Methods and compositions for rapid detection and analysis of RNA modifications

Abstract

Aspects of the present disclosure are directed to methods, compositions, and kits for detection and analysis of RNA pseudouridylation. Certain aspects include methods, compositions and kits useful in sequencing of pseudouridinylated nucleic acids, including pseudouridinylated nucleic acids from low-input samples such as cell-free RNA. Also disclosed herein are methods, compositions and kits useful in sequencing of pseudouridinylated nucleic acids with low nucleic acid damage, higher true positive results, and / or lower false negative results.

Description

METHODS AND COMPOSITIONS FOR RAPID DETECTION AND ANALYSIS OF RNA MODIFICATIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U. S. Provisional Patent Application Serial No. 63 / 712,812, filed on October 28, 2024, the contents of which are hereby incorporated by reference in their entirety.

[0002] Further applicable details may be found in U.S. Provisional Application Serial No. 63 / 694,681 filed September 13, 2024 and PCT Application No. PCT / US2025 / 046137 filed September 12, 2025 entitled “METHODS AND COMPOSITIONS FOR COOPERATIVE CATALYSIS ASSISTED SELECTIVE NUCLEIC ACID DEAMINATION,” all of which are incorporated by reference in their entireties herein.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on October 22, 2025, is named ARCD.P0860WO.xml and is 47,552 bytes in size.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under HG008935 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUNDI. Field of the Invention

[0005] Aspects of this invention relate to at least the fields of cell biology, nucleic acid sequencing, and epigenetics.II. Background

[0006] Pseudouridine (T) was the first modified ribonucleotide discovered over half a century ago, and it has since been found in most RNA species, including rRNA, tRNA, snRNA, miRNA and mRNA. In cells, pseudouridine is installed by pseudouridine synthases (PUS), which have been shown to associate with many diseases including cancer. Pseudouridine is enzymatically synthesized through the post-transcriptional conversion of uridine by eitherpseudouridine synthases (PUSs) or through a snoRNA-guided process. These enzymes can be classified into six families — RsuA, RluA, TruB, TruA, TruD, and PuslO — based on sequence homology. The presence of pseudouridine modification has been detected across all three domains of life: eukaryotes, prokaryotes, archaea, and even some viruses. Despite the development of several high-throughput detection methods for pseudouridine across the transcriptome, there remains a need for a highly efficient and unbiased method for systematically quantifying pseudouridine modification levels. Such a method is essential for advancing understanding of pseudouridine modification and facilitating its systematic study across multiple biological samples. Therefore, establishing an accurate and sensitive technique for detecting pseudouridine is highly desirable.

[0007] With the rapid advancements of functional characterization of PUS enzymes in different cell types, the demands for more sensitive and reliable pseudouridine detection methods that can map pseudouridine in all RNA species and sequence contexts have become increasingly urgent. Previous efforts have led to Pseudo-seq and CeU-seq, which rely on N- cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CMC) chemistry that generates RT-stop signals on pseudouridine sites. Bisulfite-based approaches, such as BID- seq and PRAISE, have recently been invented, which have significantly improved the resolution and quantification using a deletion-based signal. This signal deletion-based method could not map pseudouridine sites in U-rich sequences. More recently, a mutation-based approach, BACS was introduced, enabling more precise pseudouridine site detection. However, all these previous methods require harsh conditions, with high temperatures, which pose challenges such as RNA degradation and compromise chemical selectivity, ultimately reducing detection accuracy.

[0008] Thus, there exists a need for improved methods and compositions for detection and analysis of RNA pseudouridine, including improvements that allow for analysis under moderate conditions.SUMMARY

[0009] To address the aforementioned problems, the inventors created new methods and compositions that allow for more widespread applications in seeking new pseudouridine disease biomarkers or in detecting known pseudouridine biomarkers under moderate conditions (e.g., under 80 °C or under 85 °C or under 90 °C and under 10 pH or under 9 pH or under 8.8 pH) starting from low input of clinic samples. In some embodiments, the problems are solved,at least in part, through a reduction in polynucleotide damage, increased true positive modification identification, and / or decreased false positive modification identification.

[0010] The disclosure relates to enhancing selectivity and / or reactivity (and / or balancing selectivity and / or reactivity) of pseudouridine and a pseudouridine-labeling molecule. Selectivity and / or reactivity can be enhanced by, for example, leveraging the steric hindrance effect and / or use of hybridized electron-withdrawing groups.

[0011] In a particular aspect, the disclosure relates to a pseudouridine mapping method that leverages the selective interaction between pseudouridine and, for example, acrylonitrile analogs, enabling single-base resolution detection. This approach can facilitate clearer mapping by focusing on single base resolution mutation-based detection. In some aspects, the reactivity of the acrylonitrile alkene or the acrylonitrile analog alkene is augmented by introducing an electron- withdrawing group, while preserving the nitrile functional group. In such aspects, the reaction can occur under milder conditions (e.g., lower temperatures and pH, e.g., temperatures less than 80 °C, closer to 37 °C and below pH values that cause low RNA degradation).

[0012] The present disclosure provides various methods, compositions, systems, and kits for nucleic acid processing, and detection and / or analysis of pseudouridine. Certain aspects of the disclosure are directed to particular compositions useful in rapid treatment of RNA for detection and analysis of pseudouridine in RNA. The RNA can be rRNA, tRNA, snRNA, mRNA, etc. Also disclosed are RNA processing methods comprising use of the disclosed compositions for preparation RNA for sequencing and pseudouridine detection and analysis. Further disclosed are methods for pseudouridine detection, quantification, and analysis. RNA processing kits are disclosed, including kits useful in preparation of RNA for pseudouridine detection and analysis.

[0013] Aspects of the disclosure include at least pseudouridine labeling molecule solutions, RNA processing methods, methods for pseudouridine detection and analysis, nucleic acid processing kits, and RNA processing kits.

[0014] The term “pseudouridine labeling molecule solution” refers to a solution containing a pseudouridine labeling molecule. “A pseudouridine labeling molecule” refers to a molecule used to label pseudouridine for detection.

[0015] Methods of the disclosure can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more of the following steps: generating a pseudouridine labeling molecule solution, incubating an RNA molecule in a pseudouridine labeling molecule solution, removing an RNA molecule from a pseudouridine labeling molecule solution, detecting pseudouridine, quantifying pseudouridine, obtaining a sample from a subject, isolating nucleic acid molecules from a sample, sequencingan RNA molecule, detecting / quantifying / mapping an RNA molecule. Any one or more of the preceding steps may be excluded from certain aspects. Compositions (e.g., solutions) of the disclosure can include at least, or at most, 1, 2, 3, or more of the following components: a pseudouridine labeling molecule in a solution and buffer. As used herein, the term, “pseudouridine labeling molecule” is a molecule that causes a reaction when incubated with an RNA molecule comprising pseudouridine. In some aspects, the the pseudouridine labeling molecule is of the formula:(I), where R1 is or is not the electron-withdrawing group; R2 is or is not hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; and R3 and R4 are each independently H or C1-C3 alkyl.

[0016] The reaction can include 1, 2, 3, 4, 5, or more intermediate molecules / products and an end product that is detectable and / or quantifiable by standard methods in the art. The end product is also referred to throughout the disclosure as a “modified pseudouridine.” The intermediate molecules can be products of Michael addition (e.g., aza-Michael addition), aromatization, intramolecular cyclization, and / or hydrolysis reactions. An exemplary (i.e., nonlimiting) reaction mechanism is depicted below:

[0017] Exemplary pseudouridine (T) labeling molecules include Michael acceptors, for example Michael acceptors that operate through 1,4 addition. Exemplary T labeling molecules include, but are not limited to:. Any of the preceding psuedouridine molecules may be specifically excluded, in some aspects.

[0018] As used herein, any of the above molecules can be referred to by their number. For example, methyl 2-bromoacrylate, can be referred to as “8” or “reagent 8” or the like.

[0019] Any one or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more of the preceding components may be excluded from certain aspects. Kits of the disclosure can include at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more of the following components: a pseudouridine labeling molecule, a buffer, instructions for DNA processing, instructions for RNA processing, instructions for a pseudouridine labeling molecule treatment of DNA, instructions for a pseudouridine labeling molecule treatment of RNA, primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases. Any one or more of the preceding components may be excluded from certain aspects.

[0020] Some aspects of the disclosure relate to a composition comprising a pseudouridine labeling molecule solution in a buffer, which can comprise any of the pseudouridine labelingmolecules disclosed herein. In some aspects, the pseudouridine labeling molecule can include an electron-withdrawing group. Electron withdrawing groups include, but are not limited to, H, CH=CH2, -C6H5, NH2, NR2, OR, OH, I, Br, Cl, F, CONH2, COCI, COOH, COR, CHO, SO3H, CN, SO2R, NO2, NH3+and / or the like.

[0021] In some aspects, the pseudouridine labeling molecule is of the formula:whereinR1 is the electron-withdrawing group;R2 is hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; andR3 and R4 are each independently H or C1-C3 alkyl.

[0022] In some aspects, the electron-withdrawing group is nitrile, nitro, ester, carboxylic acid, ester, amide, ketone, aldehyde, sulfone, sulfonic acid, thioester, thioate, and / or the like.

[0023] In some aspects, the electron-withdrawing group is substituted. In some aspects, the electron-withdrawing group is substituted with a linking group. In some aspects, the linking group is azide, alkyne, thiol, amine, maleimide, N-hydroxysuccinimide ester, tetrazine, transcyclooctene, and / or the like.

[0024] In some aspects, the molecule further comprises a linker between the linking group and the pseudouridine labeling molecule. In some aspects, the linker is a polyethylene glycol (PEGs), a polyglycerol, a polysarcosine, polyoxazoline, peptide linker, alkyl linker and / or the like.

[0025] In some aspects, the 'P labeling molecule is selected from:

[0026] In some aspects, the pseudouridine labeling molecule comprises two electronwithdrawing groups. In some aspects, the two electron-withdrawing groups are each independently nitro, sulfonyl, sulfonate, nitrile, sulfonic acid, aldehyde, ketone, carboxylic acid, ester, acid chloride, amide, and / or the like. In some aspects, the two electron-withdrawing groups are ester and bromide.

[0027] In some aspects, the pseudouridine labeling molecule is methyl a-bromoacrylate (compound 8).

[0028] In some aspects, the buffer is or is not a Tris, phosphate, TEAB, HEPES, NasBCh, sodium cacodylate, TEAA, EDTA buffer, or the like. In some aspects, the buffer is or is notTEAB.

[0029] In some aspects, the concentration of the buffer is between about 50 mM to about 1000 mM or any derivable range or value therein, for example, between about 150 mM to about 500 mM or between about 50 mM to about 250 mM, etc. In some aspects, the buffer has a pH of about 5-10, or any derivable range or value therein (e.g., 5-9 or 7.75 to 7.95). In some aspects, the buffer has a pH of about 7.85. In some aspects, the buffer comprises an amine. In some aspects, the amine comprise NH4 , and / or the like. In some aspects, the buffer comprises NH4OH, and / or the like. In some aspects, the concentration of the amine is between about 200- 800 mM, or any derivable range or value therein In some aspects if the concentration of the amine is about 200 mM.

[0030] In some aspects, the concentration of the labeling molecule in the composition is between about 1 mM - 2000 mM, or any derivable range or value therein. In some aspects, the concentration of the labeling molecule in the composition is about 150 mM-200 mM. In some aspects, the concentration of a pseudouridine labeling molecule is at least, at most, is or is not 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500mM or any range derivable therein.

[0031] In some aspects, the pH of the composition is between about 5.5 to about 10, or any derivable range therein (e.g., 5.5 and 10, or any derivable range or value therein). In some aspects, the pH of the composition is between about 7 and 10. In some aspects, the pH of the composition is or is about 7.5-8.5. In some aspects the pH is at least, at most, is or is not 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10 or any range derivable therein.

[0032] In some aspects, the solution further comprises or does not comprise DSMO, ethanol nitrogen, DMS, or the like.

[0033] In some aspects, the composition is stable for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or any range or value derivable therein. “Stable” refers to maintaining effectivity at a temperature. The temperature can be about room temperature, about 4 °C or about -18 °C or any standard laboratory storage temperature.

[0034] In some aspects, the composition further comprises ribonucleic acid (RNA). In some aspects, the RNA comprises at least one pseudouridine modification.

[0035] In some aspects, the ratio of the pseudouridine labeling molecule to pseudouridine is between about 20: 1 to about 0.15: 1 or any derivable range or value in between, for example, about 10: 1 to about 1.25: 1, or about 5: 1 to about 2.5: 1, etc.

[0036] In some aspects, RNA is rRNA, tRNA, snRNA, and / or mRNA. In some aspects, the RNA is not rRNA, tRNA, snRNA, and / or mRNA. In some aspects, the RNA is mRNA. In some aspects, the mRNA is polyA enriched mRNA.

[0037] Some aspects of the disclosure relate to a method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising use of any of the compositions disclosed herein.

[0038] Some aspects of the invention relate to a method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, a pseudouridine labeling molecule and a buffer, under conditions sufficient to produce a modified pseudouridine that is detectable and / or quantifiable, and a buffer, wherein the pseudouridine labeling molecule comprises an electron-withdrawing group.

[0039] In some aspects, the incubation comprises an intramolecular cyclization step.

[0040] In some aspects of the method, the pseudouridine labeling molecule is of the formula:whereinR1 is the electron-withdrawing group;R2 is hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; andR3 and R4 are each independently H or C1-C3 alkyl.

[0041] In some aspects, the electron-withdrawing group is nitrile, nitro, ester, carboxylic acid, ester, amide, ketone, aldehyde, sulfone, sulfonic acid, thioester, thioate, and / or the like.

[0042] In some aspects, the electron-withdrawing group is substituted. In some aspects, the electron-withdrawing group is substituted with a linking group. In some aspects, the linking group is azide, alkyne, thiol, amine, maleimide, N-hydroxysuccinimide ester, tetrazine, or trans-cyclooctene, and / or the like.

[0043] In some aspects, the molecule further comprises a linker between the linking group and the pseudouridine labeling molecule. In some aspects, the linker is a polyethylene glycol(PEGs), a polyglycerol, a polysarcosine, polyoxazoline, peptide linker, alkyl linker, and / or the like.

[0044] In some aspects of the method, the T labeling molecule is selected from:In some aspects, the pseudouridine labeling molecule comprises or does not comprise two electron-withdrawing groups. In some aspects, the two electron-withdrawing groups are each independently nitro, sulfonyl, sulfonate, nitrile, sulfonic acid, aldehyde, ketone, carboxylic acid, ester, acid chloride, or amide. In some aspects, the wherein the two electron-withdrawing groups are ester and bromide.

[0045] In some aspects, the conditions sufficient to produce a modified pseudouridine comprise one or more of: a) the pseudouridine labeling molecule in a concentration at about 1 mM - 2000 mM, b) a pH of equal to or less than 10, c) a temperature of less than 85 °C, and / or d) an incubation time of between 2 minutes - 4 hours.

[0046] In some aspects, the conditions sufficient to produce a modified pseudouridine comprises an incubation time of 37°C for 3 hours or 70°C for 30 minutes.

[0047] In some aspects, the T labeling molecule is or comprises methyl a-bromoacrylate. In some aspects, the T labeling molecule is not (bromo) acrylonitrile.

[0048] In some aspects, the method includes one or more intermediate products. In some aspects, the one or more intermediate products is a pseudouridine methyl-propionate ester and / or a pseudouridine bromo delta lactone. In some aspects, the one or more intermediate products is not a pseudouridine propionamide and / or a pseudouridine oxazolidine.

[0049] In some aspects, the modified pseudouridine is a six-membered hydroxylactone. In some aspects, the modified pseudouridine is not a five-membered oxazolidine.

