the use of any wavelength where absorbance by AMP. CMP, UMP. and/or GMP is effective enough to generate distinguishable peaks as a function of time. In certain embodiments, determining a length of the poly(A) tail of the RNA sample comprises comparing a peak area of AMP from the RNA sample to at least two of CMP, UMP, and GMP from the RNA sample. In certain embodiments, determining a length of the poly(A) tail of the RNA sample comprises comparing a peak area of AMP from the RNA sample to all three of CMP, UMP. and GMP from the RNA sample.

[049] Methods for determining a length of a poly(A) tail may further comprise identifying a coefficient for a ratio of absorbance of AMP to at least one, at least two of, or all of CMP, UMP, and GMP in a control RNA sample. In a preferred embodiment, identifying a coefficient for a ratio of absorbance comprises identifying a coefficient for a ratio of absorbance for each of AMP to CMP, UMP, and GMP in the control RNA sample. In embodiments, identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP further comprises performing HPLC on the control RNA sample comprising a known number of C nucleobases. a known number of U nucleobases, a known number of G nucleobases. and a number of A nucleobases. While not necessary, in preferred embodiments, the HPLC on the control RNA sample is performed under the same conditions as the RNA sample for which the length of a poly(A) tail is being determined, e.g., one or more of the following conditions may be the same: temperature, column media, flow rate, buffer, column length.

[050] In embodiments, determining a length of the poly(A) tail of the RNA sample by comparing a peak area of AMP from the RNA sample to a peak area of at least one, at least two of, or all of a CMP, UMP, and GMP from the RNA sample comprises using formula 1: wherein N is the amount of a nucleobase in the RNA sample other than A nucleobase, A is the amount of A nucleobases in the non-tail portion of the RNA sample, NA is the coefficient for a ratio of absorbance for AMP with a corresponding nucleobase N from the control RNA sample, AMP is the AMP peak area in the RNA sample, and NMP is the corresponding nucleobase peak area in the RNA sample. For example, when calculating the poly(a) tail length for an RNA sample based upon comparing the peak are of AMP to the peak are of CMP, N is the amount of CMP in the RNA sample, A is the amount if AMP in the RNA sample, NA is the coefficient for ratio of absorbance for AMP: CMP, AMP is the AMP peak area in the RNA sample, and NMP is the CMP peak area in the RNA sample. In specific embodiments, NA for AMP to CMP is about 2.6 to about 1.8, or about 2.5 to about 2.0, or about 2.1 to about 2.0, or about 2.029, NA for AMP to UMP is about 1.8 to about 1.3, or about 1.7 to about 1.4, or about 1.6 to about 1.5, or about 1.514, and/or N for AMP to GMP is about 1.5 to about 1.0, or about 1.4 to about 1.1, or about 1.3 to about 1.2, or about 1.217.

[051] In embodiments, determining a length of the poly(A) tail of the RNA sample includes comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and GMP from the RNA sample using formula 1 to generate a length of poly(A) tail based upon the comparison of a peak area of AMP to CMP, a peak area of AMP to UMP, and a peak area of AMP to GMP, and averaging each of the three tail lengths determined by formula 1 to generate the length of a poly(A) tail of the RNA sample. In certain embodiments, any two of the three comparisons is used to generate two tail lengths, and those two comparisons are averaged to generate the length of a poly (A) tail of the RNA sample. For example, formula 1 is used to determine a first tail length in an RNA sample based on a comparison of peak area of AMP in the RNA sample to a peak area of CMP in the RNA sample using an NA for AMP to CMP. Optionally, formula 1 is then used to determine a second tail length in an RNA sample based on a comparison of peak area of AMP in the RNA sample to a peak area of UMP in the RNA sample using an NA for AMP to UMP. Optionally, formula 1 is then used to determine a third tail length in an RNA sample based on a comparison of peak area of AMP in the RNA sample to a peak area of GMP in the RNA sample using an NA for AMP to GMP. The first tail length, the optional second tail length, and the optional third tail length are then averaged to generate a tail length for the RNA sample. In embodiments, the first tail length, optional second tail length, and optional third tail length can each be any of AMP to CMP. AMP to UMP, and AMP to GMP. [052] The length of the poly(A) tail of the RNA sample in embodiments of the present disclosure ranges from about 5 nucleotides to about 2000 nucleotides, from about 20 nucleotides to about 2000 nucleotides, from about 30 nucleotides to about 2000 nucleotides, from about 50 nucleotides to about 2000 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 200 nucleotides to about 2000 nucleotides, or from about 300 nucleotides to about 2000 nucleotides. In certain embodiments, the length of the poly(A) tail of the RNA sample is greater than about 100 nucleotides, greater than about 200 nucleotides, greater than about 300 nucleotides, greater than about 400 nucleotides, greater than about 500 nucleotides, greater than about 600 nucleotides, greater than about 700 nucleotides, greater than about 800 nucleotides, greater than about 900 nucleotides, or greater than about 1000 nucleotides. [053] The length of the RNA sample (without the poly(A) tail) in embodiments of the present disclosure ranges from about 200 nucleotides to about 10,000 nucleotides, from about 1000 nucleotides to about 10,000 nucleotides, from about 2000 nucleotides to about 10,000 nucleotides, from about 3000 nucleotides to about 10,000 nucleotides, from about 5000 nucleotides to about 10.000 nucleotides. In certain embodiments, the length of the RNA sample (without the poly(A) tail) is greater than about 200 nucleotides, greater than about 500 nucleotides, greater than about 1000 nucleotides, greater than about 2000 nucleotides, greater than about 3000 nucleotides, greater than about 4000 nucleotides, greater than about 5000 nucleotides, or greater than about 6000 nucleotides.

