RNA gel analysis

Abstract

The present invention relates to a method for separating RNA from a sample. This method may include loading an RNA-containing sample onto an electrophoretic gel and applying an electric field across the gel to move the RNA across the gel, thereby causing linear and circular RNA present in the sample to move through the gel at different rates. The electrophoretic gel may be a urea-agarose gel. A method for separating circular and linear RNA from a sample is provided together with a method for analyzing RNA present in the sample. A kit for separating linear and circular RNA from a sample is also provided.

Description

Technical Field

[0001] This application claims priority based on UK Patent Application No. 2308668.9 filed on June 9, 2023, and its content and elements hereby form part of this specification by reference for all purposes.

[0002] The present invention relates to a gel electrophoresis method for separating circular RNA and its corresponding linear RNA in a sample.

Background Art

[0003] Circular RNA is a covalently closed single-stranded RNA molecule that does not have a free 5' end or 3' end. Circular RNA is being actively studied for the development of next-generation mRNA therapeutics, mainly due to its in vitro and in vivo stability.

[0004] There are many methods for generating synthetic circular RNA. The resulting circular RNA can be identified using various techniques including gel electrophoresis, RNase R digestion, oligonucleotide-induced RNase H digestion, single-hit hydrolysis, and high-performance liquid chromatography (HPLC). However, the characterization of circular RNA by gel electrophoresis remains difficult. The identification of circular RNA, especially for circular RNAs up to 6000 nucleotides, still requires time-consuming processing.

[0005] Native agarose gels have been found to provide good separation based on RNA length, but they cannot separate circular RNA and its linear counterpart in full length (Non-Patent Document 1). Attempts to separate RNA in a sample using native agarose gels have found that circular RNA may migrate together with its linear counterpart.

[0006] Modified polyacrylamide gels (PAGE) have been found to distinguish circular RNA from its linear counterpart because circular RNA moves much slower than its linear counterpart. However, this system is not suitable for RNA longer than approximately 500 nucleotides. Non-patent document 2 shows that the E-gel system (a pre-packaged native agarose gel system, Thermo Fisher) can separate circular RNA from its linear counterpart, but unfortunately, these gels exhibit batch-to-batch variability. Non-patent document 3 also considers the use of native agarose gel systems for analyzing the migration patterns of circular RNA.

[0007] In some systems, formaldehyde is used as a denaturing agent to provide a denatured gel. However, formaldehyde is toxic, and formaldehyde-agarose gels can be difficult to handle.

[0008] Urea is also used as a denaturing agent, and is thought to function well as a denaturing agent in PAGE. However, urea is a weak denaturing agent and requires high concentrations (over 4M) to function, so it is not considered suitable for use in agarose gels (Non-Patent Literature 4, Non-Patent Literature 5). High concentrations of urea are known to reduce the physical strength of agarose. Urea-agarose gels containing high concentrations of urea are considered brittle and difficult to handle, and therefore are not often used. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Zhang Y et al (2016) [Non-Patent Document 2] Wesselhoeft R et al (2018) [Non-Patent Document 3] Abe BT et al (2022) [Non-Patent Document 4] Masek, T et al (2005) [Non-Patent Document 5] Sumitomo, K et al (2009) [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, there is a need for alternative systems for circular RNA analysis, particularly methods suitable for distinguishing between circular and linear RNA over various lengths. [Means for solving the problem]

[0011] Therefore, a first aspect of the present invention is a method for separating RNA present in a sample, (a) Load the RNA-containing sample onto an electrophoresis gel, and the electrophoresis gel is a urea-agarose gel; (b) Applying an electric field across the gel to move RNA across the gel, with linear RNA and circular RNA present in the sample moving through the gel at different rates; This provides a method that includes [something].

[0012] In a preferred embodiment of the present invention, the urea-agarose gel contains urea at a concentration of about 1 M to about 8 M. In a further embodiment of the present invention, the urea-agarose gel is a 0.5% to 6% agarose gel.

[0013] A second aspect of the present invention provides a method for separating circular RNA from linear RNA in a sample, the method comprising separating the RNA in the sample by electrophoresis on a gel, wherein the gel is a denatured urea-agarose gel and is placed in a vertical position.

[0014] A third aspect of the present invention provides a method for analyzing RNA present in a sample, the method comprising analyzing the sample by gel electrophoresis, wherein the gel is a denatured urea-agarose gel, and the method comprises using a vertical gel electrophoresis system.

[0015] In a fourth aspect, the present invention relates to a kit for separating linear RNA and circular RNA in a sample, (a) Components for performing gel electrophoresis of an RNA-containing sample, comprising agarose and urea for the preparation of a urea-agarose gel as defined herein, (b) A loading buffer containing urea as a denaturing agent for mixing with the RNA sample, (c) Standard molecular weight control sample, We provide a kit that includes this.

[0016] Further aspects and embodiments of the present invention are described below. [Brief explanation of the drawing]

[0017] [Figure 1]Figure showing the analysis of circular CVB3-EGFP and its linear counterpart in 0.8% native agarose gel. (A) In vitro transcription (IVT) of CVB3-EGFP was performed in the presence of 10 mM, 14 mM, 18 mM, 24 mM Mg2+. Approximately 500 ng of each IVT product was loaded onto a 0.8% native agarose gel and electrophoresed at 25 W for 70 minutes. The full-length precursor of CVB3-EGFP is 1963 nucleotides, and the circular / nicked product is 1638 nucleotides. Two bands can be seen at the position of circular / nicked CVB3-EGFP. (B) Full-length CVB3-EGFP and circularized CVB3-EGFP were treated with RNase R, loaded onto a 0.8% native agarose gel, and electrophoresed at 25 W for 70 minutes. (C) Gel-purified circular CVB3-EGFP was linearized using RNase H in the presence of a 34-nucleotide DNA primer. RNase H digests RNA hybridized to DNA. Samples were separated by electrophoresis at 25 W for 83 minutes on a 0.8% native agarose gel. (D) RT-PCR was performed on full-length CVB3-EGFP and circularized CVB3-EGFP. Circularization was confirmed by the presence of a 922-bp product from circularized CVB3-EGFP. [Figure 2] Figure showing the analysis of full-length precursor and circularized RNA in native agarose gel. The full-length precursor and circularized RNA were loaded onto (A) 0.8%, (B) 1.5%, and (C) 3.0% native agarose gels at 25 W and electrophoresed for the specified times (A) 83 minutes, (B) 85 minutes, and (C) 85 minutes. (D) Circularized RNA was digested with RNase R and separated on a 3% native agarose gel at the specified electrophoresis time (120 minutes at 25 W). [Figure 3] Figure showing the analysis of circular RNA and its linear counterpart in formaldehyde agarose gel. The full-length precursor and circularized RNA were loaded onto (A) 0.8% and (B) 1.5% denaturing formaldehyde agarose gels and electrophoresed for the specified time (90 minutes at 8 W). [Figure 4]Figure showing the analysis of circular RNAs and their linear counterparts in horizontal urea agarose gels. Full-length precursors and circularized RNAs were loaded onto a 1.5% agarose gel containing 6 M urea. Electrophoresis was performed on the gel in a horizontal electrophoresis system at the specified settings (20 W for 40 minutes). [Figure 5] Figure showing the analysis of circular RNAs and their linear counterparts in vertical urea agarose gels. Full-length precursors and circularized RNAs were loaded onto either (A) a 1.5% agarose gel containing 6 M urea, (B) a 1.5% agarose gel containing 4 M urea, (C) a 1.5% agarose gel containing 2 M urea, (D) a 0.8% agarose gel containing 6 M urea, or (E) a 0.8% agarose gel containing 2 M urea. (F) Circularized 3*Flag and its RNase R digestion product were loaded onto a 4% agarose gel containing 6 M urea. The full-length precursor and circular 3*Flag are 525 nucleotides and 212 nucleotides, respectively. Electrophoresis was performed on the gel in a vertical electrophoresis system at the specified settings (20 W for (A) 29 minutes, (B) 26 minutes, (C) 18 minutes, (D) 23 minutes, (E) 11 minutes, and (F) 25 minutes). [Figure 6] Figure showing the analysis of circular RNAs and their linear counterparts in urea agarose gels. 6 M - 1.5% urea agarose gels were tested with various electrophoresis settings. (A) and (B) The electrophoresis power was fixed at 20 W, and the electrophoresis times of 24 minutes and 28 minutes were compared. (C) and (D) The electrophoresis time was fixed at 30 minutes, and the electrophoresis powers of 20 W and 15 W were compared. The loading buffer used in (A) and (B) was a denaturing urea loading buffer, and the loading buffer used in (C) and (D) was a denaturing formamide loading buffer.