[0050] In some aspects, the modified pseudouridine is a pseudouridine hydroxy lactone. In some aspects, the modified pseudouridine is not pseudouridine oxazolidine.

[0051] In some aspects, the composition comprises a concentration of a pseudouridine labeling molecule between about 50 mM and 500 mM, or any derivable range or value therein. In some aspects, the composition comprises a concentration of a pseudouridine labeling molecule of about 150 mM-250 mM. In some aspects, the concentration of a pseudouridine labeling molecule is at least, at most, is or is not 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295,300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390,395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485,490, 495, 500mM or any range derivable therein.

[0052] In some aspects, a pH of the composition is between about 5 and about 10, or any derivable range therein, for example, between about 5.5 and about 10 or about 7 and about 9. In some aspects, the pH of the composition is or is about 7.5-8.5. In some aspects, a pH of the composition is not less than 5 or greater than 10. In some aspects the pH of a reaction or a condition described herein is at least, at most, is or is not 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10 or any range derivable therein.

[0053] In some aspects, the temperature of a reaction or condition described herein, such as a reaction to convert a psuedouridine to a modified psuedouridine or a condition sufficient to convert a psuedouridine to a modified pseudouridine, is less than about 85 °C, or equal to or less than 70 °C. In some aspects, the temperature is between about 30-45 °C, etc. In some aspects, the temperature is about 37 °C. In some aspects, the temperature is not less than 20, 25, or 30 °C, or any range or value derivable therein. In some aspects, the temperature is not greater than 45, 50, 55, 60, 65, 70, 75, 80, 85 °C, or any range or value derivable therein. In some aspects the temperature of a reaction or a condition described herein is at least, at most, is or is not 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 °C or any range derivable therein.

[0054] In some aspects, the incubation time of a reaction or condition described herein, such as a reaction to convert a psuedouridine to a modified psuedouridine or a condition sufficient to convert a psuedouridine to a modified pseudouridine, is between about 30 minutes to 4 hours, or any range or value derivable therein. For example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 hours. In some aspects, the incubation time is 3 hours at 37°C or 30 min at 70°C. In some aspects the incubation time of a reaction or a condition described herein is at least, at most, is or is not 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300 minutes or any range derivable therein.

[0055] In some aspects, the method further includes a pre-heating step. In some aspects, the pre-heating step is at a higher temperature than the sufficient conditions. In some aspects, the preheating step is equal to or less than 15, 10, 5 or 2 min (or any range or value derivable therein) at a temperature of less than about 75, 70, 65 °C, or any range or value derivable therein. In some aspects, the pre-heating temperature is less than 70 °C.

[0056] In some aspects, the RNA polynucleotide comprises a low level of damage. In some aspects, the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition not comprising the pseudouridine labeling molecule (e.g., a comprising an acrylonitrile, in some aspects). In some aspects, the damage is assessed as RNA fragmentation.

[0057] In some aspects, the pseudouridine to modified pseudouridine unconverted rate is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%. In some aspects, the pseudouridine to modified pseudouridine unconverted rate is less than about 3%.

[0058] In some aspects, greater than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any range or value derivable therein) of the pseudouridine are converted to modified pseudouridine.

[0059] In some aspects, about 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80-100% of the pseudouridine are converted to modified pseudouridine.

[0060] In some aspects, the RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or any range or value derivable therein including between about 5 ng and 15 ng of RNA polynucleotide in the composition.

[0061] In some aspects, the RNA is rRNA, tRNA, snRNA, and / or mRNA. In some aspects, the RNA is not rRNA, tRNA, snRNA, and / or mRNA. In some aspects, the RNA is mRNA. In some aspects, the mRNA is polyA enriched mRNA.

[0062] In some aspects, the modified pseudouridine is detected and / or quantified and / or mapped. The detection, quantification and / or mapping can include any conventional methods in the art such as mass spectrometry or the like. In some aspects, the modified pseudouridine is mixed with dCTP before detection and / or quantification and / or mapping.

[0063] In some aspects, RNA with modified pseudouridine is subjected to reverse transcription, cDNA ligation, PCR amplification, and / or the like.

[0064] In some aspects, RNA with modified pseudouridine is subjected to reverse transcription, 2nd strand synthesis, dsDNA ligation, PCR amplification, and / or the like.

[0065] In some aspects, the buffer is or is not a Tris, phosphate, TEAB, HEPES, NasBCh, sodium cacodylate, TEAA, and / or EDTA buffer and / or the like. In some aspects, the concentration of the buffer is between about 50 mM to about 1000 mM or between about 50 mM to about 500 mM or between about 50 mM to about 250 mM. In some aspects, the concentration of the buffer is less than about 200 mM. In some aspects, the concentration of the buffer is 150 mM TEAB. In some aspects, the concentration of the buffer has a pH of about 5-10. In some aspects, the buffer has a pH of about 8.0 to about 9.0 or 7.5-8.5. .In some aspects, the buffer comprises an amine. In some aspects, the amine comprises NHP, and / or the like. In some aspects, the buffer comprises NH4OH, and / or the like. In some aspects, the concentration of the amine is between about 200 - 800 mM. In some aspects, the concentration of the amine is about 200 mM or 500mM.

[0066] Some aspects of the disclosure relate to a method of detecting a disease or disorder in a subject, wherein the disease or disorder is associated with a pseudouridine RNA modification comprising any of the methods disclosed herein. In some aspects, the subject is a pig, mouse, monkey, or human. In some aspects, the method comprises performing one or more steps described herein on an RNA polynucleotide obtained from the subject. The RNApolynucleotide may be obtained from the subject in a biological sample, such as a blood sample or biopsy.

[0067] Some aspects of the disclosure relate to a method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, methyl a- bromoacrylate and a buffer under conditions sufficient to produce modified T that is detectable and / or quantifiable, wherein the modified pseudouridine is pseudouridine hydroxylactone, and wherein the sufficient conditions comprise one or more of: a) the pseudouridine labeling molecule concentration is about 50 mM and 500 mM, b) the pH is equal to or less than 10, c) the temperature is less than 85 °C, and d) the incubation is between 30 min-4 hours.

[0068] In some aspects, the buffer is or is not a Tris, phosphate, TEAB, HEPES, Na3BO3, sodium cacodylate, TEAA, and / or EDTA buffer. In some aspects, the concentration of the buffer is between about 50 mM to about 1000 mM (or any range or value derivable therein) or between about 75 mM to about 500 mM or between about 100 mM to about 200 mM.

[0069] In some aspects, the buffer comprises or does not comprise an amine. In some aspects, the amine comprises NHE and / or the like. In some aspects, the buffer comprises NH4OH, and / or the like. In some aspects, the concentration of the amine is between about 200 - 800 mM, or any range or value derivable therein. In some aspects, the concentration of the amine is about 200 mM or 500 mM.

[0070] In some aspects, the composition comprises a concentration of a pseudouridine labeling molecule between about 100 mM and 300 mM, or any range or value derivable therein. In some aspects, the composition comprises a concentration of a pseudouridine labeling molecule of about 150mM - 200 mM.

[0071] In some aspects, the pH of the composition is between about 5.5 and about 9 or any derivable range or value therein. In some aspects, the pH of the composition is between about 8 and about 9 or any derivable range or value therein. In some aspects, the pH of the composition is or is about 7.5-8.5.

[0072] In some aspects, the temperature is less than about 85 °C, 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C or any derivable range or value therein. In some aspects, the temperature is between about 25-70 °C. In some aspects, the temperature is between about 30- 45 °C. In some aspects, the temperature is about 37 °C.

[0073] In some aspects, the incubation time is 30 minutes to 4 hours or any derivable range or value therein. In some aspects, the incubation time is about 3 hours.

[0074] In some aspects, the incubation time is 70°C for 30 minutes.

[0075] In some aspects, the method further includes a pre-heating step. In some aspects, the pre-heating step is at a higher temperature than the sufficient conditions. In some aspects, the preheating step is less than 15, 10, 5 or 2 min (or any range or value derivable therein) at a temperature of less than 75, 70, 65 °C, or any range or value derivable therein. In some aspects, the pre-heating temperature is equal to or less than 70 °C.

[0076] In some aspects, the RNA polynucleotide comprises a low level of damage. In some aspects, the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition not comprising pseudouridine labeling (e.g., a composition comprising an acrylonitrile, in some aspects). In some aspects, the damage is assessed as RNA fragmentation.

[0077] In some aspects, the pseudouridine to modified pseudouridine unconverted rate is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%. In some aspects, the pseudouridine to modified pseudouridine unconverted rate is less than about 3%.

[0078] In some aspects, greater than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudouridine are converted to modified pseudouridine, or any range derivable therein. It is contemplated that a sample may be assayed for conversion in some aspects.

[0079] In some aspects, about 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80-100% of the pseudouridine are converted to modified pseudouridine.

[0080] In some aspects, the RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or any range or value derivable therein including 5 ng and 15 ng in the composition.

[0081] In some aspects, the RNA is rRNA, tRNA, snRNA, and / or mRNA. In some aspects, the RNA is or is not rRNA, tRNA, snRNA, and / or mRNA. In some aspects, the RNA is mRNA. In some aspects, the mRNA is polyA enriched mRNA.

[0082] In some aspects, the modified pseudouridine is detected, quantified, and / or mapped, the detection, quantification and / or mapping can include any conventional methods in the art such as mass spectrometry or the like.

[0083] In some aspects, RNA with modified pseudouridine is subjected to reverse transcription, cDNA ligation, PCR amplification, and / or the like.

[0084] In some aspects, RNA with modified pseudouridine is subjected to reverse transcription, 2ndstrand synthesis, dsDNA ligation, PCR amplification and / or the like.

[0085] Some aspects of disclosure relate to a method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, methyl a- bromoacrylate and a buffer under conditions sufficient to produce modified pseudouridine that is detectable and / or quantifiable, wherein the modified pseudouridine is pseudouridine hydroxylactone, and wherein the sufficient conditions comprise: a) the pH is 7-9; b) the temperature is equal or less than 70°C, and; c) the incubation is between 0.5-3 hours, the incubation time is 37°C for 3 hours or 70°C for 30 minutes. In some aspects of the method, the buffer is TEAB. In some aspects of the method, the concentration of the buffer is between about 100 mM to about 200 mM. In some aspects of the method, the buffer comprises an amine. In some aspects of the method, concentration of the amine is between about 100 - 300 mM. In some aspects of the method, the concentration of the amine is about 200 mM. In some aspects of the method, the composition comprises a concentration of a pseudouridine labeling molecule between about 100 mM and 250 mM. In some aspects of the method, the composition comprises a concentration of a pseudouridine labeling molecule of about 150-200 mM. In some aspects, the method further comprises a pre-heating step. In some aspects of the method, the preheating step is equal or less than 2 min at a temperature of less than about 70 °C. In some aspects of the method, the RNA polynucleotide comprises a low level of damage. In some aspects of the method, the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition not comprising the labeling molecule (e.g., a comprising an acrylonitrile, in some aspects). In some aspects of the method, the pseudouridine is to modified pseudouridine unconverted rate is less than about 3%. In some aspects of the method, the modified pseudouridine is detected and / or quantified by mass spectrometry. In some aspects of the method, RNA with modified pseudouridine is subjected to reverse transcription, cDNA ligation, and / or PCR amplification. In some aspects of the method, RNA with modified pseudouridine is subjected to reverse transcription, 2nd strand synthesis, dsDNA ligation, and / or PCR amplification.

[0086] Some aspects of the disclosure relate to a polynucleotide processing kit comprising any or the compositions disclosed herein and optionally instructions for processing a polynucleotide sample.

[0087] Some aspects of the disclosure relate to a polynucleotide processing kit comprising one or more of: a) a first component comprising a first solution comprising a pseudouridine labeling molecule; b) a second component comprising a buffer; and c) optionally instructions for processing a polynucleotide sample. Any of the preceding components may be specifically excluded from the kit.

[0088] In some aspects of the kit, the T labeling molecule is selected from:

[0089] In some aspects, the buffer is or is not a Tris, phosphate, TEAB, HEPES, Na3BO3, sodium cacodylate, TEAA, and / or EDTA buffer. In some aspects, the concentration of the buffer is between about 50 mM to about 1000 mM, including any range or value derivable therein including between about 50 mM to about 500 mM or between about 50 mM to about 250 mM.

[0090] In some aspects, the second component comprises one or more amines catalyst. In some aspects, the amine comprises NHE. In some aspects, the second component buffercomprises NH4OH. In some aspects, the first and second components are provided separately or as a premixed solution.

[0091] In some aspects, the premixed solution comprises a pseudouridine labeling molecule concentration between about 1 mM - 2000 mM, or any derivable range or value therein.

[0092] In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the first and second components or the premixed solution at a temperature equal to or less than about 80 °C for a time between 2 minutes - 4 hours. In some aspects, the temperature is between about 30-45 °C. In some aspects, the temperature is about 37 °C.

[0093] In some aspects, the kit further includes or does not include one or more buffer solutions and / or enzymes. In some aspects, the one or more buffers comprise sequencing reagents. In some aspects, the sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0094] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0095] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0096] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” “(x and z) or y,” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0097] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0098] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0099] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0100] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0101] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0102] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of the Invention, Brief Description of the Drawings, Detailed Description of the Invention, and / or Claims.

[0103] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0104] A person of ordinary skill in the art would understand that a solution that does not contain a particular chemical does not contain an added quantity of that chemical. The term added means that the chemical is exogenously supplied, i.e. supplied in amounts greater than what would be considered trace or minute amounts.

[0105] As understood by the skilled artisan, a solution of the present disclosure may be described in terms of molarity (M), percent by weight (also “weight percent”; w / w), or any other units. When described in terms of one unit (e.g., w / w) equivalent solutions expressed by other units (e.g., M) are also contemplated herein.

[0106] As utilized herein, “added” has a traditional meaning, to include, put in, or join, a component to another component, e.g., a chemical can be added to a composition or method. A component that is added can be described as being included in a form substantially as described through an active step. A component that is added may also occur without specific inclusion, for example, a component may come into existence through an in situ chemical reaction without being specifically added. A component that arises through a passive in situ chemical reaction is not considered added.

[0107] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0108] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The attached drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein. All references mentioned in the drawings are each fully incorporated by reference herein.

[0110] FIGS. 1A-1F depict a tandem chemical reaction designed for selective pseudouridine labeling. FIG. 1A Schematic illustrating the principle of PUM-seq. Standard Watson-Crick pairing prevents T detection by reverse transcription (RT). A tandem reaction involving N1 -alkylation followed by intramolecular cyclization alters the hydrogen bonding interface of T, inducing mispairing with guanine (G) during RT. FIG. IB General chemical strategy for T detection, combining a selective aza-Michael addition with an intramolecular cyclization reaction. FIG. 1C Bar graph showing the screening of 12 candidate reagents for reactivity and selectivity. Conversion ratios for the four canonical bases (A, C, G, U) and pseudouridine (T) were measured by LC-MS. Reagent 8 (methyl 2-bromoacrylate) demonstrates high reactivity and selectivity for T. FIG. ID LC-MS analysis of the reaction between T and reagent 8, showing the consumption of T and the appearance of a new product with the expected mass (m / z 314). FIG. IEJH NMR spectra of unmodified T and the final cyclized product, confirming the structural transformation. FIG. IF Example mechanism for the PUM-seq tandem reaction. The process involves a selective aza-Michael addition at the N1 position of T, hydrolysis of the bromine substituent, and a subsequent intramolecular transesterification to form a stable six-membered lactone product.