Methods Using an External Standard.

[054] The disclosures related to methods using an internal standard are equally applicable to methods for using an external standard.

[055] Referring to FIG. 1 and FIG. 4, an embodiment of the present disclosure is shown 400 that relies on the use of an external standard (reference spike) to determine the length of a poly(A) tail. In an embodiment of the present disclosure, a method for determining a length of a poly(A) tail of an RNA sample comprises simulating a tail length curve based on multiple tail lengths of a given construct 402, determining a concentration (pM) of AMP in an RNA sample following digestion with a cleaving enzyme and performance of HPLC by comparing an AMP signal (peak area) in the RNA sample to a known reference spike 404, and comparing the observed concentration (pM) of AMP with an expected concentration (pM) of AMP on the simulated tail length curve 406.

[056] In an embodiment, at least twn tail lengths of a given construct are used to generate the simulated curve in step 402. In embodiments, at least three tail lengths of a given construct, at least four tail lengths of a given construct, at least five tail lengths of a given construct, at least six tail lengths of a given construct, or at least seven tail lengths of a given construct are used to generate a simulated curve. Step 402 may be performed prior to step 404, after step 404, or simultaneous with step 404. Step 402 may be performed only once per numerous steps 404 and 406 or step 402 may be performed with every method 400. In embodiments, step 402 is performed monthly, weekly, daily, and/or with each method 400 to provide the most accurate simulated curve.

[057] The reference spike in step 404 is an external standard. In embodiments, an external standard is selected based on its ability to do one or more of the following: absorb at a wavelength of 260 nm or absorb at the wavelength used for performing HPLC on the RNA sample, the ability of the external standard to elute at a different time than any of AMP, UMP, CMP. or GMP so as not to interfere with the peak area of any of AMP, UMP, CMP, or GMP, and the ability to be stable under the conditions for performing HPLC on the RNA sample (e.g., temperature, buffer). In an embodiment, the external standard is cap-0 with a known concentration. In an embodiment, HPLC is performed on the external standard (reference spike) simultaneous with the digested RNA sample from step 102 in FIG. 1. For example, an external standard is mixed with the RNA sample following step 102, or prior to step 102 if the external standard is resistant to the cleaving enzyme, and HPLC is performed on the digested RNA sample mixed with the external standard in step 104. In embodiments, HPLC is performed on the external standard (reference spike) prior to or after performing HPLC on the digested RNA sample from step 102 in FIG. 1.

[058] Still referring to step 404 and as discussed in more detail in reference to FIG. 6 below, a concentration of AMP in the digested RNA sample is determined by comparing the peak area for AMP to the peak area for the known reference spike.

[059] In step 406, the concentration for AMP observed in step 404 by comparing the peak area of AMP to the peak are of the known reference spike is then compared with the expected concentration of AMP on the simulated curve as discussed in more detail below in reference to FIG. 7.

[060] FIG. 5 shows an embodiment of a simulated tail length curve for multiple tail lengths (8 tail lengths) of a given construct as a function of tail length (# of A) and concentration of AMP (pM). Each of the multiple (8) tail length concentrations w as generated using formula 4 and a curve was generated to best fit the multiple tail length concentrations. As disclosed, the tail length increases with the increased concentration of AMP. The simulated tail length curve in FIG. 5 is an embodiment of a tail length curve used in step 406.

[061] FIG. 6 shows an embodiment of the present disclosure where the concentration of AMP was determined in a sample by comparing the AMP signal (peak area) with a known reference spike (peak area). In this embodiment, the known reference spike was cap-0 and it was run simultaneously with the cleaved RNA sample. Cap-0 eluted at about 24.5 minutes and after CMP (about 10.5 minutes), UMP (about 1 1.75 minutes), GMP (about 18 minutes), and AMP (about 22.25 minutes). The concentration of AMP was determined to be 1106.4 pM in the cleaved RNA sample based on the peak area for AMP compared to the peak area for cap-0 (known concentration).

[062] FIG. 7 shows an embodiment of the present disclosure where the observed concentration of AMP from FIG. 6 was then compared with the expected concentration of AMP on a simulated curve from FIG. 5 to determine the length of a poly(A) tail from an RNA sample. For example, the expected concentration of AMP (1106.4 pM) was located on the simulated curve (x-axis) and resulted in a poly(A) tail length determination of 323 (y-axis) (ES cap-0 based tail).

[063] In embodiments of the present disclosure, a method for determining a length of a poly(A) tail of an RNA sample is disclosed, the method comprising hydrolyzing an RNA sample with a cleaving enzyme to generate a mixture of nucleotide monophosphates compnsing adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike at a wavelength of about 260 nm, and determining a length of a poly(A) tail of the RNA sample by comparing the concentration of AMP in the RNA sample with a reference curve.