Mode for Carrying Out the Invention

[0018] The inventors have found that when urea is used as a denaturing agent in an agarose electrophoresis gel system, circular RNAs migrate slower than their linear counterparts regardless of RNA length and agarose concentration.

[0019] Therefore, the present invention provides a method for separating circular RNA and linear RNA in a sample, the method comprising separating RNA in the sample by electrophoresis on a gel, the gel being a denatured urea-agarose gel.

[0020] In one embodiment, the present invention provides a method for separating RNA present in a sample by gel electrophoresis. The method for separating RNA present in a sample is: (a) Load the RNA-containing sample onto the electrophoresis gel, and the electrophoresis gel is a urea-agarose gel; (b) Applying an electric field across the gel to move RNA across the gel, Includes.

[0021] Linear and circular RNA present in the sample move through the gel at different rates.

[0022] This method separates circular RNA from its linear RNA counterpart in a sample. The sample to be tested is loaded into wells of a prepared denatured urea-agarose gel. Electrophoresis is then performed on the gel at a power and duration that results in the separation of the circular RNA from its linear RNA counterpart. The RNA can then be visualized by appropriate means, such as staining and imaging.

[0023] The inventors have found that a denatured urea / agarose gel is effective in separating circular RNA and its linear counterpart in a sample. Despite the conventionally known problems when using urea as a denaturant with agarose gels, when urea is used as a denaturant in the preparation of the agarose gel, the gel can separate long circular RNA and its linear counterpart in a simple and rapid manner. The method of the present invention does not require long electrophoresis times to separate circular RNA and its linear counterpart.

[0024] In some embodiments of the present invention, urea can be present in the agarose gel at concentrations ranging from about 1 M to 8 M, for example, at concentrations of about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, or about 8 M. In some embodiments, urea is present in the gel at concentrations ranging from about 2 M to about 8 M, about 2 M to 6 M, about 2 M to 4 M, or about 4 M to 6 M. In one embodiment, urea is present in the agarose gel at a concentration of about 4 M to 6 M. In one embodiment, urea is present in the agarose gel at a concentration of about 4 M. In one embodiment, urea is present in the agarose gel at a concentration of about 6 M.

[0025] The urea concentration varies depending on the size of the RNA to be separated. Lower concentrations of urea can be used when separating longer RNA. For example, in some embodiments of the present invention, when separating RNA longer than 1000 nucleotides is intended, urea can be present at a concentration of approximately 2 M. However, when separating RNA longer than 1000 nucleotides is intended, urea can still be present at higher concentrations. For example, when separating RNA longer than 1000 nucleotides, a gel with urea at a concentration of approximately 4 M or 6 M can still be used. For example, if separating shorter RNA, for example, less than 500 nucleotides, is also intended on the same gel, urea can be present at higher concentrations. When separating RNA shorter than 500 nucleotides is intended, urea can be present at a concentration of approximately 6 M.

[0026] In some embodiments, the method is for separating RNA from one or more samples on a single gel, and when one or more samples contain RNA ranging in length from less than 500 nucleotides (e.g., about 200 nucleotides) to about 6000 nucleotides, a concentration of urea at 6 M is preferably used.

[0027] In this specification, when "gels" or "gel" are used, alternative expressions such as "electrophoretic gel" may be used. Methods for preparing electrophoretic gels are known in the art. In particular, agarose electrophoretic gels for separating biomolecules such as nucleic acids are known in the art. Sources of agarose and techniques for preparing and performing electrophoresis on such gels are disclosed, for example, in the reference book by Green and Sambrook (Molecular cloning: a laboratory manual, 4th edition, New York: Cold Spring Harbor Laboratory, 2012), and the reference book edited by Ausubel et al. (Current Protocols in Molecular Biology, Wiley, 2003), as well as in the references cited herein. These contents constitute part of this specification by reference. Such techniques can be adapted for use according to the present invention, for example, to produce gels having the required agarose and urea concentrations.

[0028] In some embodiments of the present invention, agarose is present in the gel at a concentration of 0.5% to 6%. In some embodiments, agarose is present at a concentration of about 0.5% to about 5%. In some embodiments, agarose is present at a concentration of about 0.8% to about 2%. In some embodiments, agarose is present at a concentration of about 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%, or 6%. In one embodiment, agarose is present at a concentration of about 1.5%, i.e., the gel is a 1.5% agarose gel. The concentration of agarose in the gel is measured as the weight (w / v%) of agarose relative to the volume of buffer used.

[0029] The agarose concentration can be varied depending on the size of the RNA to be separated. Lower concentrations of agarose can be used when separating longer RNA. For example, when separating RNA longer than 1000 nucleotides, agarose can be present at a concentration of approximately 0.8% to 2%, preferably 1.5%. When separating shorter RNA shorter than 500 nucleotides, agarose can be present at a higher concentration. For example, when separating RNA shorter than 500 nucleotides, agarose can be present at a concentration of approximately 1.5% to 4%.

[0030] In some embodiments, the method is for separating RNA from one or more samples on a single gel, and when one or more samples contain RNA ranging in length from less than 500 nucleotides to about 6000 nucleotides, agarose is preferably present at a concentration of about 1.5%.

[0031] In some embodiments, the urea-agarose gel used in the method of the present invention may include urea in a concentration ranging from 1 M to 8 M and agarose in a concentration of about 0.5% to 6%, urea in a concentration ranging from 2 M to 8 M and agarose in a concentration ranging from 0.8% to 5%, urea in a concentration ranging from 2 M to 6 M and agarose in a concentration ranging from 0.8% to 5%, urea in a concentration ranging from 2 M to 4 M and agarose in a concentration ranging from 0.8% to 2%, urea in a concentration ranging from 4 M to 6 M and agarose in a concentration ranging from 0.8% to 2%, urea in a concentration ranging from 2 M to 4 M and agarose in a concentration of about 1.5%, or urea in a concentration ranging from 4 M to 6 M and agarose in a concentration of about 1.5%.