[0111] FIGS. 2A-2E The PUM-seq workflow is optimized and validated. FIG. 2A Schematic of the PUM-seq library construction workflow. The process involves 3' adapter ligation, chemical labeling of T, and reverse transcription, where the labeled T site is read as a T-to-C mutation in the final sequencing data. FIG. 2B Box plots comparing the U-to-C (background) and -to-C (signal) mutation rates for reagents 2, 3, and 8, against an untreated control, identifying reagent 8 as efficient and specific. FIG. 2C Box plots detailing the protocol's optimization for reagents 8. The comparison includes a mild labeling condition (37°C for 3h), an accelerated condition (70°C for 30 min), and the final PUM-seq protocol. The final protocol pairs the optimal mild labeling with the addition of 10x dCTP during reverse transcription to maximize the T-to-C signal and minimize background noise. FIG. 2D Mutation rates at individual uridine and pseudouridine sites across human rRNA under fullyoptimized conditions. The data show a high average T mutation rate (83.3%) and an extremely low background U mutation rate (0.25%). FIG. 2E Heatmap showing the mutation rates for synthetic RNA oligonucleotides containing all 256 possible NN- -NN sequence contexts. The high average conversion rate (85.6%) indicates PUM-seq has no significant sequence bias.

[0112] FIGS. 3A-3F PUM-seq is compared with existing methods for accuracy and data quality. FIG. 3A Box plots compare T-to-C (signal) and U-to-C (background) mutation rates in human rRNA for PUM-seq and BACS libraries. FIG. 3B A bar chart compares the percentage of mapped and unmapped reads for libraries prepared with an untreated control, BACS, and PUM-seq. FIG. 3C A plot shows the distribution of library insert fragment lengths for PUM-seq and BACS, with the peak length for each distribution indicated by a dashed line. FIGS. 3D Genome browser snapshots of an example rRNA region show reads from BACS. FIG. 3E Genome browser snapshots of an example rRNA region show reads from PUM-seq libraries. Specific T-to-C mutations are colored light green, while other mutations and deletions are color-coded as indicated in the legend. FIG. 3F Bar charts compare background mutation rates for an untreated control, BACS, and PUM-seq. Rates are shown for all possible mutations and deletions (y-axis, grouped by type) originating from each of the four reference bases (x-axis).

[0113] FIGS. 4A-4G Depicts that Pseudouridine is quantitatively profiled across diverse RNA families in human cells. FIG. 4A A bar chart shows the proportion of sites (T density) in various RNA families from HEK293T cells. T density is calculated as the percentage of uridine sites with a modification level >10%. FIG. 4B A histogram shows the distribution of modification stoichiometries for all detected T sites in mRNA. The total number of sites (N) exceeding specific stoichiometry cutoffs are indicated. FIG. 4C A boxplot displays the modification levels for mRNA T sites, grouped by their NN NNN sequence motif. The GU NNN motif family is highlighted. FIG. 4D A scatter plot analyzes mRNA T site sequence motifs, plotting the fraction of sites found within a given motif against that motifs average modification stoichiometry. FIG. 4E A schematic illustrates the classification of mRNA T sites into Type I (localized, high stoichiometry) and Type II (diffuse, low stoichiometry). FIG. 4F A metagene plot shows the distribution of T sites across the mRNA transcript body (5' UTR, CDS, 3' UTR). FIG. 4G Plots show the number, position, and stoichiometry of sites across individual small RNA transcripts.

[0114] FIGS. 5A-5I Cross-species analysis reveals conserved and dynamic pseudouridylation in rRNA. FIG. 5A A phylogenetic tree illustrates the evolutionary relationships among the five species analyzed: human, macaque monkey, mouse, pig, andchicken (outgroup). FIG. 5B A plot shows the distribution of T sites along human ribosomal RNA (5.8S, 18S, and 28S). Each dot represents a uridine site, with its position mapped to the human rRNA sequence. The site's color indicates its conservation status: fully conserved T sites (green), partially or non-conserved T sites (orange), and unmodified uridines (gray). The vertical position of each dot represents the average modification stoichiometry. FIG. 5C A plot shows the stoichiometry of the 86 fully conserved T sites across all species and tissues. Each point represents a single sample, color-coded by species and shape-coded by tissue. Error bars depict the variance between species (gray) versus the variance between tissues (red) for each site. FIG. 5D A line plot shows the modification stoichiometry for all 86 fully conserved T sites. Each line tracks a single site across the different samples, which are grouped by species and tissue. FIG. 5E A heatmap displays the stoichiometry of variable (partially and nonconserved) T sites. Sites are grouped by their conservation pattern and further categorized as tissue-specific or lineage-specific. FIG. 5F A line plot shows a representative highly conserved site (28S:3650). The modification level is shown for 10 samples (5 species, 2 tissue types), with data from two replicates averaged and variance shown as error bars. FIG. 5G A line plot shows an example of a tissue-specific modification (18S : 1136), with consistently higher levels in the brain than in the testis across mammals. FIG. 5H A line plot shows an example of a primate-specific site (18S:210). The corresponding modification stoichiometry is shown for all samples, highlighting the absence of the modification in mouse, pig, and chicken. FIG. 51 A line plot shows a representative mammalian-specific site that exhibits a strong tissue bias, with higher modification levels observed in the brain.

[0115] FIGS. 6A-6E The evolutionary conservation of pseudouridylation is analyzed in tRNA and snRNA. FIG. 6A A schematic of a consensus tRNA cloverleaf structure highlights the positions of highly conserved T sites. FIG. 6B A heatmap displays T stoichiometry across tRNA transcripts. Rows correspond to different species and tissues, and columns correspond to tRNA positions. FIG. 6C A plot shows the average modification level variance (y-axis) against tRNA position (x-axis), with structural domains indicated. FIG. 6D A bar chart shows the percentage of T sites that are conserved across all five species for rRNA, tRNA, and snRNA. FIG. 6E Stoichiometry plots of T sites in major spliceosomal snRNAs are shown. Each point represents the stoichiometry in a specific sample (brain or testis). Novel T sites not reported in previous studies are highlighted with a light yellow shadow.

[0116] FIGS. 7A-7E Chemical principles and proposed reaction mechanisms for T labeling. FIG. 7A Schematics show the hydrogen bond donors and acceptors for canonical Watson-Crick base pairs (U-A, C-G). FIG. 7B Schematics show the hydrogen bonding patternsfor a standard T-A pair and a proposed -G mispair that can form after modification. FIG. 7C Proposed reaction mechanisms are shown for the reaction of T with reagent 2. FIG. 7D Proposed reaction mechanisms are shown for the reaction of T with reagent 8. FIG. 7E A schematic shows the proposed competing hydrolysis side reaction that can occur under harsh conditions.

[0117] FIGS. 8A-8F NMR spectrum of labeled pseudouridine using reagent 8. FIG. 8A1H NMR spectrum. FIG. 8B13C NMR spectrum. FIG. 8C 2-D ^H^H COSY NMR spectrum. FIG. 8D DEPT13C NMR spectrum. FIG. 8E 2-DJH-13C HSQC NMR spectrum. FIG. 8F 2- DJH-13C HMBC NMR spectrum.

[0118] FIGS. 9A-9F Conversion efficiency and specificity assays for reagent 8 are shown under varying conditions. FIG. 9A Conversion rates for reagent 8 were assessed under varying pH conditions (pH 1.0 to 13.0) at 25°C for 30 minutes in sodium phosphate buffer. FIG. 9B Conversion rates for reagent 8 were evaluated across treatment temperatures (10°C to 95°C) at pH 7.5 for 30 minutes in TEAB buffer. FIG. 9C Conversion rates for reagent 8 were measured at different reaction intervals (5, 10, 30, and 60 minutes) at pH 7.5 and 37°C in TEAB buffer. FIG. 9D Conversion rates for reagent 8 in different reaction buffers are shown, where the pH values of all buffers were adjusted to pH 7.5 and nucleosides were treated at 37°C for 30 minutes. FIG. 9E MALDI-TOF MS analysis of the reaction products between a T-containing RNA oligonucleotide (AG GA) and reagent 8, showing a 70 Da mass increase. FIG. 9F MALDI-TOF MS analysis of the reaction products between a control RNA oligonucleotide (AGUGA) and reagent 8, showing no mass increase.

[0119] FIGS. 10A-10F Optimization of the PUM-seq protocol is performed using nextgeneration sequencing. FIG. 10A U-to-C and T-to-C mutation rates at different temperatures (25°C to 85°C) for samples treated with reagent 8 for 30 minutes in 150 mM TEAB buffer. FIG. 10B U-to-C and T-to-C mutation rates for various incubation times (5 min to 4 h) for samples treated with reagent 8 at 37°C in 150 mM TEAB buffer. FIG. 10C U-to-C and 'P-to- C mutation rates at different TEAB buffer concentrations (10 mM to 400 mM) for samples treated with reagent 8 at 37°C for 3 hours. FIG. 10D Comparison of U-to-C and T-to-C mutation rates in TEAB buffer versus phosphate buffer (with and without NHL) for samples treated at 37°C for 3 hours. FIG. 10E Mutation rates across rRNA sites for the standard (37°C for 3h) and accelerated (70°C for 30 min) labeling protocols. FIG. 10F Distribution of library insert sizes for the standard and accelerated protocols.

[0120] FIGS. 11A-11B Benchmarking of PUM-seq against other T sequencing methods. FIG. HA T (green) and U (gray) mutation or deletion ratios for BID-seq, PRAISE, BACS,and PUM-seq across human rRNA. FIG. 11B Comparison of T levels for 105 rRNA sites as quantified by each sequencing method versus a mass spectrometry gold standard (SILNAS). The x-axis shows the SILNAS value, and the y-axis shows the difference between the sequencing method's value and the SILNAS value.

[0121] FIGS. 12A-12D Sensitivity and specificity of PUM-seq. FIG. 12A Mutation rates of uridine sites in U4 snRNA as profiled by a previous method, with known T sites indicated by green lines. FIG. 12B Background mutation rates (mean=1.01%) across an in vitro transcribed ACTB gene sample as measured by the BACS method. FIG. 12C Mutation rates of uridine sites in U4 snRNA as profiled by PUM-seq, with known T sites shown as green dots. FIG. 12D Background mutation rates (mean=0.28%) across an in vitro transcribed ACTB gene sample as measured by PUM-seq.

[0122] FIGS. 13A-13F Analysis of T sites in human mRNA. FIG. 13A Distribution of T modification levels for all detected mRNA sites. FIG. 13B Venn diagrams showing the overlap of identified mRNA T sites with stoichiometries >10% between different sequencing methods. FIG. 13C Venn diagrams showing the overlap of identified mRNA T sites with stoichiometries >20% between different sequencing methods. FIG. 13D Normalized expression of ACTB, TRUB1, and TRUB2 in control (siCTRL) versus TRUB1 knockdown (siTRUBl) HepG2 cells. FIG. 13E Comparison of T modification levels between siCTRL and siTRUBl samples in HepG2 cells. FIG. 13F Sequence logos for all detected T sites (top) and for sites with high modification levels (>20%, bottom), showing enrichment for the GUTA motif.

[0123] FIGS. 14A-14B Examples of primate-specific and novel detected T sites in rRNA. FIG. 14A Modification stoichiometry for representative tissue-specific rRNA T sites (18S:890, 18S:897, 18S:1 I36, 18S:I3I5, 18S:1539, 28S: 1536, 28S:2839, 28S:2843, 28S:3850). Each plot shows the T rate (y-axis) for brain (black circles) and testis (green triangles) across the five analyzed species (x-axis). FIG. 14B Modification stoichiometry for representative lineage-specific and novel rRNA T sites (18S:31, 18S:890, 18S:899, 18S: 1315, 18S: 1359, 18S: 1687, 28S: 1728, 28S: 1796, 28S:4566, 28S:4969). Each plot shows the T rate (y-axis) for brain and testis across the five analyzed species.

[0124] FIG. 15 tRNA T mapping in individual species. Heatmaps of pseudouridylation stoichiometry across all analyzed tRNA transcripts. Each panel represents a specific sample (species and tissue).

[0125] FIG. 16 Location of pseudouridine sites in the human spliceosome. Schematic of the secondary structures of major human spliceosomal snRNAs (Ul, U2, U4, U5, and U6) in the context of pre-mRNA splicing, with known T sites highlighted.

[0126] FIG. 17 is a reaction scheme depicting a prior art cyanoethylation reaction that proceeds through Michael addition of two molecules of acrylonitrile to pseudouridine.

[0127] FIG. 18 is a reaction scheme depicting a prior art reaction that proceeds through nucleophilic addition of bisulfite to pseudouridine followed by E2 elimination to provide a ring-opened intermediate. The ring-opened intermediate can then react with excess bisulfite (top pathway) through an SN2' substitution to give an asymmetric bisulfite addition product. Alternatively, the ring-opened intermediate can undergo a [2,3] rearrangement reaction (bottom pathway) when subjected to heat at pH 8-9 to provide an asymmetric bisulfite addition product with stereochemistry opposite that of the asymmetric bisulfite addition product.

[0128] FIG. 19 is a reaction scheme depicting a prior art reaction that involves addition of acrylonitrile analogs to pseudouridine through an N'-aza Michael addition. The Michael addition product can then undergo an intramolecular cyclization reaction to provide a labeled pseudouridine (path a) or hydrolysis (path b).

[0129] FIG. 20 is a reaction scheme depicting a pseudouridine modification reaction as disclosed herein. Acrylonitrile analog chloroacrylonitrile reacts with pseudouridine through an N'-aza Michael addition. The Michael addition product then undergoes aromatization followed by an intramolecular cyclization reaction and subsequent chloride hydrolysis to provide a novel labeled pseudouridine.

[0130] FIG. 21 is a graph comparing reaction times compound 1 and pseudouridine, cysteine, uracil, adenine, and guanine for different reaction durations. Reactions were performed at a temperature of 37 °C in 500 mM sodium cacodylate buffer at pH 7.85.

[0131] FIG. 22 is a graph comparing reaction times compound 1 and pseudouridine, cysteine, uracil, adenine, and guanine at different temperatures. Reactions were performed in 500 mM sodium cacodylate buffer at pH 7.85 for 30 minutes.

[0132] FIG. 23 is a graph comparing reaction times compound 1 and pseudouridine, cysteine, uracil, adenine, and guanine at different pH values. Reactions were performed at a temperature of 37 °C in 500 mM sodium cacodylate buffer for 1 hour.

[0133] FIG. 24 is a graph comparing reaction times compound 1 and pseudouridine, cysteine, uracil, adenine, and guanine in different buffers.

[0134] FIG. 25 is a graph depicting reaction kinetics at different substrate ratios (i.e., varying concentrations of compound 8, 20: 1 indicates that compound 8 is present at 20 times the amount of pseudouridine). The results indicate that this reaction follows first-order kinetics.

[0135] FIG. 26 includes a series of a,P-unsaturated compounds that were tested for their ability to react with pseudouridine.

[0136] FIG. 27 is a graph depicting reactivity of compounds from FIG. 35. For each compound, the five data points correspond to conversion ratio for reaction between the compound and pseudouridine, cytosine, uracil, adenine, and guanine. Reactions were performed at a temperature of 37 °C in TEAB buffer at pH 8.5 for 2 hours. Compound 8 has high reactivity and selectivity with pseudouridine. Uracil and guanine have lower conversion rates. Converted uracil does not cause mutations, while converted guanine can lead to some mutations or deletions.

[0137] FIG. 28 is a diagram depicting the mechanism of reaction between pseudouridine and bromo methylacrylate and base pairing of unreacted and reacted pseudouridine with guanine. Pseudouridine is be converted to pseudouridine hydroxylactone, which base pairs with guanine (rather than adenine) and thus be read as a thymine to cytosine mutation (L).

[0138] FIG. 29 includes a series of graphs that depict mutation rates between various a, P- unsaturated compounds and nucleosides. Compound 8 has high reactivity and selectivity with pseudouridine.

[0139] FIGS. 30A-30B Fragment analysis of untreated (FIG. 30A) and treated (FIG. 30B) RNA shows that reaction causes minimal RNA degradation. Treatment conditions: compound 8, TEAB buffer (pH =8.5), 70 °C for 2 min, then 37 °C for 3h.