[064] In certain embodiments, a method for determining a length of a poly (A) tail of an RNA sample further comprises performing HPLC on an external standard to create a reference spike, wherein the performing HPLC on an external standard to create the reference spike is simultaneous with performing HPLC on the mixture of nucleotide monophosphates from the RNA sample. In certain embodiments, the performing HPLC on an external standard to create the reference spike is performed prior to or after performing HPLC on the mixture of nucleotide monophosphates from the RNA sample. When performing HPLC on an external standard to create the reference spike is performed prior to or after performing HPLC on the mixture of nucleotide monophosphates from the RNA sample, the HPLC of the reference spike is performed under one or more of the same conditions, including using the same column, temperature, flow rate, buffer, and/or column media. [065] In embodiments, an external standard is selected based on its ability to do one or more of the following: absorb at a wavelength of 260 nm or absorb at the wavelength used for performing HPLC on the RNA sample, the ability of the external standard to elute at a different time than any of AMP, UMP, CMP, or GMP so as not to interfere with the peak area of any of AMP, UMP, CMP, or GMP, and the ability to be stable under the conditions for performing HPLC on the RNA sample (e.g., temperature, buffer). In an embodiment, the external standard is the cap-0.

[066] In an embodiment, a method for determining a length of a poly(A) tail of an RNA sample comprises creating a reference curve, wherein the reference curve is generated by identifying an expected concentration of AMP from a number of nucleotides in a sequence for at least three different tail lengths using formula 4: where N is the number of C, U, G. or A nucleobases in the known sequence. For example, a first concentration of AMP from a first number of nucleotides in a sequence for a first tail length is calculated using formula 4. Then a second concentration of AMP from a second number of nucleotides in a sequence for a second tail length is calculated using formula 4. Then a third concentration of AMP from a third number of nucleotides in a sequence for a third tail length is calculated using formula 4. Optionally, any additional number of concentrations are calculated using formula 4. At least two, preferably three, and any additional number of concentrations are then graphed as a function of tail length versus AMP concentration (pM) as show n in FIG. 5 to generate simulated curve. In embodiments, a simulated curve is generated simultaneous with a method for determining a length of a poly(A) tail of an RNA sample. In embodiments, a simulated curve is generated prior to or after performing a method for determining a length of a poly(A) tail of an RNA sample simultaneous. In embodiments, a simulated curve is generated monthly, weekly, daily, and/or with each method 400 to provide the most accurate simulated curve based on cunent column and HPLC conditions.

[067] Hydrolyzing an RNA sample with a cleaving enzyme to generate a mixture of nucleotide monophosphates will generally occur for the time period recommended by the commercial company manufacturing the cleaving enzy me taking into account the concentration of the cleaving enzyme, the activity of the cleaving enzyme, and the concentration and length of the RNA sample that is subjected to the cleaving enzyme. In an embodiment, the length of exposure (incubation) will be for a time sufficient to substantially digest the entirety of the RNA sample. In embodiments, the length of exposure (incubation) will result in greater than about 90% digestion, greater than about 93% digestion, greater than about 95% digestion, greater than about 97% digestion, greater than about 98% digestion, greater than about 99% digestion, or about 100% digestion of the RNA sample. In embodiments, the length of exposure (incubation) time will be greater than about 3 minutes, greater than about 30 minutes, greater than about 1 hour, or greater than about 2 hours. In embodiments, the length of exposure (incubation) time will be less than about 48 hours, less than about 24 hours, less than about 12 hours, less than about 6 hours, or less than about 4 hours. In embodiments, the length of exposure (incubation) time will be from about 3 minutes to about 24 hours, from about 30 minutes to about 4 hours, from about 2 hours to about 4 hours, or about 3 hours. In embodiments, exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C, from about 35 °C to about 39 °C, or about 37 °C. Generally, the incubation temperature will be the temperature recommended by the supplier of the cleaving enzyme. In embodiments, exposing an RNA sample to a cleaving enzy me generates a mixture of nucleotide monophosphates, not a mixture of nucleosides or a mixture of nucleosides and nucleotide monophosphates. In embodiments, an RNA sample is only exposed to a single cleaving enzyme. In embodiments, an RNA sample is exposed to one or more cleaving enzymes. In embodiments, a single cleaving enzyme is used where the cleaving enzy me generates nucleotide monophosphates as opposed to nucleosides. [068] The length of the poly(A) tail of the RNA sample in embodiments of the present disclosure ranges from about 5 nucleotides to about 2000 nucleotides, from about 20 nucleotides to about 2000 nucleotides, from about 30 nucleotides to about 2000 nucleotides, from about 50 nucleotides to about 2000 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 200 nucleotides to about 2000 nucleotides, or from about 300 nucleotides to about 2000 nucleotides. In certain embodiments, the length of the poly(A) tail of the RNA sample is greater than about 100 nucleotides, greater than about 200 nucleotides, greater than about 300 nucleotides, greater than about 400 nucleotides, greater than about 500 nucleotides, greater than about 600 nucleotides, greater than about 700 nucleotides, greater than about 800 nucleotides, greater than about 900 nucleotides, or greater than about 1000 nucleotides. [069] The length of the RNA sample (without the poly(A) tail) in embodiments of the present disclosure ranges from about 200 nucleotides to about 10,000 nucleotides, from about 1000 nucleotides to about 10,000 nucleotides, from about 2000 nucleotides to about 10,000 nucleotides, from about 3000 nucleotides to about 10,000 nucleotides, from about 5000 nucleotides to about 10,000 nucleotides. In certain embodiments, the length of the RNA sample (without the poly(A) tail) is greater than about 200 nucleotides, greater than about 500 nucleotides, greater than about 1000 nucleotides, greater than about 2000 nucleotides, greater than about 3000 nucleotides, greater than about 4000 nucleotides, greater than about 5000 nucleotides, or greater than about 6000 nucleotides.