[0032] Therefore, in some embodiments of the present invention, a 2M urea / 0.8% agarose gel is used in the method. In some embodiments, a 2M urea / 1.5% agarose gel is used in the method. In some embodiments, a 4M urea / 1.5% agarose gel is used in the method. In some embodiments, a 6M urea / 1.5% agarose gel is used in the method. In some embodiments, a 6M urea / 4% agarose gel is used in the method.

[0033] The separation of RNA in a sample can be varied depending on the power and duration of electrophoresis performed on the gel. Standard systems known in the art can be used to generate an electric field across the gel. In some embodiments of the present invention, electrophoresis can be performed at a constant power in the range of 5W to 75W. However, higher or lower power can be used. In some embodiments, electrophoresis can be performed at a constant power in the range of 15W to 50W, for example, at a constant power of about 5W, 10W, 15W, 20W, 25W, 30W, 35W, 40W, 45W, or 50W. In some embodiments, electrophoresis can be performed at a constant power in the range of 15W to 40W, 15W to 30W, or 15W to 25W. Preferably, electrophoresis is performed at a constant power in the range of about 15W to 25W. In one embodiment, electrophoresis is performed at a constant power of about 15W. In another embodiment, electrophoresis is performed at a constant power of about 20W.

[0034] In some embodiments of the present invention, electrophoresis can be performed for a period of about 10 to 90 minutes. However, longer or shorter electrophoresis times can be used. In some embodiments of the present invention, electrophoresis can be performed for a period of about 10 to 60 minutes, for example, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, electrophoresis can be performed for a period of about 10 to 40 minutes, about 10 to 30 minutes, about 15 to 30 minutes, about 20 to 30 minutes, or about 10 to 20 minutes. In some embodiments, electrophoresis can be performed for a period of less than 60 minutes. Preferably, electrophoresis can be performed for a period of 10 to 30 minutes.

[0035] An advantage of the present invention is that it allows for shorter electrophoresis times while still providing sufficient separation between circular RNA and its corresponding linear RNA in the sample.

[0036] In some embodiments, electrophoresis is performed at room temperature (e.g., in the range of 20°C to 25°C). In some embodiments, electrophoresis is performed at room temperature for 10 to 30 minutes at a constant power in the range of approximately 15 W to 25 W.

[0037] The electrophoresis time and electrophoresis power can be varied depending on the size of the RNA to be separated. If shorter RNA is to be separated, a higher electrophoresis power and / or a longer electrophoresis time can be used. For example, in some embodiments of the present invention, if RNA smaller than 500 nucleotides is to be separated, the electrophoresis power can be about 25W for a longer period than about 30 minutes.

[0038] For example, if the intention is to separate RNA longer than 500 nucleotides, preferably longer than 1000 nucleotides, a shorter electrophoresis time and / or lower electrophoresis power can be used. For example, in some embodiments of the present invention, if the intention is to separate RNA longer than 1000 nucleotides, the electrophoresis power can be a constant power in the range of 15W to 25W for about 10 to 30 minutes. In some embodiments of the present invention, if the intention is to separate RNA longer than 1000 nucleotides, the electrophoresis power can be about 20W for about 10 to 30 minutes.

[0039] Urea-agarose gels are suitable for separating long circular RNAs from their linear counterparts in a sample. Terms such as "circRNA" or "circular RNA" refer to polyribonucleotides that form a circular structure through covalent or non-covalent bonds. The description of a linear counterpart sequence means that the linear RNA sequence has identical or similar nucleotide sequences, particularly with respect to the sequence encoding the target gene, but is not circularized and has two free ends. In some embodiments, the linear RNA counterpart sequence is a linear precursor sequence before circularization. "Long RNA" refers to RNA with a length greater than 500 nucleotides.

[0040] In some embodiments of the present invention, the method can separate circular RNA and its linear counterpart when the RNA has a length of up to about 7,000 nucleotides. In some embodiments, the method can separate RNA having lengths of up to about 1,000 nucleotides, up to about 2,000 nucleotides, up to about 3,000 nucleotides, up to about 4,000 nucleotides, up to about 5,000 nucleotides, up to about 6,000 nucleotides, or up to about 6,500 nucleotides. In some embodiments, the method can separate RNA having a length of up to about 6,000 nucleotides.

[0041] The method of the present invention is particularly suitable for separating long circular RNA and its linear counterpart, but in some embodiments, the method can also be used to separate short circular RNA and its linear counterpart. "Short" RNA means RNA having a length of less than 500 nucleotides. Preferably, a urea-agarose gel can be used to separate RNA having lengths longer than about 200 nucleotides, longer than about 300 nucleotides, longer than about 400 nucleotides, or longer than about 500 nucleotides.

[0042] Preferably, the method of the present invention is for separating RNA having lengths in the ranges of approximately 200 to approximately 7000 nucleotides, approximately 200 to approximately 6500 nucleotides, approximately 200 to approximately 6000 nucleotides, approximately 500 to approximately 6000 nucleotides, approximately 500 to approximately 6500 nucleotides, approximately 1000 to approximately 6000 nucleotides, approximately 1000 to approximately 6500 nucleotides, approximately 1500 to approximately 6000 nucleotides, and approximately 1500 to approximately 6500 nucleotides. In some embodiments, the method is for separating RNA having lengths in the ranges of approximately 200 to approximately 6000 nucleotides.

[0043] In some embodiments, this method is for separating RNA having a length of up to approximately 6000 nucleotides, with urea present in the gel at a concentration of 2 M to 6 M and agarose at a concentration of approximately 0.8% to 4%. Preferably, the gel is a 1.5% agarose gel.

[0044] In some embodiments of the present invention, the method is for separating RNA having a length in the range of about 500 to about 6000 nucleotides, with urea present in the gel at a concentration of 2M to 6M and agarose at a concentration of about 1.5%. In some embodiments of the present invention, the method is for separating RNA having a length in the range of about 500 to about 6000 nucleotides, and a 2M to 4M urea / 1.5% agarose gel is used. In some embodiments of the present invention, the method is for separating RNA having a length in the range of about 500 to about 6000 nucleotides, and a 4M to 6M urea / 1.5% agarose gel is used.

[0045] In some embodiments of the present invention, the method is for separating RNA having a length in the range of approximately 500 to approximately 6000 nucleotides, with urea present in the gel at a concentration of 2M to 6M, agarose present at a concentration of approximately 1.5%, electrophoresis power being a constant power in the range of 15W to 25W, and electrophoresis time being approximately 10 to 30 minutes. In some embodiments of the present invention, the method is for separating RNA having a length in the range of approximately 500 to approximately 6000 nucleotides, with a 2M to 4M urea / 1.5% agarose gel being used, electrophoresis power being a constant power in the range of 15W to 25W, and electrophoresis time being approximately 10 to 30 minutes. In some embodiments of the present invention, this method is for separating RNA having a length in the range of approximately 500 nucleotides to approximately 6000 nucleotides, using a 4M to 6M urea / 1.5% agarose gel, with a constant electrophoresis power in the range of 15W to 25W, and an electrophoresis time of approximately 10 to 30 minutes.