[0140] FIG. 31 is a workflow diagram of a first library preparation method. The workflow was designed by first ligating the 3'-end adaptor. Then, the samples were split into two groups: one for chemical treatment and the other untreated. Both groups undergo reverse transcription, cDNA ligation, and PCR amplification simultaneously.

[0141] FIG. 32 is a workflow diagram of a second library preparation method. The samples are initially split into two groups: one for chemical treatment and the other untreated. Both groups were subjected to reverse transcription (using random primers), second-strand synthesis, dsDNA ligation, and PCR amplification simultaneously.

[0142] FIG. 33 is a heat map of results obtained by applying the first workflow to 84-mer RNA oligos containing the NN'PNN motif. The results demonstrate that the workflow method achieves a high mutation rate at pseudouridine with minimal motif bias.

[0143] FIG. 34 Assay reactivity and selectivity of a,P-unsaturated compounds via T vs U sites on rRNA. The first workflow described in FIG. 41 was applied to HEK 293 T total RNA using T modifications on rRNA. Compound 8 showed the highest mutation ratios on the biological samples.

[0144] FIG. 35 Assay reactivity and selectivity of buffer pH via vs U sites on rRNA. A pH value of 9.08 resulted in the highest mutation ratio.

[0145] FIG. 36 Assay reactivity and selectivity of buffer with NH4 via T vs U sites on rRNA. NH4+ was determined to function as a catalyst for reaction between a,P-unsaturated compounds and pseudouridine.

[0146] FIG. 37 Assay reactivity and selectivity of buffer concentration via T vs U sites on rRNA. Different TEAB buffer concentrations were examined, as excessive reactivity can lead to overreaction and cause side reactions, reducing selectivity. A TEAB buffer concentration of 312.5 mM yielded highest mutation ratios.

[0147] FIGS. 38A-38B Assay reactivity and selectivity of reaction time via T vs U sites on rRNA. FIG. 38A Reaction between compound 8 and pseudouridine was examined at different reaction times. FIG. 38A Reaction between compound 2 and pseudouridine was examined at different reaction times.

[0148] FIG. 39 Assay reactivity and selectivity of reaction temperature via vs U sites on rRNA. Reaction between compound 8 and pseudouridine was examined at different reaction temperatures. Reaction temperatures of 37 °C and 50 °C yielded higher mutation ratios than a reaction temperature of 25 °C.

[0149] FIG. 40 Assay reactivity and selectivity of preheating via T vs U sites on rRNA. Preheating denatures RNA and accelerates reaction between a,P-unsaturated compounds and pseudouridine. Left - no pre-heating. Middle - preheating at 70 °C for 2 minutes. Right - preheating at 85 °C for 2 minutes.

[0150] FIG. 41 is a graph comparing insert RNA length using compounds 8 and 13 and the specified reaction conditions. The results demonstrate that the first workflow described in FIG. 41 resulted in insertion of linger RNA sequences.

[0151] FIG. 42 Assay reactivity and selectivity.

[0152] FIGS 43A-43B. FIG. 43A Ratio of T modification. FIG. 43B Average ratio.DETAILED DESCRIPTION

[0153] Aspects of the present disclosure relate to compositions, methods, and kits for detection and analysis of pseudouridine in RNA. Certain aspects are directed to compositions for treatment of RNA comprising pseudouridine. Also disclosed, in some aspects, are methods for treatment of RNA comprising pseudouridine including methods comprising incubation at moderate pHs (e.g., less than about 9) and at moderate temperatures (e.g., less than about 85 °C) using the disclosed a pseudouridine labeling molecule solutions. Kits including the disclosed compositions are also described herein, along with instructions for analysis of RNAcomprising . Aspects of the disclosure provide sequencing methods comprising rapid treatment, low background noise, and high sensitivity, enabling highly accurate sequencing of pseudouridine in RNA, starting from low-input biological RNA.

[0154] Aspects of the disclosure relate to a sequencing method comprising an intramolecular cyclization step which can lead to T-to-C mutations as a readout of pseudouridine sites. In some embodiments, the reactivity of the alkene can be increased by introducing electron-withdrawing groups. In some embodiments, the use of dual electronwithdrawing groups are used to aid in cyclization and mutation detection. Certain groups may also have the potential to form a six-membered ring through the alcoholysis of the cyano group. The formation of a six -membered ring requires less energy and speed up the reaction under milder conditions.Overview

[0155] Pseudouridine (T) is one of the most prevalent RNA modifications in diverse RNA families and across numerous species, yet its precise distribution and biological roles remain insufficiently understood. This knowledge gap largely stems from the lack of highly efficient detection methods, which have been challenged by the difficulty of developing a selective reaction for T. In some embodiments, this disclosure provides a tandem reaction for pseudouridine, which utilizes a unique intramolecular transesterification reaction, allowing for highly selective and efficient detection under mild conditions and overcome the limitation of previous methods. By detecting the incorporated base as a mutation upon reverse transcription, the inventors developed a system herein referred to as PUM-seq (PseudoUridine Mutationbased sequencing) for mapping T sites across the transcriptome at single-nucleotide resolution. PUM-seq efficiently can detect all known T sites in human rRNA, tRNA, mRNA and all other RNA families with high sensitivity. PUM-seq can also be applied to transcriptomes from diverse species and tissues, uncovering a conserved and widespread landscape of pseudouridine modification across evolutionary lineages. This approach provides a robust and unbiased platform for future studies on T modifications and their biological significance, particularly in systematically quantifying T stoichiometry across multiple samples.

[0156] Pseudouridine was the first modified ribonucleotide discovered over half a century ago1, and has since been identified in most RNA species, including rRNA, tRNA, snRNA, miRNA and mRNA2 4. In cells, T is enzymatically synthesized via the post-transcriptional isomerization of uridine by either distinct families of pseudouridine synthases (PUSs)5or a snoRNA-guided process6, and its deposition is believed to be dynamic across cell types. Thepresence of T modification has been detected across all three domains of life: eukaryotes, prokaryotes, archaea7, and even some viruses. This widespread distribution in different RNAs, cell types, and species, suggests ancient and fundamental roles. With the rapid advancements in the functional characterization of various samples, there is a growing demand for more sensitive and reliable methods to detect T. While several high-throughput techniques exist, a highly efficient, unbiased method that can accurately quantify modification levels across the transcriptome is still needed.

[0157] The fundamental principle of T detection is to transform the modification signal into a genetic readout, with mutations serving as the optimal signal for high-throughput sequencing. This strategy presents two major chemical challenges: selectively labeling T sites and converting the resulting adduct into a mutational signal. Early approaches, including Pseudo-seq8and CeU-seq9employed N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CMC) chemistry to label T sites. However, these adducts could not be converted into mutational signals and instead produced reverse transcription (RT) stop signatures. Subsequent methods, such as BID-seq4and PRAISE10, adopted bisulfite-based strategies11 15that convert T into an open-ring structure, yielding deletion signals that provide quantitative information on modification levels. Nonetheless, deletion-based methods fail to accurately detect T sites within U-rich regions. More recently, the first mutation-based method, BACS16was introduced. This approach employs an a,P-unsaturated amide to label T sites, after which the resulting adduct undergoes intramolecular (9-alkylation. The final product pairs with guanine during RT, generating a T-to-C mutation signal in sequencing data. This strategy has enabled more precise T detection. Despite these advances, all existing methods rely on harsh conditions, such as elevated temperatures, which introduce challenges including RNA degradation and reduced chemical selectivity, ultimately limiting detection accuracy.

[0158] Leveraging the high selectivity of pseudouridine cyanoethylation17,18together with a previously unexploited intramolecular transesterification reaction, PUM-seq, a mutationbased T detection strategy, overcomes the limitations of existing approaches. This approach enables highly sensitive and accurate T detection across the transcriptome under mild conditions, a significant advantage over methods requiring harsh treatments. At the chemical level, cyanoethylation provides site-specific activation of T, while the subsequent transesterification reaction induces a polarity switch in hydrogen-bonding capacity, thereby converting T into a mutagenic analog during reverse transcription (in some aspects). This tandem mechanism establishes a chemical pathway that couples selective T labeling with efficient mutational readout, creating the basis for a transcriptome-wide sequencing strategy.I. RNA Processing Methods

[0159] Aspects of the present disclosure relate to compositions and methods for RNA processing to, for example, facilitate detection of T. Particular aspects relate to compositions comprising a pseudouridine labeling molecule and a buffer and methods for use of such compositions in treatment of RNA. Accordingly, disclosed herein, in some aspects, are methods for RNA processing comprising incubating a solution comprising an RNA molecule and a T labeling molecule under conditions sufficient to modify pseudouridine. Modifications of T can include Micheal addition (e.g., aza-Michael addition), aromatization, intramolecular cyclization, hydrolysis, and / or the like. As disclosed herein, incubating one or more RNA molecules in a pseudouridine labeling molecule solution of the disclosure under appropriate conditions results in extremely rapid modification pseudouridine of with low RNA degradation, leading to identification of pseudouridine with very low false positive rate. In some aspects, methods disclosed herein result in a reduced level of background noise (e.g., unconverted pseudouridine) relative to canonical treatments.

[0160] In some aspects, RNA processing methods of the disclosure include incubating one or more RNA molecules in a pseudouridine labeling molecule solution, where the a pseudouridine labeling molecule solution comprises a pseudouridine labeling molecule. The reaction can include 1, 2, 3, 4, 5, or more intermediate molecules and an end product that is detectable and / or quantifiable by standard methods in the art. The intermediate molecules can relate to Michael addition (e.g. Aza-Michael addition), aromatization, intramolecular cyclization, and / or hydrolysis. An exemplary (i.e., nonlimiting) reaction mechanism is depicted below:

[0161] The pseudouridine labeling molecule comprises an electron-withdrawing group.

[0162] The pseudouridine labeling molecule can be of the formula:whereinR1 is the electron-withdrawing group;R2 is hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; andR3 and R4 are each independently H or C1-C3 alkyl.

[0163] Electron-withdrawing groups can be, but are not limited to, nitrile, nitro, ester, carboxylic acid, ester, amide, ketone, aldehyde, sulfone, sulfonic acid, thioester, thioate, and / or the like. The pseudouridine labeling molecule of claim 1.3, wherein the electron-withdrawing group can be substituted with a linking group. The linking group, can be, but is not limited to azide, alkyne, thiol, amine, maleimide, N-hydroxysuccinimide ester, tetrazine, transcyclooctene and / or the like. The pseudouridine labeling molecule can also include a linker between the linking group and the pseudouridine labeling molecule. Examples of linkers can be, but are not limited to polyethylene glycols (PEGs), polyglycerols, polysarcosines, polyoxazolines, peptide linkers, alkyl linkers, and / or the like.

[0164] Exemplary T labeling molecules include, but are not limited to:

[0165] In certain aspects, the method does not include any of the following intermediate products and / or end product in the following:

[0166] In certain aspects, a solution suspected of comprising pseudouridine can be incubated with a buffer. Examples of buffers suitable for use can include, but are not limited to a Tris, phosphate, triethylammonium bicarbonate (TEAB), HEPES, NasBCh, sodium cacodylate, triethylammonium acetate (TEAA), ethylenediaminetetraacetic acid (EDTA) buffer, and / or the like. In certain aspects, the buffer is TEAB. The buffer can be used at 0. IM, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1.0M, 1.5M, 2.0M, 2.5M, 3.0M, or any derivable range or value in between. The pH of the buffer can be a pH of 1.0 to 13.0; for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5 or 13. In certain aspects, the reaction pH is 7.5-8.5. In some aspects, the pH of the buffer is 8. In some aspects, the reaction time can be between 0.5 and 4 hours; for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4 hours. In some aspects, the temperature of the reaction can be equal to or less than a temperature in the range of 25°C -80°C; for example, 25, 30, 37, 40, 45, 50, 55, 60, 65, 70, 75, or 80°C. In some aspects, the temperature of the reaction is allowed to cool, to, for example, 37, 40, 45, 50, 55, or 60 C if used at a high temperature. For example, the reaction is allowed to cool for at least 1, 2 or 3 hrs. In some aspects, the method can include a step of incubating the mixture in a first step at in a first temperature and first period of time and a second step at a second temperature at a second period of time (for example in a preheating step). In some aspects, the first temperature is higher than the second temperature. In some aspects, the first period of time is shorter than the second period of time. In some aspects, the reaction is at 70 °C for 2min, followed by 37 °C for 3h. In some aspects, the reaction is at 37C for 3 hours. The pseudouridine labeling molecule of the composition, methods, or kits described herein can be used at various concentrations, for example, at 10 mM - 1000 mM, and any value or derivable range in between, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360,365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455,460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550,555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645,650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740,745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835,840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930,935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or lOOOmM, or any derivable range in between.

[0167] The concentration of the buffer of the composition, methods, or kits described herein can be, for example, between 10 - 1000 mM, and any value or derivable range in between, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290,295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480,485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575,580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955,960, 965, 970, 975, 980, 985, 990, 995, or 1000 mM, or any derivable range in between.

[0168] The incubation time of any of the methods disclosed herein can be between 2 - 240 min, or any derivable range in between; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 min, or any derivable range in between.

[0169] The incubation temperature of any of the methods disclosed herein can be equal to or less than a temperature between 20 - 85°C, or any derivable range in between; for example, less than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 °C.

[0170] The pH of the composition, method or kit can be equal to or less than a pH between 3 - 10; for example equal to or less than about 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3, or any derivable range in between.II. Modification and Detection of Pseudouridine

[0171] Aspects of the present disclosure are directed to methods for modification of pseudouridine. In some embodiments, such methods comprise incubating a pseudouridine, an RNA molecule comprising a pseudouridine, and / or a population of RNA molecules comprising pseudouridines with a pseudouridine labeling molecule under conditions sufficient to modify the pseudouridine. As used herein, a “modified pseudouridine” or “a modified ” describes a pseudouridine that has been chemically modified, e.g., by addition or removal of a chemical moiety. In some embodiments, a modified pseudouridine is generated by addition of a chemical moiety to a pseudouridine. In some embodiments, a modified pseudouridine is generated by treatment with a pseudouridine labeling molecule. In some embodiments, the modified pseudouridine is pseudouridine hydroxylactone, or the like. In some embodiments, a modified pseudouridine is generated by treatment with methyl a-bromoacrylate and the modified pseudouridine is pseudouridine hydroxylactone. In some aspects, the modified pseudouridine capable of being detected and / or quantified (the modified pseudouridine is detectable and / or quantifiable). The term “detectable” means the ability to be detected and quantified by standard methods in the art. The term “quantifiable” means the ability to be quantified by standard methods in the art. As used herein the terms “modified pseudouridine” and “labeled pseudouridine” can be used interchangeably.