Kits for Determining Poly(A) Tail Length.

[070] The present disclosure contemplates kits for practicing methods disclosed herein, including both methods for determining a poly(A) tail length using an internal standard and methods for determining a poly(A) tail length using an external standard. [071] In an embodiment, kits for performing methods according to any method of the present disclosure are contemplated, the kit comprising a cleaving enzyme capable of hydrolyzing an RNA sample to a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), optionally, either (a) a first RNA sample comprising known amounts of C nucleobases, U nucleobases, G nucleobases, and A nucleobase that is capable of generating a coefficient indicative of a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP, or is capable of acting as an external standard by generating a 3 to 7 point calibration curve for molarity of CMP, UMP, GMP, and AMP or (b) an external standard capable of eluting at a time different than at least one of CMP, UMP, GMP, and AMP, and instructions for performing high-performance liquid chromatography (HPLC) comprising one or more of a mobile phase, a column, a mobile phase gradient, a flow rate, a run length, a column temperature, a detector, or any combination thereof.

Products

[072] The present disclosure contemplates products made by practicing methods disclosed herein, including both methods for determining a poly(A) tail length using an internal standard and methods for determining a poly(A) tail length using an external standard. [073] Embodiments of the present disclosure include an RNA sample having a poly(A) tail is disclosed where the length of the poly(A) tail is determined by a process comprising the methods disclosed herein. For example, in an embodiment, an RNA sample having a poly(A) tail where the length of the poly(A) tail is determined by exposing an RNA sample to a cleaving enzy me to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates, and determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample. In embodiments, the length of the poly(A) tail of the RNA sample is determined by comparing a peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and GMP from the RNA sample using formula 1: , wherein N is the amount of a nucleobase in the RNA sample other than A nucleobase, A is the amount of A nucleobases in the non-tail portion of the RNA sample, NA is the coefficient for a ratio of absorbance for AMP with a corresponding nucleobase N from the control RNA sample, AMP is the AMP peak area in the RNA sample, and NMP is the corresponding nucleobase peak area in the RNA sample.

[074] Embodiments of the present disclosure include an RNA sample having a poly(A) tail where the length of the poly(A) tail is detennined by hydrolyzing an RNA sample with a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP), performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates; determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike at a wavelength of about 260 nm; and determining a length of a poly(A) tail of the RNA sample by comparing the concentration of AMP in the RNA sample with a reference curve. In embodiments, the length of the poly(A) tail is determined by further creating a reference curve, wherein the reference curve is generated by identifying an expected concentration of AMP from a number of nucleotides in a sequence for at least three different tail lengths using formula 4: the number of C, U, G, or A nucleobases in the known sequence.

[075] Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the disclosure.

EXAMPLES

[076] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how⁷ to make and use the methods and compositions of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, molecular w eight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1: Preparation of Base Ratio Assay RNA Tail Length.

[077] Each RNA sample was digested with 0.4 pL nuclease Pl (New England Biolabs, Inc., catalog M0660S (100,000 units/ml)). In addition to nuclease Pl, each sample included 40 pg RNA and water. for a total solution volume of 50 pL per sample. The cleavage reaction was incubated for 30 minutes at 37 °C. In the HPLC, the RNase Pl -digested mRNA sample was mixed with tetraethylammonium bromide (TEAB) and then eluted with acetonitrile (ACN). 10 pL of sample was injected on the column for HPLC analysis.

[078] HPLC experiments were performed using a Thermo Dionex UltiMate 3000 as set forth in Table 1 A and Table IB. Mobile phase A was 25 mM TEAB at pH 7.4 (± 0.2) in a clean glass bottle. In certain embodiments. Mobile phase A has a pH of 8.5. Mobile phase B was ACN. The separation column was a YMC-Pack Pro Cl 8, 250 x 4.6 mm, 5 uni. 120 A. The flow rate was 1.0 mL/min, column temperature was 25 °C, and UV detection wavelength was 260 nm and 280 nm. Mobile phase B gradient was 15% B. The run time was approximately 39 minutes, and the column was then washed with mobile phase A and equilibrated at initial gradient conditions for 12 minutes. No cleanup was necessary.

[079] RNA samples in this example included DasherGFP® mRNA from Aldevron (catalogue #3870-0200). DasherGFP® mRNA encodes an IP-free, fluorescent protein optimized for expression in mammalian cells. It mimics mature mRNA with 5’ Cap 1 structure and 3’ poly(A) tail, and it is ideal for studying transfection and expression using a variety' of assays. DasherGFP® was used to confirm the disclosed invention would work effectively with both traditional mRNA and mRNA having an enzymatic tail.

Table 1A: HPLC Instrument Method

Table IB: Mobile Phase Gradient for Each Sample Injection.

Example 2: Preparation of Intrinsic Internal Standard (ISS).