[0046] In some embodiments of the present invention, the method is for separating RNA having a length in the range of about 1000 nucleotides to about 6000 nucleotides, using a 4M to 6M urea / 0.8% to 1.5% agarose gel, with a constant electrophoresis power in the range of 15W to 25W, and an electrophoresis time of about 10 to 30 minutes. In some embodiments of the present invention, the method is for separating RNA having a length in the range of about 1000 nucleotides to about 6000 nucleotides, using a 4M urea / 1.5% agarose gel, with an electrophoresis power of about 20W, and an electrophoresis time of about 10 to 30 minutes.

[0047] Electrophoretic gels can be run in a vertical or horizontal orientation. Typically, agarose gels are operated in a horizontal orientation. Horizontal gel electrophoresis systems are known in the art. Examples of systems for running gels in a horizontal orientation include, but are not limited to, the E-Gel® Power Snap Plus Electrophoresis Systems (Invitrogen). However, in some embodiments of the present invention, the urea-agarose gel is placed in a vertical position during electrophoresis. The method may include using a vertical electrophoresis gel system. Placing the gel in a vertical orientation improves band resolution and enhances the separation of circular RNA and linear RNA in the sample compared to running gels in a horizontal orientation. The improved resolution allows for sharper or narrower bands of molecules that are more spaced apart or separated from each other compared to bands achieved by other separation methods.

[0048] Vertical gel electrophoresis systems are known in the art, and such systems include Bio-Rad's MiniProtean Tetra vertical electrophoresis cell, the Owl® Dual-Gel vertical electrophoresis system (Thermo Scientific), and the XCell SureLock® MiniCell (Invitrogen). In a vertical gel electrophoresis system, the gel is supported in a frame between a bottom plate and a cover plate in a substantially vertical position during electrophoresis. An electric field is generated perpendicularly across the gel. Typically, vertical gel electrophoresis systems are intended for use in PAGE electrophoresis. These systems can be adapted to improve their use with agarose gels. For example, in some embodiments, a stopper can be positioned at the bottom edge of the bottom plate. The stopper can help prevent the gel from sliding during electrophoresis.

[0049] In some embodiments, this method Perform electrophoresis of a control sample containing a molecular weight size marker in the first lane of the electrophoresis gel, Performing electrophoresis of the RNA sample in the second lane of the electrophoresis gel, Visualizing the bands, It also includes.

[0050] This method may further include comparing the mobility and band formation patterns of the first lane and the second lane to determine whether circular RNA and / or linear RNA are present.

[0051] The bands in the gel can be visualized by appropriate means known in the art. The means for visualizing the bands vary depending on the dye used in this method. The bands can be visualized using UV imaging techniques. Visualizing the bands may include staining the gel and visualizing the bands using UV imaging techniques. Methods for staining gels and imaging gels are known in the art.

[0052] One aspect of the present invention is a method for separating circular RNA from linear RNA in a sample, the method comprising separating the RNA in the sample by electrophoresis on a gel, the gel being a denatured urea-agarose gel and placed in a vertical position. The gel and operating conditions for use in electrophoresis are as described above. For example, in some embodiments, the urea-agarose gel contains urea at a concentration of 1 M to 8 M and agarose at a concentration of about 0.5% to 6%. Preferably, the urea-agarose gel contains urea at a concentration of 4 M to 6 M and agarose at a concentration of about 1.5%. Preferably, the method is for separating RNA having a length of up to about 6000 nucleotides.

[0053] A further aspect of the present invention is a method for analyzing RNA present in a sample. This method involves analyzing the sample by gel electrophoresis, wherein the gel is a denatured urea-agarose gel, and the method involves using a vertical gel electrophoresis system. The gel and operating conditions for use in electrophoresis are as described in the embodiments above. For example, in some embodiments, the urea-agarose gel contains urea at a concentration of 1 M to 8 M and agarose at a concentration of about 0.5% to 6%. Preferably, the urea-agarose gel contains urea at a concentration of 4 M to 6 M and agarose at a concentration of about 1.5%. Preferably, this method is for analyzing RNA having a length of up to about 6000 nucleotides.

[0054] In some embodiments, this method is for RNA length analysis and / or morphological analysis. Morphological analysis means determining whether circular RNA and / or linear RNA are present.

[0055] The method of the present invention is particularly useful for samples containing complex RNA, such as samples containing different types of RNA species having multiple lengths. For example, a sample may include circular RNA and linear RNA. A sample may also contain RNA of multiple lengths. The method of the present invention can separate circular RNA and linear RNA that may be present in a sample. Therefore, in some embodiments of the present invention, a sample may include linear RNA and circular RNA. A sample may also contain RNA of different lengths.

[0056] Multiple types or species of RNA refer to two or more different forms of RNA, such as circular RNA and linear RNA. The presence of circular RNA in a sample can be identified because circular RNA moves through the gel at a different rate than other RNA species in the sample. Therefore, the method of the present invention can be used to detect whether or not circular RNA is present in a sample.

[0057] A further aspect of the present invention is a method for determining the presence of circular RNA in a sample. This method involves analyzing the sample by gel electrophoresis, wherein the gel is a denatured urea-agarose gel. The gel can be placed in a vertical position during electrophoresis. The gel and operating conditions for use in electrophoresis are as described in the embodiments above. For example, in some embodiments, the urea-agarose gel contains urea at a concentration of 1 M to 8 M and agarose at a concentration of about 0.5% to 6%. Preferably, the urea-agarose gel contains urea at a concentration of 4 M to 6 M and agarose at a concentration of about 1.5%. Preferably, this method is for determining whether circular RNA having a length of up to about 6000 nucleotides is present in the sample.

[0058] This method can be used to detect whether circular RNA has been produced after in vitro or in vivo production of circular RNA using standard methods in the art. Whether circular RNA has been produced can be determined by analyzing a sample of the product obtained from the production method using the method of the present invention to detect the presence of circular RNA in the sample. Circular RNA moves at a different rate than its corresponding linear RNA, enabling its detection. On a urea-agarose gel, circular RNA moves slower than its corresponding linear counterpart.

[0059] Therefore, in one embodiment, the present invention includes a method for determining the presence of circular RNA in a sample. This method is (a) Load the sample onto the electrophoresis gel, which is a urea-agarose gel; (b) Applying an electric field across the gel to move RNA present in the sample across the gel, (c) Analyze the mobility and band pattern of the gel to determine the presence of circular RNA in the sample, Includes.

[0060] The gel and operating conditions for use in this method are as described in the embodiments above.

[0061] The urea-agarose gel for use in the method of the present invention can be prepared by known techniques as described above.

[0062] Agarose gel can be formed by suspending dry agarose in an aqueous, usually buffered, medium and heating the mixture to dissolve the agarose in the usual manner. The agarose-containing medium is mixed with urea at an appropriate concentration. The urea-agarose medium can be mixed with further buffer and aqueous medium, then injected into a cassette and cooled to solidify the gel. Multiple wells can be introduced into the gel by using a "comb" with a row of protruding teeth, positioned so that the teeth protrude into the gel layer while the gel layer solidifies. Cooling can be done at room temperature (e.g., in the range of 20°C to 25°C) or at a lower temperature, such as in the range of 4°C to 6°C.

[0063] In some embodiments, the gel is prepared by mixing agarose with an aqueous medium at a suitable concentration to provide a mixture containing agarose of a desired concentration. In some embodiments, the aqueous medium is DEPC-treated water. Other suitable aqueous mediums can be used.