[0172] Further aspects of the disclosure are directed to methods for detection and / or quantification of pseudouridine in RNA. In some embodiments, the disclosed methods comprise generating a modified pseudouridine in an RNA molecule, followed by subjecting the RNA molecule to reverse transcription and / or cDNA ligation-based library construction. As disclosed herein, reverse transcription of an RNA molecule comprising a modified pseudouridine may result in a deletion in the resulting DNA molecule. The deletion may be a one nucleotide deletion, where the one nucleotide corresponds to the pseudouridine in the original RNA molecule. The deletion may be a deletion of two or more nucleotides, where the deletion corresponds to the pseudouridine in the original RNA molecule plus one or more nucleotides adjacent to the pseudouridine. Pseudouridine sites are read as mutations in the sequencing data. In some embodiments, T-to-C mutations are used as a readout of pseudouridine sites.III. General Assay Methods1. LC-MS / MS

[0173] Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) is an high-sensitivity approach to HPLC-UV, which requires much smaller quantities of the hydrolyzed DNA sample. In the case of mammalian DNA, of which ~2%-5% of all cytosine residues are methylated, LC-MS / MS has been validated for detecting levels of methylation levels ranging from 0.05%-10%, and it can confidently detect differences between samples as small as -0.25% of the total cytosine residues, which corresponds to -5% differences in global DNA methylation. The procedure routinely requires 50-100 ng of DNA sample, although much smaller amounts (as low as 5 ng) have been successfully profiled.2. MALDI TOF-Based Methods

[0174] MALDI mass spectrometry can be attractive when a time-of-flight (TOF) configuration is used as a mass analyzer (see, Hillenkamp et al. (1990) pp 49-60 in “Matrix Assisted UV-Laser Desorption / Ionization: A New Approach to Mass Spectrometry of Large Biomolecules,” Biological Mass Spectrometry, Burlingame and McCloskey, editors, Elsevier Science Publishers, Amsterdam). In most cases, MALDLTOF does not produce multiple molecular ion peaks, and the mass spectra, in principle, look simpler compared to other types mass spectrometry. MALDI TOF MS can be used for determining oligonucleotide identity (e.g., cytosine to cytosine-bi sulfite conversion, or pseudouridine to modified pseudouridine).B. Sequencing1. DNA SequencingIn some aspects, DNA may be analyzed by sequencing. The DNA may be prepared for sequencing by any method known in the art, such as library preparation, hybrid capture, sample quality control, product-utilized ligation-based library preparation, or a combination thereof. The DNA may be prepared for any sequencing technique. In some aspects, a unique genetic readout for each sample may be generated by genotyping one or more highly polymorphic SNPs. In some aspects, sequencing, such as base pair and / or paired-end sequencing, may be performed to cover approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater percentage of target oligonucleotides at, or at more than 20*, 25*, 30*, 35*, 40*, 45*, 50*, or greater than 50* coverage (or any range derivable therein). In certain aspects, mutations, SNPS, INDELS, copynumber alterations (somatic and / or germline), or other genetic differences may be identified from the sequencing using at least one bioinformatics tool, including but not limited to, VarScan2, any R package (including CopywriteR) and / or Annovar.2. RNA Sequencing

[0175] In some aspects, RNA may be analyzed by sequencing. The RNA may be prepared for sequencing by any method known in the art, such as but not limited to, poly-A selection, cDNA synthesis, stranded or nonstranded library preparation, or a combination thereof. The RNA may be prepared for any type of RNA sequencing technique, including but not limited to, stranded specific RNA sequencing. In some aspects, sequencing may be performed to generate approximately 10M, 15M, 20M, 25M, 30M, 35M, 40M or more reads, including paired reads. In some aspects, the sequencing may be performed at a read length of at least, at most, approximately, or exactly 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 105 bp, 110 bp, or longer (or any range derivable therein). In some aspects, raw sequencing data may be converted to estimated read counts (RSEM), fragments per kilobase of transcript per million mapped reads (FPKM), and / or reads per kilobase of transcript per million mapped reads (RPKM).3. Example Sequencing Methods

[0176] DNA (including bisulfite-converted DNA) and / or RNA (including modified RNA) may be used for amplification of one or more regions of interest followed by sequencing. Accordingly, aspects of the disclosure may include sequencing nucleic acids to detect and / or quantify methylation of nucleic acids biomarkers. In some aspects, the methods of the disclosure include a sequencing method. Sequencing may be excluded from certain methods of the disclosure. Example sequencing methods include, but are not limited to, those described below.

[0001]

[0002] Further applicable details may be found in U.S. Provisional Application Serial No. 63 / 694,681 filed September 13, 2024 and PCT Application No. PCT / US2025 / 046137 filed September 12, 2025 entitled “METHODS AND COMPOSITIONS FOR COOPERATIVE CATALYSIS ASSISTED SELECTIVE NUCLEIC ACID DEAMINATION,” all of which are incorporated by reference in their entireties herein. a. Massively parallel signature sequencing (MPSS).

[0177] The first of the next-generation sequencing technologies, massively parallel signature sequencing (or MPSS), was developed in the 1990s at Lynx Therapeutics. MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapterdecoding, reading the sequence in increments of four nucleotides. This method made it susceptible to sequence-specific bias or loss of specific sequences. b. Polony sequencing.

[0178] The Polony sequencing method, developed in the laboratory of George M. Church at Harvard, was among the first next-generation sequencing systems and was used to sequence a full genome in 2005. It combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E coli genome at an accuracy of >99.9999% and a cost approximately 1 / 9 that of Sanger sequencing. c. 454 pyrosequencing™.

[0179] A parallelized version of pyrosequencing was developed by 454 Life Sciences™, which has since been acquired by Roche Diagnostics™. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picoliter-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD™ on the other. d. Illumina™ (Solexa) sequencing.

[0180] Solexa developed a sequencing method based on reversible dye-terminators technology, and engineered polymerases, that it developed internally. The terminated chemistry was developed internally at Solexa and the concept of the Solexa system was invented by Balasubramanian and Klennerman from Cambridge University's chemistry department. In 2004, Solexa acquired the company Manteia Predictive Medicine in order to gain a massively parallel sequencing technology based on "DNA Clusters", which involves the clonal amplification of DNA on a surface. The cluster technology was co-acquired with Lynx Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.

[0181] In this method, DNA molecules and primers are first attached on a slide and amplified with polymerase so that local clonal DNA colonies, later coined "DNA clusters", are formed. To determine the sequence, four types of reversible terminator bases (RT -bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides, then the dye, along with the terminal 3' blocker, is chemicallyremoved from the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.

[0182] Decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity. With an optimal configuration, the ultimately reachable instrument throughput is thus dictated solely by the analog-to-digital conversion rate of the camera, multiplied by the number of cameras and divided by the number of pixels per DNA colony required for visualizing them optimally (approximately 10 pixels / colony). In 2012, with cameras operating at more than 10 MHz A / D conversion rates and available optics, fluidics and enzymatics, throughput can be multiples of 1 million nucleotides / second, corresponding roughly to one human genome equivalent at 1 * coverage per hour per instrument, and one human genome re-sequenced (at approx. 30*) per day per instrument (equipped with a single camera). e. SOLiD™ sequencing.

[0183] SOLiD™ technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina™ sequencing. f. Ion Torrent™ semiconductor sequencing.

[0184] Ion Torrent™ Systems Inc. developed a system based on using standard sequencing chemistry, but with a novel, semiconductor-based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in asingle cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. g. DNA Nanoballs™ sequencing.

[0185] DNA Nanoballs™ sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The company Complete Genomics® uses this technology to sequence samples submitted by independent researchers. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run and at low reagent costs compared to other next generation sequencing platforms. However, only short sequences of DNA are determined from each DNA nanoball which can make mapping the short reads to a reference genome difficult. This technology has been used for multiple genome sequencing projects. h. Heliscope single molecule sequencing.

[0186] Heliscope sequencing is a method of single-molecule sequencing developed by Helicos Biosciences. It uses DNA fragments with added poly-A tail adapters which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Heliscope sequencer. The reads are short, up to 55 bases per run, but recent improvements allow for more accurate reads of stretches of one type of nucleotides. This sequencing method and equipment were used to sequence the genome of the M13 bacteriophage. i. Single molecule real time (SMRT) sequencing.

[0187] SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMRT technology developer, this methodology allows detection of nucleotidemodifications. This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases.C. Additional Assay Methods

[0188] In some aspects, methods involve amplifying and / or sequencing one or more target genomic regions using at least one pair of primers specific to the target genomic regions. In certain aspects, the primers are heptamers. In certain aspects, enzymes are added such as primases or primase / polymerase combination enzyme to the amplification step to synthesize primers.

[0189] In some aspects, arrays can be used to detect nucleic acids of the disclosure. An array comprises a solid support with nucleic acid probes attached to the support. Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodorc / ., 1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is used in certain aspects, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.

[0190] In addition to the use of arrays and microarrays, it is contemplated that a number of difference assays could be employed to analyze nucleic acids. Such assays include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, digital PCR, ddPCR (droplet digital PCR), nCounter® (nanoString®), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged- Amplicon deep sequencing), PAP (Pyrophosphorolysis-activation polymerization), next generation RNA sequencing, northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and / or Bridge Litigation Assay (Genaco).

[0191] Amplification primers or hybridization probes can be prepared to be complementary to a genomic region, biomarker, probe, or oligo described herein. The term "primer" as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process and / or pairing with a single strand of an oligo of the disclosure, or portion thereof. Typically, primers are oligonucleotides from ten to twenty and / or thirty nucleic acids in length, but longer sequences can be employed. Primers may be provided in double-stranded and / or single-stranded form, although the singlestranded form is preferred.

[0192] The use of a primer of between 13 and 100 nucleotides, particularly between 17 and 100 nucleotides in length, or in some aspects up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length may be used to increase stability and / or selectivity of the hybrid molecules obtained. One may design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

[0193] In some aspects, each probe / primer comprises at least 15 nucleotides. For instance, each probe can comprise at least, at most, approximately, or exactly 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). They may have these lengths and have a sequence that is identical or complementary to a gene described herein. Particularly, each probe / primer has relatively high sequence complexity and does not have any ambiguous residue (undetermined "n" residues). The probes / primers can hybridize to the target gene, including its RNA transcripts, under stringent or highly stringent conditions. It is contemplated that probes or primers may have inosine or other design implementations that accommodate recognition of more than one human sequence for a particular biomarker.

[0194] For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50 °C to about 70 °C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. Itis generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0195] In some aspects, quantitative RT-PCR (such as but not limited to TaqMan™, AB I) is used for detecting and comparing the levels or abundance of nucleic acids in samples. The concentration of the target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before the PCR was begun. By determining the concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. This direct proportionality between the concentration of the PCR products and the relative abundances in the starting material is true in the linear range portion of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the sampling and quantifying of the amplified PCR products may be carried out when the PCR reactions are in the linear portion of their curves. In addition, relative concentrations of the amplifiable DNAs may be normalized to some independent standard / control, which may be based on either internally existing DNA species or externally introduced DNA species. The abundance of a particular DNA species may also be determined relative to the average abundance of all DNA species in the sample.

[0196] In some aspects, the PCR amplification utilizes one or more internal PCR standards. The internal standard may be an abundant housekeeping gene in the cell or it can specifically be GAPDH, GUSB and P-2 microglobulin. These standards may be used to normalize expression levels so that the expression levels of different gene products can be compared directly. A person of ordinary skill in the art would know how to use an internal standard to normalize expression levels.

[0197] A problem inherent in some samples is that they are of variable quantity and / or quality. This problem can be overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable DNA fragment that is similar or larger than the target DNA fragment and in which the abundance of the DNA representing the internal standard is roughly 5-100 fold higher than the DNA representing the target nucleic acid region.

[0198] In some aspects, the relative quantitative RT-PCR uses an external standard protocol. Under this protocol, the PCR products are sampled in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling can beempirically determined for each target DNA fragment. In addition, the nucleic acids isolated from the various samples can be normalized for equal concentrations of amplifiable DNAs.

[0199] A nucleic acid array can comprise at least, at most, approximately, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes, which may hybridize to different and / or the same biomarkers. Multiple probes for the same gene can be used on a single nucleic acid array. Probes for other disease genes can also be included in the nucleic acid array. The probe density on the array can be in any range. In some aspects, the density may be at least, at most, approximately, or exactly 50, 100, 200, 300, 400, 500 or more probes / cm2(or any range derivable therein).

[0200] Specifically contemplated are chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization (see also, Pease et al., 1994; and Fodor et al, 1991). It is contemplated that this technology may be used in conjunction with evaluating the expression level of one or more cancer biomarkers with respect to diagnostic, prognostic, and treatment methods.

[0201] Certain aspects may involve the use of arrays or data generated from an array. Data may be readily available. Moreover, an array may be prepared in order to generate data that may then be used in correlation studies.

[0202] In some embodiments, the conversion rate of T to -BS and C-to-U conversion rates can be quantified by comparing the product yield, e.g., MS peak, with the starting material yield, e.g., MS peak. In some embodiments, the MS peak height of the product is divided by the height of the starting material MS peak to quantify the conversion rate.IV. Methods of UseA. Clinical and diagnostic applications

[0203] The methods of the disclosure may be useful for evaluating DNA and / or RNA for clinical and / or diagnostic purposes. Certain aspects relate to methods for evaluating DNA. Certain aspects relate to methods for evaluating RNA. Certain aspects relate to a method for evaluating a sample comprising DNA molecules and / or RNA molecules. The evaluation may be the detection or determination of a particular pseudouridine modification or the differential detection or determination of a particular modification.

[0204] The sample may be from a biopsy such as from fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In certain aspects, the sample is obtained from a biopsy from cancerous tissue by any of the biopsy methods previously mentioned. In certain aspects, the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm. In certain aspects, the cyst, tumor or neoplasm is colorectal. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, in certain aspects the biological sample can be obtained without the assistance of a medical professional.

[0205] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. In some aspects, the sample comprises cell-free DNA. In some aspects, the sample comprises a fertilized egg, a zygote, a blastocyst, or a blastomere. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[0206] In some aspects, the methods of the disclosure can be used in the discovery of novel biomarkers for a disease or condition. In some aspects, the methods of the disclosure can performed on a sample from a patient to provide a prognosis for a certain disease or condition in the patient. In some aspects, the methods of the disclosure can be performed on a sample from a patient to predict the patient’s response to a particular therapy. In some aspects, the disease comprises a cancer. For example, the cancer may be pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumorsbrain tumor, visual pathway and hypothalamic glioma, breast cancer, lymphoid cancer, bronchial adenomas / carcinoids, tracheal cancer, Burkitt lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal carcinoma of unknown primary, central nervous system lymphoma, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's, childhood extragonadal Germ cell tumor, extrahepatic bile duct cancer, eye Cancer, intraocular melanoma eye Cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, glioma of the brain stem, glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer, leukemia, acute lymphoblastic (also called acute lymphocytic leukemia) leukemia, acute myeloid (also called acute myelogenous leukemia) leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia) leukemia, chronic myelogenous (also called chronic myeloid leukemia) leukemia, hairy cell lip and oral cavity cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphomas, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, Non-Hodgkin (an old classification of all lymphomas except Hodgkin's) lymphoma, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, childhood medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant, fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial- stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreaticcancer, islet cell paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, childhood Salivary gland cancer Sarcoma, Ewing family of tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sezary syndrome sarcoma, skin cancer (nonmelanoma), skin cancer (melanoma), skin carcinoma, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, metastatic stomach cancer, supratentorial primitive neuroectodermal tumor, childhood T-cell lymphoma, testicular cancer, throat cancer, thymoma, childhood thymoma, thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, childhood vulvar cancer, and wilms tumor (kidney cancer).

[0207] In some aspects, the cancer comprises ovarian, prostate, colon, or lung cancer. In some aspects, the method is for determining novel biomarkers for ovarian, prostate, colon, or lung cancer by evaluating RNA using methods of the disclosure. In some embodiments, the methods of the disclosure may be used on fetal RNA isolated from a pregnant female. In some aspects, the methods of the disclosure may be used for prenatal diagnostics using fetal RNA isolated from a pregnant female. In some aspects, the methods of the disclosure may be used for the evaluation of a fertilized embryo, such as a zygote or a blastocyst for the determination of embryo quality or for the presence or absence of a particular disease marker.

[0208] In some aspects, methods disclosed herein are performed on RNA that is at a low input concentration. In some aspects, a low input RNA concentration is at least, at most, approximately, or exactly 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15 nanograms, or any range derivable therein. In some aspects, a low input RNA concentration is at least, at most, approximately, or exactly 1 to 10 ng, 5 to 10 ng, 10 to 50 ng, 10 to 100, or 100 to 200 ng total RNA. In some aspects, a low input concentration of RNA is obtained from about or less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 cells.V. Sample Preparation

[0209] In certain aspects, methods involve obtaining a sample (also “biological sample”) from a subject. The methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, liquid biopsy, or skin biopsy. In certain aspects the sample is obtained from a biopsy from tissue by any of the biopsy methods previously mentioned. In certain aspects the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0210] A biological sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. In some aspects, a biological sample comprises extracellular vesicles such as exosomes. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. A biological sample may be a cell-free sample. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, cerebrospinal fluid collection, urine collection, feces collection, collection of menses, tears, or semen.