[080] The creation of an internal intrinsic standard is necessary because bases do not have the same absorbance spectra. Use of the intrinsic internal control standard requires knowing the number of A nucleobases, C, nucleobases, U, nucleobases, and G nucleobases in the untailed region of the poly(A) tailed mRNA sample. Intrinsic internal standards were prepared by combining 49.6 pL water, 40 pL RNA sample (1.8 mg/mL). 10 pL lOx Pl buffer, and 0.4 pL nuclease Pl for a total solution volume of 100 pL. The RNase Pl-digested mRNA sample was vortexed for three seconds and incubated for about 3 hours at 37 °C. The sample was injected on the column for HPLC analysis as described in Example 1. The chromatogram resulting from the HPLC is disclosed in FIG. 3, where the HPLC profile is shown as absorbance (mAU) at 260 nm as a function of time (minutes). 1=CMP, 2=UMP, 3=GMP, and 4= AMP.

Example 3: Calculating Tail Length based on Intrinsic Internal Standard (IIS).

[081] The resulting chromatogram from Example 2 generates four peaks corresponding to absorbance at 260 nm for one of each CMP, UMP, GMP, and AMP (FIG. 3). The tail length can be estimated from the area ratio of AMP versus each of CMP, UMP, and GMP. For example, the retention time (Min), area under the peak (mAU*min), the height (mAU), relative area (%), and relative height (%) for each of CMP (1), UMP (2), GMP (3), and AMP (4) are shown in Table 2.

Table 2: Analysis of chromatogram from FIG. 3

[082] An average tail length can be found by averaging the tail lengths calculated for AMP versus each of CMP, UMP, and GMP. For each intrinsic internal standard, the tail length was calculated based on the following equations: the universal multipliers (coefficients) provided in Table 3.

[083] In the intrinsic internal standard, the known amounts of C, U, G and A bases in the untailed region of the sample act as built-in standards such that an externally added spike is not necessary. To calculate tail length, the ratio of absorbance of A to each of C, U, and G was measured at 260 nm. Table 3 demonstrates universal multipliers of ratio of absorbance of A to each base.

Table 3: Universal multipliers (coefficients) .

[084] For example, as illustrated in Table 2. the area (at 260 nm) was about 30.5 for CMP, about 33.6 for UMP, about 58.0 for GMP, and about 157.0 for AMP. In a known untailed Dasher sequence, the number of C nucleobases is 181, U nucleobases is 150, G nucleobases is 208, and A nucleobases is 215. Based on these respective areas, the universal multipliers, and the nucleobases in an untailed known sequence, the calculated tail length for a C-based tail is 244 A’s, for a U-based tail is 248 A’s, and for a G-based tail is 247 A’s. Accordingly, the average tail length based on Eq.’s 1-3 is 246.3 A’s.

Example 4: Preparation of External Standard.

[085] In addition to internal intrinsic standard (ISS) discussed above, external standards were prepared by combining 49.6 pL water, 40 pL RNA sample (1.8 mg/mL), and pUMP, or cap-0. Cap-0 (New England Biolabs, Inc.) and pUMP (New England Biolabs, Inc.) were selected because they are molecularly similar to target analytes and commercially available. Cap-0 is a N7-methyl guanosine connected to the 5’ nucleotide through a 5’ to 5’ triphosphate linkage. Cap-0 elutes far from other bases and will not co-elute with native cap-1. The RNase Pl -digested mRNA sample was mixed for several seconds and incubated for approximately 3 hours at 37 °C. Each sample was injected on the column for HPLC analysis as described in Example 1.

Example 5: Calculating Tail Length based on External Standard.

[086] The tail length w as calculated based on an external standard as disclosed in

FIG. 4. The tail length curve w as simulated for multiple tail lengths of a given construct, where the number of C. U, and G nucleobases were known as disclosed in Table 4.

Table 4: Nucleotides in sequence for multiple tail lengths

[087] The expected concentration (pM) in a reaction mixture for each nucleotide was calculated using Eq. 4. , the number of C, U, G, or A nucleobases in the known sequence. [088] Based on the formula in Eq. 4, the expected concentration (in pM) in the reaction mixture for each nucleobase was calculated and is disclosed in Table 5.

Table 5: Expected concentration (pM) in reaction mixture for each nucleotide.

[089] A simulated tail length curve for multiple tail lengths of a given construct was generated as a function of tail length (# of A) and concentration of AMP (pM) in FIG. 5. The concentration of AMP was determined in each sample by comparing the AMP signal with the know n reference spike (cap-0) as disclosed in FIG. 6. The observed concentration of AMP was then compared with the expected concentration of AMP on a simulated curve in FIG. 7.

Example 6: Calculating Tail Length based on External Standard

[090] In another embodiment, the tail length was calculated based on an external standard. The concentration of cap-0 (pM) has the following relationship with absorbance (260 nm) for each of CMP, UMP. GMP, AMP. and cap-0.

Table 6: Cap-0 concentration comparison to absorbance signal (mAU).

[091] The above absorbance signals were then used to calculate cap-0 calibration constants by dividing the ratio of cap-0 signal by NMP signal to reach a cap-0 calibration constant for each NMP as disclosed in Table 7.

Table 7: Cap-0 calibration constants.

[092] An equalized signal for each NMP at each cap-0 concentration was established using formula 5 : (NMP signal / cap-0 signal) x cap-0 calibration constant, as shown in Table 8.

Table 8: Equalized signal for each NMP based on cap-0 calibration constants.