[0064] Therefore, in some aspects of the present invention, the modified urea / agarose gel is (a) Agarose is mixed with DEPC-treated water, and the mixture is heated to dissolve the agarose, forming an aqueous agarose mixture. In addition, an appropriate amount of agarose is mixed in order to provide an agarose gel having the desired agarose concentration; (b) Mixing urea with the aqueous agarose mixture of (a) to provide a urea-agarose mixture, and furthermore, an appropriate amount of urea is mixed in order to provide a urea / agarose gel having a desired urea concentration; (c) The urea-agarose mixture from (b) is mixed with electrophoresis buffer and further DEPC-treated water, (d) The mixture obtained from (c) is injected into a gel plate, (e) Solidifying the gel over a certain period of time, Prepared by, The resulting gel contains urea in concentrations ranging from 1 M to 8 M and agarose in concentrations ranging from 0.5% to 6%.

[0065] In one embodiment, in step (c), a dye capable of binding to RNA is also mixed with the urea-agarose mixture.

[0066] In some embodiments, the RNA-binding dye is not mixed with the urea-agarose mixture before casting the gel. In such embodiments, the gel is stained with a mixture of electrophoresis buffer and RNA-binding dye after electrophoresis.

[0067] The buffer used during staining can be a urea-free buffer. Urea-free means that the buffer does not contain urea. Using a urea-free buffer during staining can help improve the strength of the gel. Buffers for use in staining urea-free gels are known in the art, but are not limited to, TBE (tris-borate-EDTA buffer) and TAE (tris-acetic acid-EDTA buffer).

[0068] In some embodiments, the gel plate is preheated before the gel mixture is injected into it. Preheating the plate helps to avoid gelation during the injection of the gel mixture. Preferably, the plate is preheated when the gel contains urea at a concentration of about 6 M and agarose at a concentration of about 4%.

[0069] The test sample can be any sample containing RNA. Methods for preparing test samples are known in the art.

[0070] Test samples can be denatured before being loaded onto the gel. RNA-containing samples can be prepared by mixing the sample with a loading buffer, preferably a denaturing loading buffer. Examples of denaturing loading buffers are known in the art and include, for example, denaturing formamide loading buffer and denaturing urea loading buffer. Preferably, the loading buffer contains urea as a denaturing agent. Examples of urea loading buffers are known in the art. In some embodiments of the present invention, an RNA sample can be mixed with an equal volume of urea loading buffer. The mixture can then be denatured at 95°C for about 2 minutes before loading the sample into the gel wells.

[0071] Suitable electrophoresis buffers, dyes, loading buffers, and other aqueous media known in the art can be used to prepare urea-agarose gels. Examples of electrophoresis buffers include, but are not limited to, TAE and TBE, and examples of RNA-binding dyes include, but are not limited to, SYBR Green and ethidium bromide.

[0072] In some embodiments, the gel is not formed from acrylamide; for example, the gel is not a composite polyacrylamide-agarose gel. In some embodiments, the gel does not contain formaldehyde as a denaturing agent. In some embodiments, formaldehyde and / or formamide may also not be present in the electrophoresis buffer or loading buffer.

[0073] The present invention also provides a kit for separating linear RNA and circular RNA in a sample, and this kit is (a) Components for performing gel electrophoresis of an RNA-containing sample, comprising agarose and urea for the preparation of a urea-agarose gel as described above, (b) A buffer for mixing with the RNA sample, containing urea as a denaturing agent, (c) Standard molecular weight control sample, Includes.

[0074] The standard molecular weight control sample includes an appropriate molecular weight size marker. The kit may further include urea-free staining buffer. This kit may further include apparatus for vertical gel electrophoresis.

[0075] Wherever these terms appear herein, they may be replaced by "consisting of," "consists of," "consisting essentially of," or "consists essentially of," and vice versa.

[0076] A range may be expressed herein as “about” one particular value and / or “about” another particular value. Where such a range is expressed, another embodiment includes one particular value and / or another particular value. Similarly, where the preceding use of “about” represents a value as an approximation, it will be understood that a particular value forms another embodiment. The term “about” with respect to a number is arbitrary and means, for example, ±10%.

[0077] All publications cited herein, by reference to provide a more complete explanation of the state of the art to which the present invention belongs, constitute an entirety of this specification.

[0078] The present invention will be further understood by referring to the following embodiments. [Examples]

[0079] The examples provided below demonstrate that the use of urea as a denaturing agent improved the separation of circular RNA and its linear counterpart in agarose gel electrophoresis.

[0080] Materials and methods plasmid A circularized PIE Ana 3.0 construct without spacers, internal homologous arms, and the target gene (GOI) was synthesized from IDT (Integrated DNA Technologies, pUCIDT-PIE-Ana3.0-empty) by placing it between the T7 promoter and the Not I and EcoR V cleavage sites (Non-Patent Literature 2). The GOI and the corresponding backbone vector were individually PCR amplified and combined using the Gibson assembly master mix (NEB). All plasmids were Sanger sequenced and amplified using homemade TOP10 competent cells. For CVB3-EGFP (pUCIDT-PIE-Ana3.0-CVB3-EGFP), the exon, spacer, internal homologous arms, and gene block containing CVB3-EGFP were synthesized from IDT and amplified using primers CVB3-EGFP-gF and CVB3-EGFP-gR. The vector for CVB3-EGFP was amplified from pUCIDT-PIE-Ana3.0-empty using primers empty-vF and empty-vR. Subsequently, pUCIDT-PIE-Ana3.0-CVB3-EGFP was used as a backbone vector for firefly luciferase, Spike protein, Cas9, and T2A-EGFP. (Circular 3) * Plasmids for the flag were synthesized by IDT. All plasmids were scaled up using the plasmid plus maxi kit (QIAGEN) and then linearized using EcoR V (NEB) or Not I (NEB) for subsequent in vitro transcription (IVT). The vectors and primers for these genes are listed in Tables 1 and 2.

[0081] [Table 1]

[0082] [Table 2]

[0083] The GOI used in the following experiment has the following precursor length / ring length.

[0084] [Table 3]

[0085] Synthesis of circular RNA IVT was performed using a 50 ng / μl DNA template, 14 μg / μl of homemade T7 polymerase, 0.04 U / μl of an RNase inhibitor (Promega), 6 mM of each NTP, and 1× IVT buffer. For IVTs that allowed cotranscription splicing, the 1× IVT buffer contained 80 mM Tris-HCl (pH 7.4), 2 mM spermidine, 40 mM DTT, and 24 mM MgCl2. To test the MgCl2 dependence of cotranscription splicing, the MgCl2 concentration in the 1× IVT buffer was set to 10 mM, 14 mM, 18 mM, or 24 mM. When cotranscription splicing should be suppressed, the MgCl2 concentration in the 1× IVT buffer was 14 mM. The IVT reaction was incubated at 37°C for 3 to 5 hours, followed by digestion with RNase-free DNase I for 20 minutes. Subsequently, 100 mM EDTA was added to a concentration of 25 mM to remove all precipitate. Then, an equal volume of 7.5 M lithium chloride was added, and the RNA was precipitated at -20°C for 30 minutes to overnight. The precipitate was then centrifuged at 13,000 rpm / min for at least 20 minutes. The RNA pellet was washed with 75% ethanol, air-dried, and dissolved in DEPC-treated H2O.