[0211] The sample may be obtained by methods known in the art. In certain aspects the samples are obtained by biopsy. In certain aspects the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue and one or more samples from another specimen (for example serum) may be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type and one or more samples from another specimen (e.g. serum) may be obtained at the same or different times. Samples may be obtained at different times are stored and / oranalyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[0212] In some aspects, the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0213] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, endoscopy, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some aspects, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0214] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In some aspects, the sample is a fine needle aspirate of a tissue or a suspected tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[0215] In some aspects of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0216] In some aspects of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling servicesare included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and / or adequacy are provided.

[0217] In some aspects, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample.VI. Chemical Definitions

[0218] The term “alkyl” includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), heteroatom -unsubstituted alkyl, heteroatom-substituted alkyl, heteroatom- unsubstituted Cn-alkyl, and heteroatom-substituted Cn-alkyl. In certain embodiments, lower alkyls are contemplated. The term “lower alkyl” refers to alkyls of 1-6 carbon atoms (that is, 1, 2, 3, 4, 5 or 6 carbon atoms). The term “heteroatom-unsubstituted Cn-alkyl” refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted Ci- Cio-alkyl has 1 to 10 carbon atoms. The groups, — CH3 (Me), — CH2CH3 (Et), — CH2CH2CH3 (n-Pr), — CH(CH3)2(iso-Pr), — CH(CH2)2(cyclopropyl), — CH2CH2CH2CH3 (n-Bu), — CH(CH3)CH2CH3 (sec-butyl), — CH2CH(CH3)2 (iso-butyl), — C(CH3)3 (tent-butyl), — CH2C(CH3)3 (neo-pentyl), cyclobutyl, cyclopentyl, and cyclohexyl, are all non-limiting examples of heteroatom -unsubstituted alkyl groups. The term “heteroatom-substituted Cn- alkyl” refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom- substituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The following groups are all non-limiting examples of heteroatom-substituted alkyl groups: trifluoromethyl, — CH2F, — CH2CI, — CH2Br, piperidinyl, — CH2OH, — CH2OCH3, — CH2OCH2CF3, — CH2OC(O)CH3, —CH2NH2, — CH2NHCH3, — CH2N(CH3)2, — CH2CH2CI, — CH2CH2OH,CH2CH2OC(O)CH3, — CH2CH2NHCO2C(CH3)3, and — CH2Si(CH3)3.

[0219] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, and thiol. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0220] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

[0221] The claimed invention is also intended to encompass salts of any of the compounds of the present invention. The term “salt(s)” as used herein, is understood as being acidic and / or basic salts formed with inorganic and / or organic acids and bases. Zwitterions (internal or inner salts) are understood as being included within the term “salt(s)” as used herein, as are quaternary ammonium salts such as alkylammonium salts. Nontoxic, pharmaceutically acceptable salts are preferred, although other salts may be useful, as for example in isolation or purification steps during synthesis. Salts include, but are not limited to, sodium, lithium, potassium, amines, tartrates, citrates, hydrohalides, phosphates and the like. A salt may be a pharmaceutically acceptable salt, for example. Thus, pharmaceutically acceptable salts of compounds of the present invention are contemplated.

[0222] Compounds employed in methods of the invention may contain one or more asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and allgeometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S- or the R-configuration, as defined by the IUPAC 1974 Recommendations. Compounds may be of the D- or L-form, for example. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic form, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers.

[0223] In addition, atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C.VII. Kits

[0224] Also disclosed herein are kits, which may be useful for performing the methods of the disclosure. The contents of a kit can include one or more reagents described throughout the disclosure and / or one or more reagents known in the art for performing one or more steps described throughout the disclosure, a solution comprising a pseudouridine labeling molecule, a buffer, nuclease-free water, one or more primers, polyethylene glycol, magnetic beads, DNA polymerase, taq polymerase, DNA ligase, RNA ligase, a reverse transcriptase, dNTPs, DNA polymerase buffer, RNA polymerase, DTT, redox reagent, Mg2+, K+, adaptors, DNA adaptors, DNA comprising an RNA promoter, a protease, an alkaline solution, a sodium hydroxide solution, and NTPs. Any one or more of the preceding components may be excluded from a kit in certain aspects of the present disclosure.

[0225] One or more reagent is preferably supplied in a solid form or liquid buffer that is suitable for inventory storage, and later for addition into the reaction medium when the method of using the reagent is performed. Suitable packaging is provided. The kit may provide additional components that are useful in the procedure. These additional components may include buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.

[0226] Any components of a kit described herein may be used in a method disclosed herein. Further, components described in the context of a disclosed method may be provided in a kit of the present disclosure.Examples

[0227] The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute certain modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1

[0228] The Examples provides a tandem reaction for pseudouridine, which utilizes a unique intramolecular transesterification reaction, allowing for highly selective and efficient detection under mild conditions and overcome the limitation of previous methods. By detecting the incorporated base as a mutation upon reverse transcription, the inventors developed a system herein referred to as PUM-seq (PseudoUridine Mutation-based sequencing) for mapping T sites across the transcriptome at single-nucleotide resolution. PUM-seq efficiently detected all known T sites in human rRNA, tRNA, mRNA and all other RNA families with high sensitivity. PUM-seq can also be applied to transcriptomes from diverse species and tissues, uncovering a conserved and widespread landscape of pseudouridine modification across evolutionary lineages. This approach provides a robust and unbiased platform for future studies on T modifications and their biological significance, particularly in systematically quantifying T stoichiometry across multiple samples.

[0229] Pseudouridine was the first modified ribonucleotide discovered over half a century ago1, and has since been indentified in most RNA species, including rRNA, tRNA, snRNA, miRNA and mRNA2 4. In cells, T is enzymatically synthesized via the post-transcriptional isomerization of uridine by either distinct families of pseudouridine synthases (PUSs)5or a snoRNA-guided process6, and its deposition is believed to be dynamic across cell types. The presence of T modification has been detected across all three domains of life: eukaryotes, prokaryotes, archaea7, and even some viruses. This widespread distribution in different RNAs, cell types, and species, suggests ancient and fundamental roles. With the rapid advancementsin the functional characterization of various samples, there is a growing demand for more sensitive and reliable methods to detect T. While several high-throughput techniques exist, a highly efficient, unbiased method that can accurately quantify modification levels across the transcriptome is still needed.

[0230] The fundamental principle of T detection is to transform the modification signal into a genetic readout, with mutations serving as the optimal signal for high-throughput sequencing. This strategy presents two major chemical challenges: selectively labeling T sites and converting the resulting adduct into a mutational signal. Early approaches, including Pseudo-seq8and CeU-seq9employed N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate (CMC) chemistry to label T sites. However, these adducts could not be converted into mutational signals and instead produced reverse transcription (RT) stop signatures. Subsequent methods, such as BID-seq4and PRAISE10, adopted bisulfite-based strategies11 15that convert T into an open-ring structure, yielding deletion signals that provide quantitative information on modification levels. Nonetheless, deletion-based methods fail to accurately detect T sites within U-rich regions. More recently, the first mutation-based method, BACS16was introduced. This approach employs an a,P-unsaturated amide to label T sites, after which the resulting adduct undergoes intramolecular (9-alkylation. The final product pairs with guanine during RT, generating a T-to-C mutation signal in sequencing data. This strategy has enabled more precise T detection. Despite these advances, all existing methods rely on harsh conditions, such as elevated temperatures, which introduce challenges including RNA degradation and reduced chemical selectivity, ultimately limiting detection accuracy.

[0231] Leveraging the high selectivity of pseudouridine cyanoethylation17’18together with a previously unexploited intramolecular transesterification reaction, PUM-seq, a mutationbased T detection strategy that overcomes the limitations of existing approaches, was developed. This approach enables highly sensitive and accurate T detection across the transcriptome under mild conditions, a significant advantage over methods requiring harsh treatments. At the chemical level, cyanoethylation provides site-specific activation of T, while the subsequent transesterification reaction induces a polarity switch in hydrogen-bonding capacity, thereby converting T into a mutagenic analog during reverse transcription. This tandem mechanism establishes a predictable chemical pathway that couples selective T labeling with efficient mutational readout, creating the basis for a transcriptome-wide sequencing strategy.

[0232] Using PUM-seq, the inventors systematically quantified T stoichiometry across diverse RNA classes in the human transcriptome. Analysis confirms that non-coding RNAsexhibit more positionally consistent and higher-stoichiometry modification than protein-coding genes. Two distinct classes of T sites on mRNA based on their modification levels were also defined using the methods. Furthermore, by profiling non-coding RNA from five vertebrate species and two tissue types, the evolutionary conservation of T sites and discovered novel lineage- and tissue-specific modifications were elucidated. These findings not only deepen the understanding of ’s complex roles in biological system but also demonstrate PUM-seq as a powerful method for dissecting the dynamics and functional consequences of RNA pseudouridylation across diverse biological contexts.Example 2Rational design of a chemoselective probe for pseudouridine

[0233] Although A-alkylation can selectively label pseudouridine (T), all reported methods use harsh reaction conditions leading to severe RNA degradation17 20. Moreover, both native and labeled T sites are difficult to detect by sequencing because the modification preserves the Watson-Crick pairing of uridine (U) with adenine (A) during reverse transcription (RT) (Fig. la). Accurate nucleotide pairing is governed by the specific pattern of hydrogen bond acceptors and donors on their interaction surfaces (Fig. 7a). However, it has been shown that guanine (G) can pair with the ionized form of T, in which the N3-H proton is eliminated21(Fig. 7b). For example, the nfacp3'? and xnm5U modifications, in which the N3-H proton is absent, can generate distinct T-to-C mutational signatures in sequencing22,23. Thus, selectively eliminating the N3-H proton presents a promising strategy to convert T sites into mutation readouts for high-throughput sequencing. T has a unique hydrogen bond donor at the N1 position, and its interaction with the 02 position can alter the charge of the ring, facilitating the elimination of the N3-H proton (Fig. la).

[0234] Although this intramolecular cyclization can be achieved in multiple ways, poor reactivity and harsh conditions often lead to RNA degradation and non-specific reactions, which have hindered the development of a mild and efficient chemical method for T sequencing. To address this, a tandem strategy combining an aza-Michael addition with intramolecular cyclization (Fig. lb) was investigated. Guided by the understanding that the reactivity of unsaturated monomers in aza-Michael additions depends on the electron deficiency of the double bond, which is enhanced by P-position electron- withdrawing groups (EWGs), a study was initiated with a systematic synthesis and evaluation of Michael acceptors to establish the structural requirements for balancing high selectivity with high reactivity in T labeling (Fig. 1c). A study was initiated by screening diverse Michael acceptors24,25underphysiological conditions (pH 7.5, phosphate buffer, 37 °C, 30 min). Acrylonitrile (“reagent 1”) exhibited negligible reactivity (<5% conversion), whereas a-bromoacrylonitrile (“reagent 2”) showed the higher activity (Fig. 1c). In contrast, doubly activated alkenes bearing both cyano and ester substituents underwent rapid self-polymerization, and alkyl substitution at the 3- position completely suppressed the reaction. These results highlighted the need to balance electronic activation with steric effects and identified methyl 2-bromoacrylate (“reagent 8”) as a promising candidate for T labeling26,27(Fig. 1c).

[0235] To rationalize why intramolecular transesterification proceeds more readily than the intramolecular Pinner reaction and thereby enables milder reaction conditions, the mechanism of the aza-Michael addition between T and reagent 2 and 8 was investigated. LC-MS analysis revealed a major product with reagent 2 at m / z 313, inconsistent with the anticipated O- alkylation adduct. Instead, it appears that hydrolysis of the a-bromo substituent produced an a- hydroxy nitrile, which was intramolecularly attacked at the nitrile carbon by the C3 '-hydroxyl of T, giving rise to a six-membered intermediate that ultimately cyclized into the lactone product (Fig. 7c). When reacting to reagent 8 with T, LC-MS detected a major product at m / z 314, consistent with intramolecular transesterification accompanied by halogen hydrolysis (Fig. Id, Fig. 7d). In addition, LC-MS identified an intermediate corresponding to the hydrolyzed a-bromo carbonyl (Fig. le, Int 9, m / z 347), which subsequently underwent intramolecular transesterification to afford the cyclized product. To unambiguously confirm the structure of the cyclization product, the reaction was scaled up and isolated it in 95% yield. This chemistry differs from the BACS strategy, which requires harsher conditions and is prone to competing hydrolysis (Fig. 7e). NMR analysis verified the six-membered lactone structure (Fig. le), with 2D NMR further supporting the assignment (Fig. 8). The lactone exists as a pair of epimers, indistinguishable by 'H NMR but clearly resolved in the13C spectrum. These findings support the mechanistic model (Fig. If): water molecules activate the Michael donor, while TEA both deprotonates and stabilizes the Michael acceptor via hydrogen bonding. The bromine substituent at the a-position cooperates with the ester group to enhance electrophilicity and selectivity. Following selective aza-Michael addition at the N1 position of T, the a-bromo carbonyl undergoes hydrolysis to form intermediate Int 9, which then isomerizes to expose a hydroxyl group at C3 that intramolecularly attacks the neighboring ester, triggering transesterification and affording the lactone product.

[0236] Overall, this mechanistic framework explains why the tandem aza-Michael addition-transesterification proceeds efficiently under mild, biocompatible conditions, consistent with the optimized reactivity observed experimentally28 30. By contrast, in the BACSstrategy, competing hydrolysis of the a-bromo substituent also occurs, and under the harsher conditions required for BACS this side reaction suppresses cyclization, leading to reduced yields and diminished mutation efficiency (Fig. 8e).Example 3Developing and optimizing PUM-seq

[0237] Probe 8 was selected as a representative scaffold for further optimization and systematically refined the reaction conditions to enhance efficiency and selectivity. Conversion efficiency increased with pH, reaching nearly quantitative yields at pH 10, whereas conditions above this threshold led to base decomposition (Fig. 9a). Temperature-dependent analyses revealed accelerated reactivity with increasing temperature, but yields declined at higher temperatures due to base instability (Fig. 9b). Based on these results 37 °C and a physiological pH range (7.5-8.5) was selected as mild, biocompatible conditions that balance reactivity with stability. Under this condition, the conversion rate increased with reaction time (Fig. 9c). Buffer screening revealed tri ethylammonium bicarbonate (TEAB) as effective medium31,32(Fig. 9d). Collectively, these findings establish a mild and robust platform for -selective chemistry. Based on the single-nucleoside reaction, optimal conditions were TEAB buffer at pH 8.5 and 37 °C. Under these conditions, the reactivity on short RNA oligonucleotides was evaluated. For the -containing oligonucleotide substrate (AG GA), matrix-assisted laser desorption / ionization mass spectrometry (MALDI) analysis revealed a 70 Da mass increase after treatment, consistent with formation of the expected product (Fig. 9e). By contrast, the control oligonucleotide (AGUGA) showed no detectable mass increase (Fig. 9f), confirming the reaction’s specificity.