[093] A base ratio for each of CMP, UMP, GMP, and AMP was determined by using formula 6: equalized mAU for each NMP / Total mAU for each NMP, as shown in Table 9.

Table 9: Base ratio for each NMP.

[094] The observed tail length was then calculated as shown in Table 10 and

Table 11.

Table 10: Formula for tail length based on observed base ratio.

Table 11: Calculated tail length.

[095] Referring to Tables 6-11, the same process was implemented for pUMP as well, where the pUMP calibration constants were determined to be 0.672 for C_PUMP, 0.502 for Upuwp, 0.404 for G_PUMP, and 0.331 for A_PUMP.

Example 7: Identifying Repeatability with a Calibration Constant from Intrinsic Internal Standard (IIS).

[096] Signal values can differ across instruments and even within an instrument based on the date of each analysis. To confirm the repeatability of the present disclosure, signal values (absorbance at 260 nm) were measured for each of CMP, UMP, GMP, and AMP according to Example 1 on various instruments and on various dates as disclosed in Table 11.

Table 11: Signal values across multiple instruments and dates.

[097] The relative standard deviation (RSD) was calculated for each of CMP, UMP, GMP, and AMP and resulted in roughly 48-49% RSD. The ratio of signal to total signal value was then calculated for each of the samples in Table 11 and are shown in Table 12.

Table 12: Ratio of signal to total for Table 6 data.

[098] The RSD was recalculated for each of CMP, UMP, GMP, and AMP based on the signal to total signal value and as shown in Table 12. Notably, despite a high signal variation of roughly 48-49% RSD in Table 6, the final ratio of signal to total signal value were low at roughly 1% RSD. The calibration constant was also determined for the samples in Table 1 1 by calculating the ration of AMP absorbance to CMP, UMP, and GMP as shown in Table 13.

Table 13: Ratio of AMP absorbance to CMP, UMP, and GMP for Table 6 data.

[099] The RSD was recalculated for the calibration constants as shown in Table 12. Notably, despite a high signal variation of roughly 48-49% RSD in Table 11. the calibration constant values were low at roughly 1.7-1.9% RSD.

[0100] It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.

[0101] Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other methods, kits, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.

[0102] The following numbered clauses define further example aspects and features of the present disclosure:

1. A method for determining a length of a poly (A) tail comprising: exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP); performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates; and determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample.

2. The method of clause 2, wherein comparing the peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and a GMP in the RNA sample is at a wavelength of about 260 nm.

3. The method of any one of clauses 1-2. wherein determining a length of the poly(A) tail of the RNA sample comprises comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and a GMP from the RNA sample.

4. The method of any one of clauses 1 -3, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time ranging from about 3 minutes to about 24 hours.

5. The method of any one of clauses 1-4, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time range of about 30 minutes to about 4 hours.

6. The method of any one of clauses 1-5, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time of about 3 hours. 7. The method of any one of clauses 1-6, wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about

45 °C.

8. The method of any one of clauses 1-7, wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 35 °C to about 39 °C.

9. The method of any one of clauses 1-8, wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature of about 37 °C.

10. The method of any one of clauses 1-9, wherein the length of the poly (A) tail of the RNA sample ranges from about 20 nucleotides to about 2000 nucleotides.

11. The method of any one of clauses 1-10, wherein the length of the RNA sample ranges from about 200 nucleotides to about 10,000 nucleotides.

12. The method of any one of clauses 1-11, further comprises performing HPLC on a control RNA sample comprising a known number of C nucleobases, a known number of U nucleobases, a known number of G nucleobases. and a known number of A nucleobases; and identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP in the control RNA sample.

13. The method of clause 12, wherein identifying a coefficient for a ratio of absorbance comprises identifying a coefficient for a ratio of absorbance for each of AMP to CMP. UMP, and GMP in the control RNA sample.

14. The method of any one of clauses 1-13, wherein the cleaving enzyme comprises nuclease Pl, nuclease SI, benzonase, phosphodiesterase, or any combination thereof.

15. The method of any one of clauses 1-14, wherein determining a length of the poly(A) tail of the RNA sample by comparing a peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and GMP from the RNA sample comprises using formula 1: , wherein A is the amount of a nucleobase in the RNA sample other than A nucleobase, A is the amount of A nucleobases in the non-tail portion of the RNA sample, NA is the coefficient for a ratio of absorbance for AMP with a corresponding nucleobase N from a control RNA sample, AMP is the AMP peak area in the RNA sample, and NMP is the corresponding nucleobase peak area in the RNA sample.

16. The method of clause 15, wherein determining a length of the poly(A) tail of the RNA sample includes comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and GMP from the RNA sample using formula 1 to generate a length of poly(A) tail based upon the comparison of a peak area of AMP to CMP, a peak area of AMP to UMP. and a peak area of AMP to GMP; and averaging each of the three tail lengths determined by formula 1 to generate the length of a poly(A) tail of the RNA sample.

17. The method of clause 16, wherein NA for AMP to CMP is about 2.029, NA for AMP to UMP is about 1.514, and/or NA for AMP to GMP is about 1.217.

18. A method for determining a length of a poly(A) tail of an RNA sample, the method comprising: hydrolyzing an RNA sample to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP); performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates; determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike; and determining a length of a poly(A) tail of the RNA sample by comparing the concentration of AMP in the RNA sample with a reference curve.