[0086] For cyclization, the RNA precursor was first diluted to approximately 700 ng / μl. Then, 90 μl of each RNA was denatured at 95°C for 2 minutes and annealed on ice for 3 minutes. Subsequently, 10 μl of 10× cyclization buffer (500 mM Tris-HCl, pH 7.4, 100 mM MgCl2, 10 mM DTT, 20 mM GTP) was added to the annealed RNA, and the mixture was heated at 55°C for 20 minutes. Circulation was stopped by adding 20 μl of 100 mM EDTA.

[0087] RNase R digestion and RNase H digestion To perform RNase R digestion, the cyclic CVB3-EGFP was first column-washed (ZYMO RESEARCH). Then, 5 μg of full-length precursor and 5 μg of cyclic RNA were digested with 10 U of RNase R (antibodies-online) at 37°C for 15 minutes. The digested RNA was loaded onto a native agarose gel as described below.

[0088] To perform RNase H digestion, 100 μg of full-length precursor RNA of CVB3-EGFP was circularized and isolated on a 0.8% native agarose gel for 80 minutes. Subsequently, the circular RNA band was excised and extracted using an RNA gel recovery kit (ZYMO RESEARCH). The circular RNA was eluted in DEPC H2O. Then, 5 μg of purified circular CVB3-EGFP was heated at 65°C for 5 minutes in the presence of 5x molar volume of CVB3-Fluci / Spike / Cas9-vR primer. After cooling on ice for 3 minutes, RNase H (NEB) and buffer were added, and digestion was performed at 37°C for 20 minutes. The digested RNA was loaded onto a native agarose gel as described below.

[0089] RT-PCR The reverse transcriptase and DNA polymerase used here were SuperScrip IV Reverse Transcriptase (Thermo Fisher) and Q5 High-Fidelity DNA Polymerase (NEB). Reverse transcription and PCR were performed according to the manufacturer's manual. Full-length CVB3-EGFP RNA and circularized CVB3-EGFP RNA were used as templates for reverse transcription using CVB3-Fluci / Spike / Cas9-vR(R) as the reverse primer. Subsequently, the reverse transcripts were subjected to PCR using either forward primer CVB3-EGFP-gF(F1) or forward primer CVB3-Fluci-vF(F2).

[0090] Native Agarose Gel 100 ml of 0.8% to 3.0% agarose gels were prepared in 1 × TBE (89 mM Tris, 89 mM borate, 3 mM EDTA) in the presence of 10 μl of SYBR Safe (Thermo Fisher). The gels were run horizontally on an Owl Easy Cast B2 Mini Gel Electrophoresis System (Thermo Scientific) at a constant 25 W at room temperature for 70 to 120 minutes using 1 × TBE as the electrophoresis buffer. For sample loading, each RNA sample (approximately 500 ng) was mixed with an equal volume of formamide loading buffer (Thermo Fisher) and denatured at 95°C for 2 minutes. The same sample loading procedure was used for the formaldehyde agarose gels and urea polyacrylamide gels described below. All gels (including those described below) were imaged using the Bio-Rad Chemidoc XRS+ Imaging System.

[0091] Formaldehyde agarose gel Formaldehyde agarose gels containing 0.8% and 1.5% were prepared as previously described (Rio, DC et al, (2015)). Notably, SYBR Safe was mixed with the gel and electrophoresis was performed at a constant temperature of 4°C and 8W for 90 minutes.

[0092] Urea agarose gel For horizontal urea agarose gels, a horizontal Owl Easy Cast B1 Mini Gel electrophoresis system (Thermo Scientific) was used. To prepare a 1.5% agarose gel containing 6M urea, 0.75g of agarose was first boiled in 40ml of DEPC H2O and mixed with 18g of urea. Then, 5ml of 10×TBE and 5μl of SYBR Green were added, and DEPC H2O was added up to 50ml. The gel was then injected into the B1 Mini Gel cassette and gelled at 4°C for several hours. For sample loading, each RNA sample (approximately 500ng) was mixed with an equal volume of urea loading buffer (NEB) and denatured at 95°C for 2 minutes. Electrophoresis was performed on the gel at room temperature and 15W for 40 minutes.

[0093] For vertical urea agarose gels, a Bio-Rad MiniProtean Tetra vertical electrophoresis cell system was used. Agarose was first boiled in DEPC H2O, mixed with varying amounts of urea, 10×TBE and DEPC H2O were added, and then injected into a gel plate fitted with a 1.5 mm spacer. A 0.75 mm stopper was attached to the bottom edge of the bottom plate to prevent gel slippage during electrophoresis. The gel was allowed to gel at 4°C for several hours. To avoid damaging the gel, the comb must be removed by sliding the cover plate downwards to expose it. At this point, the gel wells were washed with the pipette tip, and then the cover plate was slid back into place. For 4% agarose gels containing 6M urea, the gel plate was preheated to help prevent gel formation during injection. Electrophoresis was performed on the gel at room temperature, 15W-25W, for 15-30 minutes. For sample loading, each RNA sample (approximately 100 ng) was mixed with an equal volume of urea loading buffer (NEB) and denatured at 95°C for 2 minutes. Formamide loading buffer causes shadowing of RNA bands and is therefore unsuitable for urea agarose gels. The gels were stained with SYBR Green in 10 ml of 1×TBE for 10 minutes prior to imaging.

[0094] Example 1 It has been reported that separation of circular RNA and its linear counterpart cannot be obtained on native agarose gels (Non-Patent Literature 1). To test this, CVB3-EGFP was cloned into a PIE construct. First, the in vitro transcribed RNA was checked by electrophoresis on a 0.8% native agarose gel at a constant 25W for 70 minutes (Figure 1A, lane 1). Due to cotranscription splicing, multiple RNA species are already visible. Of these, the full-length precursor (1963 nucleotides) can be easily identified, but the circular / nicked RNA (1638 nucleotides) cannot be identified because there are two major bands around the size of the circular / nicked RNA. To reduce the complexity of the IVT product, as previously described (Zaug AL et al, 1993), Mg in IVT was used to suppress cotranscription splicing of ribozymes. 2+ The concentration of was reduced. As a result, the IVT product here is mainly the full-length precursor (Figure 1A, lanes 2-4).

[0095] The full-length precursor was subjected to a cyclization reaction, and the product was loaded onto a 0.8% native agarose gel (Figure 1B, lane 3). The post-transcriptional cyclization procedure yielded far fewer RNA species. As described above, there are still two bands near the position of the circular / nicked CVB3-EGFP. The lower band migrates to the same position as the 1500 nucleotide marker, while the upper band migrates to approximately 1600 nt. Therefore, the upper band appears to be circular / nicked CVB3-EGFP (1638 nucleotides). When the full-length precursor and cyclized samples were digested with the exonuclease RNase R, only the lower band was found to be resistant to RNase R digestion, indicating that the lower band, not the upper band, contains circular CVB3-EGFP (Figure 1B, lanes 1-4).

[0096] Because the lower band migrated faster than expected for linear CVB3-EGFP, we hypothesized that the lower band was circular and the upper band was nicked circular CVB3-EGFP. The lower band was purified by RNase R digestion following gel extraction (Figure 1C, lane 1). The identity of the circular CVB3-EGFP was further confirmed by RT-PCR. Circularization was confirmed by the presence of a 922 bp product from the circularized CVB3-EGFP (Figure 1D). Subsequently, when the circular CVB3-EGFP was digested with RNase H induced by a 34-mer DNA oligo, linearized CVB3-EGFP was found to migrate similarly to the upper band (Figure 1C, lane 2), indicating that the upper band is nicked CVB3-EGFP. The slight difference between the nicked RNA and the RNA linearized by RNase H is a result of the removal of additional nucleotides by RNase H digestion.