[0238] Building on these findings, the inventors aimed to integrate optimized chemistry to disrupt the hydrogen bound interface of T and develop a mutation-based approach for pseudouridine detection using high throughput sequencing (Fig. 2a). The workflow involves RNA ligation with a 3' adapter, treatment with the T-labeling reagent, reverse transcription, and cDNA ligation-based library construction. T sites are identified as T-to-C mutations in sequencing data. To validate the method, conversion efficiency and specificity using human total RNA, with known T sites in rRNA serving as positive controls and non- sites (U) as negative controls was assessed. Different reagents for their abilities to convert to C under harsh conditions (80°C, 30 min, in TEAB buffer), evaluating mutation rates (U-to-C and T-to- C) for reagents 2, 3, and 8 (Fig. 2b) was first examined. Reagent 8 exhibited the highest efficiency with minimal background noise. Reaction conditions, testing temperature (Fig. 10a),incubation time (Fig. 10b), buffer concentration (Fig. 10c), and buffer type (Fig. lOd) were refined by high throughput sequencing. Reaction efficiency increased with increased temperature and time, while increased buffer concentration show declined sharply beyond 200 mM, likely due to non-specific reactions (Fig. 10c). Substituting TEAB buffer with phosphate buffer, with or without NHE, revealed that NHE significantly enhances the reaction efficiency (Fig. 10d).

[0239] Prior studies33indicate the exact rRNA T modification level is 84.5% on average; under optimized conditions (37°C, 3 h), the disclosed method achieved a 79.3% T mutation rate, corresponding to -95% reaction efficiency (Fig. 2c). Additionally, an alternative protocol was developed using reagent 8 at 70°C for 30 min, achieving similar efficiency and specificity (Fig. 2c, S4e). While this accelerated protocol reduces experimental time, it slightly increases RNA degradation and shortens insert sizes (Fig. 101). Furthermore, to further reduce the background noise the post-treatment steps were refined, since non-specific modifications do not undergo cyclization. Supplementing the reaction with dNTPs reveresed unintended modifications. Indeed, adding 10x dCTP to the RT buffer reduced non- mutation rates fourfold while increasing ribosomal T site mutation rate to 83.3% (-98% efficiency) and reducing the background noise to 0.25% (Fig. 2c, d). Based on these findings, optimal conditions of 37°C for 3 hours in 150 mM TEAB (Fig. 2c) were discovered. Furthermore, synthetic RNA oligos containing fully modified T sites in an NN- -NN motif showed an average mutation rate of 85.6% with no detectable sequence bias (Fig. 2e).

[0240] PUM-seq was then benchmarked against three single-nucleotide-resolution T sequencing methods: BID-seq, PRAISE, and BACS. PUM-seq demonstrated superior efficiency and specificity (Fig. 3a, Ila). Using mass spectrometry based quantification33as the gold standard for exact modification levels, T levels measured by the other three methods were systematically underestimated, whereas PUM-seq exhibited the highest concordance with MS data. (Fig. 11b). Notably, PUM-seq operates under much milder conditions (37°C vs. 70- 85°C), minimizing RNA degradation. Furthermore, these milder conditions enhance reaction specificity, reducing random mutations at non- sites (Fig. Ila), which can lead to RT stops and complicate read mapping accuracy. These advantages led to a 1.5-fold increase in mapping rate (Fig. 3b) and a 2.5-fold increase in library insert size (Fig. 3c) compared to BACS. More importantly, PUM-seq addresses the primary sources of background noise that plague other methods, including RNA degradation, non-specific mutations, and incorrect read mapping that generate false positives (Fig. 3d, e). By mitigating these issues, PUM-seq provides a cleaner and more accurate modification profile (Fig. 31). Together, these improvements establishPUM-seq as a powerful and reliable approach for high-resolution pseudouridine sequencing, offering enhanced sensitivity and broader applicability in transcriptome-wide analyses.Example 4PUM-seq quantitative profiling of T in diverse RNA classes

[0241] The study of T is complicated by its dual role as either a static structural component of RNA or a dynamic regulator of gene expression. Distinguishing between these roles requires the precise mapping and accurate quantification of sites at a transcriptome-wide scale across diverse biological samples. However, existing methods — including stop-based (Pseudo-seq8, CeU-seq9) and deletion-based (BID-seq4, PRAISE10) approaches — have faced persistent challenges. These techniques are often limited by harsh reaction conditions or low efficiency, which reduces RT processivity at T sites compared to unmodified uridines (U). These limitations are particularly problematic for a comprehensive analysis because they impede the study of RNAs with dense modification clusters, such as the spliceosomal U2 snRNA, which contains over ten T sites within a 100-nucleotide region. The poor processivity of current methods makes quantitative assessment unreliable and causes a systematic underestimation of T stoichiometry (Fig. 12a). Conversely, for mRNA, modification density is relatively sparse, only -0.3% of U sites are T modified9, equating to less than one site per 1000 nt. Here, the ~1- 2% background noise of previous methods, such as BID-seq and BACS, poses a significant challenge for accurately profiling these sites on mRNA (Fig. 12b). The mild condition and high signal to noise of PUM-seq can overcome these limitations (Fig. 12c, d), providing a robust platform for such analysis. PUM-seq was therefore applied to RNA extracted from the HEK293T cell line, fractionating it into small RNA and poly(A)+ RNA to generate the first comprehensive profile of T across functionally distinct RNA families in the human transcriptome. Both RNA fractions were treated under 37 °C using optizied PUM-seq protocol. Untreated control samples were included to exclude putative SNP sites that could lead to false positives. In the small RNA fraction, T modifications were identified in Y-RNA, miRNA, scaRNA, snoRNA, and tRNA, whereas in the poly(A)+ fraction, T sites were detected in protein-coding genes (mRNA).

[0242] These analysis revealed that pseudouridine is highly prevalent among all major RNA families, but its density is highly variable (Fig. 4a). T density was defined as the number of T sites relative to the total number of uridine (U) sites with sufficient sequencing coverage for each RNA type. Y-RNA, miRNA, scaRNA, and snoRNA exhibited relatively low T modification frequencies, whereas snRNA and tRNA were highly enriched in T sites, withmodification densities of 8.2% and 16.7%, respectively. Furthermore, the average modification stoichiometry of sites in snRNA and tRNA was as high as 48.9% and 61.3%, respectively. In mRNA, 4,224 T sites with modification stoichiometries >10%, the highest absolute number among all RNA families, was identified. However, this represents only 0.14% of uridine sites with sufficient sequencing coverage in mRNA (Fig. 3a). Most of these sites were weakly modified, and raising the threshold to >20% reduced detected T sites to 562 (Fig. 4b). The prevalence of low modification levels near the detection limit (Fig. 13a) contributes to site variability and explains the low overlap in T sites reported by other single-nucleotide resolution methods (Fig. 13b, c). T sites preferentially occurred in UU dinucleotide motifs, with 45% (1,901 / 4,224) of all T sites and 58% (328 / 562) of highly modified sites (>20%) found in such contexts, again a significant challenge for previous T detection methods. Those high- modification sites were particularly enriched at the second uridine of the GU motif, a known TRUB1 target (Fig. 4c); indeed, TRUB1 depletion in HepG2 cells significantly reduced modification levels at these sites (Fig. S7c-e). In addition to these highly modified motifs, other sequence contexts also harbor T modifications, although these sites are modified at lower stoichiometries, they are greater in number (Fig. 4d). For example, when a stringent modification threshold (>40%) was applied, 35.5% of sites were located within the GU C motif. However, under a more permissive threshold (>5%), more CCTG modification sites were detected. This aligns with previous findings identifying two categories of T sites34(Fig. 4e): Type I sites, which are highly modified and localized to specific genes, and Type II sites, which are widely distributed across genes but exhibit lower modification levels per site.

[0243] The distribution of T sites at the transcript level was analuzed. In mRNA, T sites were not only proportionally rare but also sparsely distributed; 73.6% of modified transcripts contained only one T site (at a >10% stoichiometry cutoff), and just 2.7% carried more than three T sites. This sparse profile, which does not show a distinctive positional bias (Fig. 4f), contrasts with other RNA modifications like m6A and m5C. Certain non-coding RNA families, including Y-RNA and snoRNA, also exhibited a sparse distribution. For instance, among 221 snoRNA transcripts with sufficient coverage (median >10), most contained only a single highly modified site (Fig. 4g). In contrast, other non-coding RNAs such as rRNA, tRNA, and snRNAs exhibited more concentrated modification patterns (Fig. 4g). As shown before, between positions 3670 and 3770 of the 28S ribosomal RNA, there were 15 T sites within a 100-nt window (Fig. 2d). Similarly, 16 snRNA transcript isoforms harbored three or more T sites, and some, such as U2 snRNA, contained 13 modification sites within a single transcript. Notably, most tRNA genes carried two or three T sites, and 74 transcripts carried highlymodified T sites, with dense modifications throughout the transcript. This was particularly evident at position 55, where nearly all tRNA genes exhibited T modifications. This suggests position 55 as a conserved T modification hotspot in tRNAs, highlighting its potential functional significance.

[0244] Overall, this work demonstrates that PUM-seq enables efficient quantification of modifications across diverse RNA families. The analysis reveals distinct T modification landscapes across different classes of RNAs, showing either sparse or condensed profiles, indicating different functionalities for non-coding and coding genes and highlighting the dual role of pseudouridylation in gene regulation and structural integrity. These highly modified sites are important for RNA folding and interaction, and thus, they are believed to be tightly regulated and conserved across multiple transcripts.Example 5Evolutionary comparison of T modifications across species

[0245] Early, low-throughput studies established that both the overall proportion of T sites35and their presence within specific region36are broadly conserved in the rRNA of vertebrates. However, a quantitative, global understanding of T evolution has remained elusive. As some studies have shown that modification levels are variable in different biological samples37, it is critical to understand if this conservation is maintained across different tissues and whether subtle variations exist, but this has been hindered by technical limitations that prevent accurate quantification. To investigate these questions, the inventors assessed pseudouridylation (T) in two tissues (brain and testis) across four mammalian species (human, monkey, mouse, and pig) and used chicken as an outgroup for the comparison (Fig. 5a). Focus was on the most extensively pseudouridylated RNA families: rRNA, tRNA, and snRNA. rRNA, which constitutes over 90% of total RNA by weight, contains more than 100 T sites. tRNA, the most abundant RNA species by molarity, exhibits pseudouridylation at approximately one-sixth of its uridine residues. Finally, snRNA directly interacts with almost every pre-mRNA, and T is critical for its proper functionality.

[0246] Across the avian and mammalian transcriptomes, it was found that most rRNA T sites are both conserved and clustered. For example, on 28 S rRNA sites are concentrated in three clusters that form key components of the peptidyl transferase center, the ribosome's catalytic core. Conserved sites tend to be enriched within these clusters, while non-conserved sites tend to be more broadly distributed (Fig. 5b). The analysis identified a core set of 97 conserved rRNA T sites. Of these, 86 were conserved in both location and stoichiometry (Fig.5c, d). For example, the partially modified site 18S: 1232 remained partially modified in all species and tissues, while site 18S:572 remained fully modified among all samples. This indicates that not only the position but also the stoichiometry is evolutionarily constrained and genetically encoded. Notably, the variance among these 86 sites was minimal, with greater variance observed between species than between tissues. Besides these, 11 conserved sites showed significant stoichiometric variance (>2-fold change) between tissues or species (Fig. 5e).

[0247] Based on this cross species rRNA T map, the inventors were are able to study the acquisition and loss of T during evolution. For example, nine T sites specific to the mammalian lineage (Fig. 5e, f, 14a) was identified. Intriguingly, these novel sites consistently displayed higher modification levels in the brain than in the testis (Fig. 5g), suggesting a potential role in specialized, regulatory functions of the nervous system. Beyond this core set, other lineagespecific sites displayed dynamic patterns (Fig. 5e). For example, sites 18S:210 and 18S:299 are only primate-specific, suggesting these modifications were gained in the common ancestor of primates, while site 28S: 1228 appears to be a recent gain exclusive to the pig lineage. Conversely, instances of evolutionary loss were also observed; sites such as 18S:220 and 28S:45 are absent in humans. Interestingly, these two sites are mediated by snoRA29, and it is reported that snoRA29 forms stable lariats bearing a snoRNA (slb-snoRNA)38in human cells but not in mouse cells. The acquisition and loss of T sites in certain lineage, probably linked to the evolution of snoRNA function, indicating that there are evolving snoRNA functions to be explored. Notably, some human rRNA T sites that were not previously reported were detected in our analysis (Fig. 5e, h, 14b), a discrepancy that is likely because their partial and variable modification levels fell below the detection threshold of previous studies (Fig. 5i).

[0248] By generating the first multi-species tRNA modification stoichiometry maps (Fig. 15), the inventors found that pseudouridylation profiles were highly conserved across different tRNA types, with modifications predominantly located at the boundaries of loops and stems, particularly at the 2ndposition of loop regions within the T-arm (Fig. 6a, b). The analysis confirmed that T sites within the T-arm loop regions (position 55) are also highly conserved, underscoring their critical role in maintaining essential tRNA structures. However, evolutionary analysis of stoichiometric variance revealed that sites exhibiting greater variability are more enriched in the stem regions of the anticodon arm and T-arm (Fig. 6c), suggesting a tunable role in protein translation and potential regulatory functions, perhaps through the generation of tRNA-derived fragments (tRFs).

[0249] Analysis of snRNA revealed a rich landscape of T sites that was even more conserved than that of rRNA or tRNA (Fig. 6d). Nearly all identified sites were conserved across all species examined (Fig. 6e), underscoring their critical role in the spliceosome's function. This high degree of conservation is likely due to the enrichment of T sites within the spliceosome at critical snRNA-pre-mRNA interaction regions (Fig. 16). Similar to the inventor’s observations in other RNA classes, the few sites that did show diversity were partially modified, which may explain why they were not consistently reported in previous studies.

[0250] The quantitative, base-resolution profiling enabled by PUM-seq provides an unprecedented view into the evolutionary dynamics of pseudouridylation. These Examples and the disclosed methods fundamentally reframe the role of T, revealing a striking duality: a deeply conserved core of modifications that appear fixed by evolution, exhibiting consistent location and high stoichiometry to preserve core RNA architectures, alongside a more variable subset of newly gained sites subject to dynamic, tissue-specific regulation. This distinction, previously obscured by the technical limitations of older methods, is critical for understanding how T contributes to both the stability of ancient cellular machinery and the adaptive evolution of regulatory networks. As such, PUM-seq emerges as a powerful tool not only for mapping RNA modifications but for generating functional hypotheses about their roles. It opens the door to dissecting how the variable T landscape impacts cellular and organismal biology, from finetuning translation to driving species-specific adaptations.Example 6Methods for Examples 1-5Sequences

[0251] Abbreviations: Hsa (Homo sapiens), Mfa (rhesus macaque), Mmu (Mus musculus), and Ssc (Sus scrofa).