19. The method of clause 18, further comprising performing HPLC on an external standard to create the reference spike.

20. The method of clause 19, wherein performing HPLC on the external standard is prior to performing HPLC on the mixture of nucleotide monophosphates from the RNA sample, simultaneous with performing HPLC on the mixture of nucleotide monophosphates from the RNA sample, or after performing HPLC on the mixture of nucleotide monophosphates from the RNA sample.

21. The method of any one of clauses 18-20, further comprising creating a reference curve.

22. The method of clause 21, wherein creating the reference curve comprises simulating a reference curve. 23. The method of any one of clauses 21-22, wherein the reference curve is a number of A nucleobases on one axis versus a concentration of AMP on a second axis.

24. The method of any one of clauses 21-23, wherein the reference curve is generated by identifying an expected concentration of AMP from a number of nucleotides in a sequence for at least three different tail lengths using formula 4: the number of C, U, G, or A nucleobases in the know n sequence.

25. The method of any one of clauses 18-24, wherein determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike is at a wavelength of about 260 nm.

26. The method of any one of clauses 18-25, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time ranging from about 3 minutes to about 24 hours.

27. The method of any one of clauses 18-26, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time range of about 30 minutes to about 4 hours.

28. The method of any one of clauses 18-27, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time of about 3 hours.

29. The method of any one of clauses 18-28, wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C.

30. The method of any one of clauses 18-29, wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 35 °C to about 39 °C.

31. The method of any one of clauses 18-30, wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature of about 37 °C.

32. The method of any one of clauses 18-31, wherein the length of the poly (A) tail of the RNA sample ranges from about 20 nucleotides to about 2000 nucleotides.

33. The method of any one of clauses 18-32, wherein the length of the RNA sample ranges from about 200 nucleotides to about 10,000 nucleotides. 34. The method of any one of clauses 18-33, wherein the cleaving enzyme comprises nuclease Pl, nuclease SI. benzonase. phosphodiesterase, or any combination thereof.

35. A kit for performing the method according to any one of clauses 1-33, the kit comprising: a cleaving enzyme capable of hydrolyzing the RNA sample to a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP); and either (a) a first RNA sample comprising known amounts of C nucleobases, U nucleobases. G nucleobases. and A nucleobase and/or (b) an external standard capable of eluting at a time different than at least one of CMP, UMP, GMP, and AMP.

36. The kit of clause 35, further comprising at least one second RNA sample comprising a poly(A) tail of unknown length.

37. The kit of any one of clauses 35-36, wherein the first RNA sample is capable of generating a coefficient indicative of a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP.

38. The kit of any one of clauses 36-37, wherein the second RNA sample comprising a poly (A) tail of unknown length further comprises a modified base or an artificial base.

39. The kit of any one of clauses 35-38, wherein the cleaving enzyme comprises nuclease Pl, nuclease SI, benzonase, phosphodiesterase, or any combination thereof.

40. The kit of any one of clauses 35-39, further comprising instructions for performing high-performance liquid chromatography (HPLC) comprising one or more of a mobile phase, a column, a mobile phase gradient, a flow rate, a run length, a column temperature, a detector, or any combination thereof.

41. The kit of any one of clauses 35-40, further comprising instructions for performing any one of clauses 1-34.

42. The kit of any one of clauses 35-41. wherein each component of the kit is housed in a separate container.

43. An RNA sample having a poly(A) tail where the length of the poly(A) tail is determined by a process comprising any one of clauses 1-34.

Claims

CLAIMS What is claimed is:

1. A method for determining a length of a poly(A) tail comprising: exposing an RNA sample to a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP); performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates; and determining a length of the poly(A) tail of the RNA sample by comparing a peak area of adenosine monophosphate (AMP) from the RNA sample to a peak area of at least one of a cytidine monophosphate (CMP), a uridine monophosphate (UMP), and a guanosine monophosphate (GMP) from the RNA sample.

2. The method of claim 1 , wherein comparing the peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and a GMP in the RNA sample is at a wavelength of about 260 nm.

3. The method of claim 2, wherein determining a length of the poly(A) tail of the RNA sample comprises comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and a GMP from the RNA sample.

4. The method of claim 3, wherein exposing the RNA sample to the cleaving enzyme is for an incubation time ranging from about 3 minutes to about 24 hours, from about 30 minutes to about 4 hours, or about 3 hours; and wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C, from about 35 °C to about 39 °C, or about 37 °C.

5. The method of claim 4, wherein the length of the poly (A) tail of the RNA sample ranges from about 5 nucleotides to about 2000 nucleotides, from about 20 nucleotides to about 2000 nucleotides, from about 30 nucleotides to about 2000 nucleotides, from about 50 nucleotides to about 2000 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 200 nucleotides to about 2000 nucleotides, or from about 300 nucleotides to about

2000 nucleotides.

6. The method of claim 4, wherein the length of the RNA sample ranges from about 200 nucleotides to about 10,000 nucleotides, from about 1,000 nucleotides to about 10,000 nucleotides, from about 2,000 nucleotides to about 10,000 nucleotides, from about 3.000 nucleotides to about 10,000 nucleotides, from about 5,000 nucleotides to about 10,000 nucleotides.

7. The method of claim 5, further comprises identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP in a control RNA sample.