[0097] Overall, these results demonstrate that circular CVB3-EGFP migrates faster than its linear counterpart on a 0.8% native agarose gel, instead of migrating together with it. This is similar to the observation (Non-Patent Literature 2) that under native conditions, circular RNA has a longer retention time than its linear counterpart on a Sepax gel filtration column.

[0098] Example 2 Four other constructs were prepared to produce cyclic EGFP (813 nucleotides), CVB3-firefly luciferase (Fluci, 2600 nucleotides), SARS-CoV-2 CVB3-Spike protein fused with EGPF (Spike, 5468 nucleotides), and CVB3-spCas9 fused with EGFP (Cas9, 5756 nucleotides). First, IVT was performed, and these samples were loaded onto a 0.8% agarose gel for separation (pre- and post-cyclization). As shown in Figure 2A, on this 0.8% gel, cyclic T2A-EGFP, CVB3-EGFP, and CVB3-Fluci migrated faster than their nicked counterparts. Cyclic CVB3-Spike migrated similarly to its nicked product, and cyclic CVB3-Cas9 migrated even slower than its full-length precursor. Consistently, circular CVB3-EGFP and nicked CVB3-EGFP migrate as two separate bands (Figure 2A). For circular EGFP and Fluci, the two RNA species migrate faster than the full-length precursor, and the lower band is stronger than the upper band, indicating that circular RNA migrates faster than these two RNAs. For Spike, two partially overlapping bands can be seen below the full-length precursor, but it is difficult to determine which is the circular RNA. However, for Cas9, a new band appears above the precursor (Figure 2A). We assume that the uppermost band of Cas9 is the circular RNA (Figure 2A). Samples were loaded onto 1.5% and 3% agarose gels (Figures 2B and 2C). As shown in Figure 2B, in a 1.5% gel, circular T2A-EGFP and CVB3-EGFP migrate faster than their linear counterparts, but with much lower separation, while circular CVB3-Fluci migrates similarly to its linear counterpart. In a 1.5% gel, the separation between circular RNA and nicked RNA is lower for EGFP and CVB3-EGFP, and for Fluci, circular RNA migrates slightly slower than nicked RNA.Here, circular CVB3-Spike migrates much slower than its precursor, and the separation between circular CVB3-Cas9 and its linear precursor is greater. For Cas9, the upper band moves even further away from the precursor. Interestingly, a new band appears above the Spike precursor. As shown in Figure 2(C), in a 3% gel, all circular RNAs migrate slower than their linear counterparts.

[0099] To further confirm that these upper bands are circular RNA, RNase R digestion was performed and these samples were loaded onto a 3% agarose gel (Figure 2D). This clearly shows that only the upper bands are resistant to RNase R digestion. In the gel in Figure 2D, these upper bands are confirmed to be circular RNA.

[0100] These results demonstrate that the separation of circular RNA and its linear counterpart can be regulated by altering the agarose concentration in the native agarose gel system.

[0101] Example 3 To investigate how circular RNAs behave on a common denatured agarose gel using formaldehyde as a denaturant, these samples were first loaded onto a 0.8% formaldehyde-agarose gel. It was found that for all five RNAs, the circular RNAs and their linear counterparts were not separated (Figure 3A). Subsequently, when these samples were loaded onto a 1.5% formaldehyde-agarose gel, it was found that the circular Spike and Cas9 migrated slower than their linear counterparts (Figure 3B), similar to the case on native agarose gels. However, no separation of circRNAs and their linear counterparts for EGFP, CVB3-EGFP, and Fluc was observed. It was hypothesized that loading these samples onto a higher concentration agarose gel, e.g., 3%, would allow for separation of these three short RNAs. However, 3% formaldehyde-agarose gels are difficult to handle. These results suggest that circular RNA behaves similarly in formaldehyde-agarose gels as it does in native agarose gels.

[0102] Example 4 Due to the toxicity of formaldehyde, urea is sometimes used as a substitute (Rosen, JM et al (1975), Reijnders L et al (1973)). However, it is generally believed that urea is not a good denaturant in agarose gel systems for nucleic acids (Non-Patent Literature 4). To test the ability of urea as a denaturant in agarose gels, samples were loaded onto 6M-1.5% urea-agarose gels in a horizontal electrophoresis system. As shown in Figure 4, the separation of circular RNA and its linear counterpart using the horizontal electrophoresis system in which the urea-agarose gel was tested here was not very clear.

[0103] We also used a vertical gel electrophoresis system originally designed for protein PAGE.

[0104] As shown in Figure 5A, in a 6M-1.5% urea-agarose gel, all circular RNA species migrate much slower than their linear counterparts, rather than faster. For example, circular EGFP migrates to the same position as its full-length precursor, while all other circular RNAs migrate slower than their full-length precursors (Figure 5A).

[0105] The agarose concentration was fixed at 1.5%, and the urea concentration was reduced to 4M and 2M. The degree of separation between circular RNA and its linear counterpart also decreased (Figures 5B and 5C). For example, when the urea concentration was 4M, circular EGFP migrated slower than its linear counterpart but faster than its full-length precursor. On the other hand, when the urea concentration was 2M, circular EGFP migrated together with its linear counterpart. These results clearly demonstrate that adding urea to the agarose gel system can separate circular RNA from its linear counterpart. Considering that circular RNA behaves similarly on native agarose gels and denatured formaldehyde gels, the difference in mobility between circular RNA and its linear counterpart on denatured urea-agarose gels must be due to urea, not RNA denaturation.

[0106] To test whether the agarose concentration affects the separation of circular RNA and its linear counterpart in a urea-agarose gel system, these samples were loaded onto a 0.8% agarose gel containing 6M urea. Compared to a 1.5% agarose gel, the separation of circular RNA and its linear counterpart was found to be lower (Figure 5D). Further reductions in the urea concentration in the 0.8% agarose gel resulted in even lower separation (Figure 5E).

[0107] Therefore, given a given agarose concentration, the higher the urea concentration, the greater the separation between circular RNA and its linear counterpart. Furthermore, it can be observed that, for a given urea-agarose gel, the longer the circular RNA, the greater the separation between circular RNA and its linear counterpart. Thus, these results indicate that long circular RNA is better separated using a low-concentration urea-agarose gel, and vice versa.

[0108] These results demonstrate that circular RNAs in the range of approximately 800 to 6000 nucleotides can be easily separated using appropriate concentrations of agarose and urea.

[0109] To test whether this system works for short circular RNAs, we used a 212-nucleotide circular 3 * A novel vector producing Flag RNA was constructed. When the circularized sample was loaded onto a 4% agarose gel containing 6M urea, circular 3 * The Flag and its linear counterpart were found to be well separated (Figure 5F). The urea-agarose gel system can also separate short circular RNAs.

[0110] Therefore, these results demonstrate that circular RNAs ranging from approximately 200 to 6000 nucleotides can be separated from their linear counterparts on an agarose gel using urea as a denaturing agent.