8. The method of claim 7, wherein identifying a coefficient for a ratio of absorbance of AMP to at least one of CMP, UMP, and GMP further comprises perforating HPLC on the control RNA sample comprising a known number of C nucleobases, a known number of U nucleobases, a know n number of G nucleobases, and a number of A nucleobases.

9. The method of claim 7, wherein identifying a coefficient for a ratio of absorbance comprises identifying a coefficient for a ratio of absorbance for each of AMP to CMP, UMP, and GMP in the control RNA sample.

10. The method of claim 9, wherein determining a length of the poly(A) tail of the RNA sample by comparing a peak area of AMP from the RNA sample to a peak area of at least one of a CMP, UMP, and GMP from the RNA sample comprises using formula 1: wherein N is the amount of a nucleobase in the RNA sample other than A nucleobase, A is the amount of A nucleobases in the non-tail portion of the RNA sample, NA is the coefficient for a ratio of absorbance for AMP with a corresponding nucleobase N from the control RNA sample, AMP is the AMP peak area in the RNA sample, and NMP is the corresponding nucleobase peak area in the RNA sample.

11. The method of claim 10, wherein determining a length of the poly(A) tail of the RNA sample includes comparing a peak area of AMP from the RNA sample to a peak area for each of CMP, UMP, and GMP from the RNA sample using formula 1 to generate a length of poly(A) tail based upon the comparison of a peak area of AMP to CMP, a peak area of AMP to UMP, and a peak area of AMP to GMP; and averaging each of the three tail lengths determined by formula 1 to generate the length of a poly(A) tail of the RNA sample.

12. The method of claim 1 1, wherein NA for AMP to CMP is about 2.029, NA for AMP to UMP is about 1.514, and/or NA for AMP to GMP is about 1.217.

13. The method of claim 12, wherein the cleaving enzyme comprises nuclease Pl, nuclease SI, benzonase, phosphodiesterase, or any combination thereof.

14. A method for determining a length of a poly(A) tail of an RNA sample, the method comprising: hydrolyzing an RNA sample with a cleaving enzyme to generate a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP); performing reverse phase high-performance liquid chromatography (HPLC) on the mixture of nucleotide monophosphates; determining a concentration of AMP in the RNA sample by comparing a peak area for AMP to a reference spike at a wavelength of about 260 nm; and determining a length of a poly(A) tail of the RNA sample by comparing the concentration of AMP in the RNA sample with a reference curve.

15. The method of claim 14, further comprising performing HPLC on an external standard to create the reference spike, wherein the performing HPLC on an external standard to create the reference spike is simultaneous with performing HPLC on the mixture of nucleotide monophosphates from the RNA sample; and wherein the external standard is cap-0.

16. The method of claim 15, further comprising creating a reference curve, wherein the reference curve is generated by identifying an expected concentration of AMP from a number of nucleotides in a sequence for at least three different tail lengths using formula 4: , , the number of C, U, G, or A nucleobases in the known sequence.

17. The method of claim 17, wherein the cleaving enzyme comprises nuclease Pl, nuclease S 1 , benzonase, phosphodiesterase, or any combination thereof; wherein exposing the RNA sample to the cleaving enzyme is for an incubation time ranging from about 3 minutes to about 24 hours, from about 30 minutes to about 4 hours, or about 3 hours; and wherein exposing the RNA sample to the cleaving enzyme is at an incubation temperature ranging from about 25 °C to about 45 °C. from about 35 °C to about 39 °C, or about 37 °C.

18. The method of claim 4, wherein the length of the poly(A) tail of the RNA sample ranges from about 5 nucleotides to about 2000 nucleotides, from about 20 nucleotides to about 2000 nucleotides, from about 30 nucleotides to about 2000 nucleotides, from about 50 nucleotides to about 2000 nucleotides, from about 100 nucleotides to about 2000 nucleotides, from about 200 nucleotides to about 2000 nucleotides, or from about 300 nucleotides to about 2000 nucleotides.

19. The method of claim 4, wherein the length of the RNA sample ranges from about 200 nucleotides to about 10,000 nucleotides, from about 1,000 nucleotides to about 10,000 nucleotides, from about 2,000 nucleotides to about 10,000 nucleotides, from about 3.000 nucleotides to about 10,000 nucleotides, from about 5,000 nucleotides to about 10,000 nucleotides.

20. An RNA sample having a poly(A) tail where the length of the poly(A) tail is determined by a process comprising any one of claims 1-19.

21. A kit for performing the method according to claim 1, the kit comprising: a cleaving enzyme capable of hydrolyzing the RNA sample to a mixture of nucleotide monophosphates comprising adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and guanosine monophosphate (GMP); either (a) a first RNA sample comprising known amounts of C nucleobases, U nucleobases, G nucleobases, and A nucleobase that is capable of generating a coefficient indicative of a ratio of absorbance of AMP to at least one of CMP, UMP. and GMP, or is capable of acting as an external standard by generating a 3 to 7 point calibration curve for molarity of CMP, UMP, GMP, and AMP or (b) an external standard capable of eluting at a time different than at least one of CMP, UMP, GMP, and AMP; and instructions for performing high-performance liquid chromatography (HPLC) comprising one or more of a mobile phase, a column, a mobile phase gradient, a flow rate, a run length, a column temperature, a detector, or any combination thereof.