[0111] Example 5 To investigate how gel electrophoresis parameters affect the separation of circular RNA and its linear counterpart, different combinations of electrophoresis time and electrophoresis power were tested.

[0112] When using a 1.5% agarose gel containing 6M urea, it was found that the separation between circular RNA and its linear counterpart improved with longer electrophoretic time or higher electrophoretic power (Figure 6).

[0113] As shown in Figures 6A and 6B, with fixed electrophoresis power, the separation of circular RNA and its linear counterpart increases with longer electrophoresis time. As shown in Figures 6C and 6D, with fixed electrophoresis power, the separation of circular RNA and its linear counterpart increases with higher electrophoresis power. For the gels in Figures 6C and 6D, denatured formamide loading buffer was used. Formamide loading buffer causes shadowing of RNA bands. For example, various RNA markers have an upper shadow band.

[0114] These results demonstrate that the separation of circular RNAs in the range of approximately 200 to 6000 nucleotides from their linear counterparts can be adjusted by changing the urea / agarose concentration and electrophoretic parameters.

[0115] The examples provided above demonstrate that an agarose gel using urea as a denaturant effectively separates circular RNA and its linear counterpart in a sample. These results illustrate a novel technique for identifying circular RNA up to 6000 nucleotides in RNA-containing samples from its linear counterpart. The present invention provides a simple method for identifying circular RNA and may be useful in developing better circular RNA analysis strategies for circular RNA-based therapeutics.

[0116] While this disclosure is described in detail with reference to certain features, it will be apparent to those skilled in the art that this description relates only to preferred embodiments and does not limit the scope of this disclosure.

[0117] array The sequences referenced in this specification are listed in the table below.

[0118]

Table 4

[0119] References - Zaug AJ et al, McEvoy MM and Cech TR. Self-splicing of the Group I Intron from Anabaena Pre-tRNA Requirement for Base-Pairing of the Exons in the Anticodon Stem. Biochemistry vol. 32(31) p7946-53 (1993) - Wesselhoeft, R. A., Kowalski, P. S. and Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun vol.9, p2629 (2018) - Abe BT, Wesselhoeft RA, Chen R, Anderson DG and Chang HY. Circular RNA migration in agarose gel electrophoresis. Molecular Cell vol. 82 p1768-1777 (2022) - Zhang Y, Yang L and Chen LL. Characterization of Circular RNAs. In Feny Y and Zhang L (eds) Long Non-Coding RNA: Methods and Protocol, Methods in Molecular Biology, vol 1402 (2016) - Masek, T., Vopalensky, V., Suchomelova, P. & Pospisek, M. Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem 336, (2005) - Sumitomo, K., Sasaki, M. & Yamaguchi, Y. Acetic acid denaturing for RNA capillary polymer electrophoresis. Electrophoresis 30, 1538-1543, (2009). - Rosen, J. M., Woo, S. L., Holder, J. W., Means, A. R. & O'Malley, B. W. Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct. Biochemistry-Us 14, 69-78 (1975). - Reijnders, L., Sloof, P., Sival, J. & Borst, P. Gel electrophoresis of RNA under denaturing conditions. Biochim Biophys Acta 324, 320-333 (1973) - Rio, D. C. Denaturation and electrophoresis of RNA with formaldehyde. Cold Spring Harbor Protocols, p219-222, (2015)

Claims

1. A method for separating RNA present in a sample, (a) Loading the sample containing the RNA onto an electrophoresis gel, wherein the electrophoresis gel is a urea-agarose gel; (b) Applying an electric field across the gel to move the RNA across the gel, wherein linear RNA and circular RNA present in the sample move through the gel at different rates; Methods that include...

2. The method according to claim 1, wherein the gel contains urea at a concentration of about 1 M to about 8 M.

3. The method according to claim 1 or 2, wherein the gel contains urea at a concentration of about 4 M to 6 M.

4. The method according to any one of claims 1 to 3, wherein the agarose is present at a concentration of about 0.5% to 6%.

5. The method according to any one of claims 1 to 4, wherein the agarose is present at a concentration of about 1.5% agarose.

6. The method according to any one of claims 1 to 5, wherein the electric field is applied at approximately 15W to 40W.

7. The method according to any one of claims 1 to 6, wherein the electric field is applied at approximately 15W to 25W.

8. The method according to any one of claims 1 to 7, wherein the electric field is applied for a period of approximately 10 to 40 minutes.

9. The method according to any one of claims 1 to 8, wherein the electric field is applied for a period of approximately 10 to 30 minutes.

10. The method according to any one of claims 1 to 9, wherein the gel is a 6M urea / 1.5% agarose gel.

11. The method according to any one of claims 1 to 10, wherein the electric field is applied at a power of approximately 15 W to 25 W for a period of approximately 10 minutes to 30 minutes.

12. The method according to any one of claims 1 to 11, wherein the method is for separating RNA having a length of up to approximately 6,000 nucleotides.

13. The method according to any one of claims 1 to 12, wherein the method is for separating RNA having a length in the range of about 200 nucleotides to about 6000 nucleotides.

14. The method according to any one of claims 1 to 9, wherein the method is for separating RNA having a length in the range of about 500 nucleotides to about 6000 nucleotides, the gel is a 4 M urea / 1.5% agarose gel, and the method comprises applying the electric field at a constant power of about 20 W for 10 to 30 minutes.

15. The method according to any one of claims 1 to 14, wherein a vertical electrophoresis gel system is used.

16. (a) Performing electrophoresis of a control sample containing a molecular weight marker in the first lane of the electrophoresis gel, (b) Performing electrophoresis of the RNA sample in the second lane of the electrophoresis gel, (c) Visualizing the bands, The method according to any one of claims 1 to 15, further comprising:

17. The method according to any one of claims 1 to 16, wherein the band of the gel is visualized by UV imaging technology.

18. The method according to any one of claims 1 to 17, further comprising the step of mixing the RNA sample with a loading buffer before loading the sample onto the gel, wherein the loading buffer contains a denaturant.

19. The method according to claim 18, wherein the denaturing agent of the loading buffer is urea.

20. A method for separating circular RNA and linear RNA in a sample, comprising separating the RNA in the sample by electrophoresis on a gel, wherein the gel is a denatured urea-agarose gel and is placed in a vertical position.

21. (a) The urea-agarose gel is as defined in any one of claims 1 to 19, and / or (b) Separating RNA in a sample by electrophoresis on a gel includes performing electrophoresis on a gel according to any one of claims 1 to 19. The method according to claim 20.

22. A method for analyzing RNA present in a sample, wherein the method comprises analyzing the sample by gel electrophoresis, the gel being a denatured urea-agarose gel, and the method comprising using a vertical gel electrophoresis system.

23. (a) The urea-agarose gel is as defined in any one of claims 1 to 19, and / or (b) Analyzing the sample by gel electrophoresis includes performing electrophoresis with the gel described in any one of claims 1 to 19. The method according to claim 22.

24. A kit for separating linear RNA and circular RNA in a sample, (a) A component for performing gel electrophoresis of an RNA-containing sample, comprising agarose and urea for the preparation of the urea-agarose gel according to any one of claims 1 to 19, (b) A loading buffer for mixing with the RNA sample, which contains urea as a denaturing agent, (c) Standard molecular weight control sample, A kit that includes this.

25. The kit according to claim 24, further comprising an apparatus for vertical gel electrophoresis.

Images