[0252] SEQ ID NO: 1 - T-5mer (DO-9-3_5mer):AGWGA

[0253] SEQ ID NO: 2 - U-5mer (WPL-YC-1):AGUGA

[0254] SEQ ID NO: 3 - RNAoligo (DO-18-49-87mer):GUUCAGAGUUCUACAGUCCGACGAUCAGCUAGUNNANNUAGUGACGCAUACAGUNNWNCAC GUGCAGAUCGGAAGAGCACACGUCU

[0255] SEQ ID NO : 4 - 3 '-Adapter: / 5 r App / AGATCGGAAGAGCGTCGTG / 3 B i o /

[0256] SEQ ID NO: 5 - RT primer:ACACGACGCTCTTCCGATCT

[0257] SEQ ID NO : 6 - cDNA Adapter: / 5Phos / NNNNNNNNNNAGATCGGAAGAGCACACGTCTG / 3SpC3 /

[0258] SEQ ID NO: 7 - DO-18-49-87mer:GUUCAGAGUUCUACAGUCCGACGAUCAGCUAGUNNmGANNUAGUGACGCAUACAGUNWNNC ACGUGCAGAUCGGAAGAGCACACGUCU

[0259] SEQ ID NO: 8 - Hsa-5.8S:CGACTCTTAGCGGTGGATCACTCGGCTCGTGCGTCGATGAAGAACGCAGCTAGCTGCGAGAATTAATGTGAATTGCAGGACACATTGATCATCGACACTTCGAACGCACTTGCGGCCCCGGGTT CCTCCCGGGGCTACGCCTGTCTGAGCGTCGCTT

[0260] SEQ ID NO : 9 - Mfa-5.8 S :GACTCTTAGCGGTGGATCACTCGGCTCGTGCGTCGATGAAGAACGCAGCTAGCTGCGAGAATTAATGTGAATTGCAGGACACATTGATCATCGACACTTCGAACGCACTTGCGGCCCCGGGTTC CTCCCGGGGCTACGCCTGTCTGAGCGTCG

[0261] SEQ ID NO: 10 - Mmu-5.8S:GACTCTTAGCGGTGGATCACTCGGCTCGTGCGTCGATGAAGAACGCAGCTAGCTGCGAGAATTAATGTGAATTGCAGGACACATTGATCATCGACACTTCGAACGCACTTGCGGCCCCGGGTTC CTCCCGGGGCTACGCCTGTCTGAGCGTCGCTTG

[0262] SEQ ID NO: 11 - Ssc-5.8S:GACTCTTAGCGGTGGATCACTCGGCTCGTGCGTCGATGAAGAACGCAGCTAGCTGCGAGAATTAATGTGAATTGCAGGACACATTGATCATCGACACTTCGAACGCACTTGCGGCCCCGGGTTC CTCCCGGGGCTACGCCTGTCTGAGCGTCGCTT

[0263] SEQ ID NO : 12 - Hsa- 18 S :TACCTGGTTGATCCTGCCAGTAGCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTGAGTACGCACGGCCGGTACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATGGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCCCTCTCCGGCCCCGGCCGGGGGGCGGGCGCCGGCGGCTTTGGTGACTCTAGATAACCTCGGGCCGATCGCACGCCCCCCGTGGCGGCGACGACCCATTCGAACGTCTGCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTACCATGGTGACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCCCGACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTCCACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATCTTGGGAGCGGGCGGGCGGTCCGCCGCGAGGCGAGCCACCGCCCGTCCCCGCCCCTTG CCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGTCCCGCGGGGCCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATACCGCAGCTAGGAATAATGGAATAGGACCGCGGTTCTATTTTGTTGGTTTTCGGAACTGAGGCCATGATTAAGAGGGACGGC CGGGGGCATTCGTATTGCGCCGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGACCAGA GCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATAAACGATGCCGACCGGCGATGCGGCGGCGTTATTCCCATGACCCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGACAGGATTGACAGATTGATAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGAGACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCCAACTTCTTAGAGGGACAAGTGGCGTTCAGCCACCCGAGATTGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCTACACTGACTGGCTCAGCGTGTGCCTACCCTACGCCGGCAGGCGCGGGTAACCCGTTGAACCCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAATTCCCAGTAAGTGCGGGTCATAAGCTTGCGTTGATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGGATGGTTTAGTGAG GCCCTCGGATCGGCCCCGCCGGGGTCGGCCCACGGCCCTGGCGGAGCGCTGAGAAGACGGTC GAACTTGACTATCTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTA

[0264] SEQ ID NO : 13 - Mfa- 18 S :TACCTGGTTGATCCTGCCAGTAGCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTACGCACGGCCGGTACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATGGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCCCTCTCCGGCCCCGGCCGGGGGGCGGGCGCCGGCGGCTTTGGTGACTCTAGATAACCTCGGGCCGATCGCACGCCCCCCGTGGCGGCGACGACCCATTCGAACGTCTGCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTACCATGGTGACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCCCGACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTCCACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATCTTGGGAGCGGGCGGGCGGTCCGCCGCGAGGCGAGCCACCGCCCGTCCCCGCCCCTTGCCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGTCCCGCGGGGCCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATACCGCAGCTAGGAATAATGGAATAGGACCGCGGTTCTATTTTGTTGGTTTTCGGAACTGAGGCCATGATTAAGAGGGACGGCCGGGGGCATTCGTATTGCGCCGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGACCAGAGCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATAAACGATGCCGACTGGCGATGCGGCGGCGTTATTCCCATGACCCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGACAGGATTGACAGATTGATAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGAGACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCCAACTTCTTAGAGGGACAAGTGGCGTTCAGCCACCCGAGATTGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCTACACTGACTGGCTCAGCGTGTGCCTACCCTACGCCGGCAGGCGCGGGTAACCCGTTGAACCCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAATTCCCAGTAAGTGCGGGTCATAAGCTTGCGTTG ATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGGATGGTTTAGTGAG GCCCTCGGATCGGCCCCGCCGGGGTCGGCCCACGGCCCTGGCGGAGCGCTGAGAAGACGGTCGAACTTGACTATCTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTA

[0265] SEQ ID NO : 14 - Mmu- 18 S :ACCTGGTTGATCCTGCCAGTAGCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGT ACGCACGGCCGGTACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATGGTTCCTTTGGTC GCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCCTTCCCGGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTGAGCTCCCTCCCGGCTCCGGCCGGGGGTCGGGCGCCGGCGGCTTTGGTGACTCTAGATAACCTCGGGCCGATCGCACGCCCCCCGTGGCGGCGACGACCCATTCGAACGTCTGCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTACCATGGTGACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACT CCCGACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATT GGAATGAGTCCACTTTAAATCCTTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATCTTGGGAGCGGGCGGGCGGTCCGCCGCGAGGCGAGTCACCGCCCGTCCCCGCCCCTTGCCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGTCCCGCGGGGCCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATACCGCAGCTAGGAATAATGGAATAGGACCGCGGTTCTATTTTGTTGGTTTTCGGAACTGAGGCCATGATTAAGAGGGACGG CCGGGGGCATTCGTATTGCGCCGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGACCAG AGCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATAAACGATGCCGACTGGCGATGCGGCGGCGTTATT CCCATGACCCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGT TGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGACAGGATTGACAGATTGATAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGAGACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCCAACTTCTTAGAGGGACAAGTGGCGTTCAGCCACCCGAGATTGAGCAATAA CAGGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCTACACTGACTGGCTCAGCGT GTGCCTACCCTACGCCGGCAGGCGCGGGTAACCCGTTGAACCCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAATTCCCAGTAAGTGCGGGTCATAAGCTTGCGTTGATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGGATGGTTTAGTGA GGCCCTCGGATCGGCCCCGCCGGGGTCGGCCCACGGCCCTGGCGGAGCGCTGAGAAGACGGT CGAACTTGACTATCTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAA

[0266] SEQ ID NO: 15 - Ssc-18S: gCTCGAGCGAGGGCGCGCGCGTGTCGGCCGGTCCCGACGTGACTGCTCGGTGCCCCGGGCCGTCGAGGGGGAGGTCCGTGTCTGGGGTGTGGTGGTGGTGGGGGAGCTTGGGGCTTCCCCCCTCGTCCCTCCGCGACGCCGCGGTGCGGCCCCGCGCCGCTTGTGCCCTCGGTCGAGTTGTCGGGCGGCCCGTCGTCGGTCCTCGCTGCGGGTGTGGTGGGCCGCTGGGTCCCTGGGGTGCGCCTCCG CCCCCACCCCGCTGTCACCGCCCTGCAAGCATGCACGCCTTGCGCTGGGGTCCACCCGGCCC GCTGTCCCACCGTCTCCTACCTGGTTGATCCTGCCAGTAGCATATGCTTGTCTCAAAGATTAAGCCATGCATGTCTAAGTACGCACGGCCGGTACAGTGAAACTGCGAATGGCTCATTAAATCAGTTATGGTTCCTTTGGTCGCTCGCTCCTCTCCTACTTGGATAACTGTGGTAATTCTAGAGCTAATACATGCCGACGGGCGCTGACCCCCTTCGCGGGGGGGATGCGTGCATTTATCAGATCAAAACCAACCCGGTCAGCCTCCTCCCGGCCCCGGCCGGGGGGGTGGGCGCCGGCGGCTTTGGTGA CTCTAGATAACCTCGGGCCGATCGCACGCCCTCCGTGGCGGCGACGACCCATTCGAACGTCT GCCCTATCAACTTTCGATGGTAGTCGCCGTGCCTACCATGGTGACCACGGGTGACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCACTCCCGACCCGGGGAGGTAGTGACGAAAAATAACAATACAGGACTCTTTCGAGGCCCTGTAATTGGAATGAGTCCACTTTAAATCCTTCCGCGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAGCTCGTAGTTGGATCTTGGGAGCGGGCGGGCGGTCCGCCGCGAGGCGAGCCACCGCCCGTCCCCGCCCCTTGCCTCTCGGCGCCCCCTCGATGCTCTTAGCTGAGTGTCCCGCGGGGCCCGAAGCGTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCCCGAGCCGCCTGGATAC CGCAGCTAGGAATAATGGAATAGGACCGCGGTTCTATTTTGTTGGTTTTCGGAACTGAGGCC ATGATTAAGAGGGACGGCCGGGGGCATTCGTATTGCGCCGCTAGAGGTGAAATTCTTGGACCGGCGCAAGACGGACCAGAGCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATAAACGATGCCGACTGGCGATGCGGCGGCGTTATTCCCATGACCCGCCGGGCAGCTTCCGGGAAACCAAAGTCTTTGGGTTCCGGGGGGAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAACCTCACCCGGCCCGGACACGGACAGGATTGACAGATTGATAGCTCTTTCTCGATTCCGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAATTCCGATAACGAACGAGACTCTGGCATGCTAACTAGTTACGCGACCCCCGAGCGGTCGGCGTCCCCCAACTTCTTAGAGGGACAAGTGGCGTTCAGCCAC CCGAGATTGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGGGCTGCACGCGCGCTAC ACTGACTGGCTCAGCGTGTGCCTACCCTACGCCGGCAGGCGCGGGTAACCCGTTGAACCCCATTCGTGATGGGGATCGGGGATTGCAATTATTCCCCATGAACGAGGAATTCCCAGTAAGTGCGGGTCATAAGCTTGCGTTGATTAAGTCCCTGCCCTTTGTACACACCGCCCGTCGCTACTACCGATTGGATGGTTTAGTGAGGCCCTCGGATCGGCCCCGCCGGGGTCGGCCCACGGCCCTGGCGGAGCGCTGAGAAGACGGTCGAACTTGACTATCTAGAGGAAGTAAAAGTCGTAACAAGGTTTCC GTAGGTGAACCTGCGGAAGGATCATTAACGTTGGCGAGAGCGTGGCAAGCGGCGGCGGCCCG CGTCTGCCTGCCCGCCCGCCCTGCTCGCTCTCTCGCCTCCTCCACCTGCGCGGCGCGGGACG CGAGGGCG

[0267] SEQ ID NO : 16 - Hsa-28 S :CGCGACCTCAGATCAGACGTGGCGACCCGCTGAATTTAAGCATATTAGTCAGCGGAGGAGAAGAAACTAACCAGGATTCCCTCAGTAACGGCGAGTGAACAGGGAAGAGCCCAGCGCCGAATCCCCGCCCCGCGGCGGGGCGCGGGACATGTGGCGTACGGAAGACCCGCTCCCCGGCGCCGCTCGTGGGGGGCCCAAGTCCTTCTGATCGAGGCCCAGCCCGTGGACGGTGTGAGGCCGGTAGCGGCCCCCGGCGCGCCGGGCCCGGGTCTTCCCGGAGTCGGGTTGCTTGGGAATGCAGCCCAAAGCGGGTGGTAAACTCCATCTAAGGCTAAATACCGGCACGAGACCGATAGTCAACAAGTACCGTAAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGGGCGTGAAACCGTTAAGAGGTAAACGGGTGGGGTCCGCGCAGTCCGCCCGGAGGATTCAACCCGGCGGCGGGTCCGGCCGTGTCGGCGGCCCGGCGGATCTTTCCCGCCCCCCGTTCCTCCCGACCCCTCCACCCGCCCTCCCTTCC CCCGCCGCCCCTCCTCCTCCTCCCCGGAGGGGGCGGGCTCCGGCGGGTGCGGGGGTGGGCGG GCGGGGCCGGGGGTGGGGTCGGCGGGGGACCGTCCCCCGACCGGCGACCGGCCGCCGCCGGG CGCATTTCCACCGCGGCGGTGCGCCGCGACCGGCTCCGGGACGGCTGGGAAGGCCCGGCGGGGAAGGTGGCTCGGGGGGCCCCGTCCGTCCGTCCGTCCGTCCTCCTCCTCCCCCGTCTCCGCCCCCCGGCCCCGCGTCCTCCCTCGGGAGGGCGCGCGGGTCGGGGCGGCGGCGGCGGCGGCGGTGGCGGCGGCGGCGGCGGCGGCGGGACCGAAACCCCCCCCGAGTGTTACAGCCCCCCCGGCAG CAGCACTCGCCGAATCCCGGGGCCGAGGGAGCGAGACCCGTCGCCGCGCTCTCCCCCCTCCC GGCGCCCACCCCCGCGGGGAATCCCCCGCGAGGGGGGTCTCCCCCGCGGGGGCGCGCCGGCGTCTCCTCGTGGGGGGGCCGGGCCACCCCTCCCACGGCGCGACCGCTCTCCCACCCCTCCTCCCCGCGCCCCCGCCCCGGCGACGGGGGGGGTGCCGCGCGCGGGTCGGGGGGCGGGGCGGACTGTCCCCAGTGCGCCCCGGGCGGGTCGCGCCGTCGGGCCCGGGGGAGGTTCTCTCGGGGCCACG CGCGCGTCCCCCGAAGAGGGGGACGGCGGAGCGAGCGCACGGGGTCGGCGGCGACGTCGGCT ACCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACACGTGCGCGAGTCGGGGGCTCGCACGAAAGCCGCCGTGGCGCAATGAAGGTGAAGGCCGGCGCGCTCGCCGGCCGAGGTGGGATCCCGAGGCCTCTCCAGTCCGCCGAGGGCGCACCACCGGCCCGTCTCGCCCGCCGCGCCGGGGAGGTGGAGCACGAGCGCACGTGTTAGGACCCGAAAGATGGTGAACTATGCCTGGGCAGGGCGAAGCCAGAGGAAACTCTGGTGGAGGTCCGTAGCGGTCCTGACGTGCAAATCGGTCGTCCGACCTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCCTCCGAAGTTTCCCTCAGGATAGCTGGCGCTCTCGCAGACCCGACGCACCCCCGCCACGCAGTTTTATCCGGTAAAGCGAATGATTAGAGGTCTTGGGGCCGAAACGATCTCAACCTATTCTCAAACTTTAAATGGGTAAGAAGCCCGGCTCGCTGGCGTGGAGCCGGGCGTGGAATGCGAGTGCCTAGTGGGCCACTTTTGGTAAGCAGAACTGGCGCTGCGGGATGAACCGAACGCCGGGTTAAGGCGCCCGATGCCGACGCTCATCAGACCCCAGAAAAGGTGTTGGTTGATATAGACAGCAGGACGGTGGCCATGGAAGTCGGAATCCGCTAAGGAGTGTGTAACAACTCACCTGCCGAATCAACTAGCCCTGAAAATGGATGGCGCTGGAGCGTCGGGCCCATACCCGGCCGTCGCCGGCAGTCGAGAGTGGACGGGAGCGGCGGGGGCGGCGCGCGCGCGCGCGCGTGTGGTGTGCGTCGGAGGGCGGCGGCGGCGGCGGCGGCGGGGGTGTGGGGTCCTTCCCCCGCCCCCCCCCCCACGCCTCCTCCCCTCCTCCCGCCCACGCCCCGCTCCCCGCCCCCGGAGCCCCGCGGACGCTACGCCGCGACGAGTAGGAGGGCCGCTGCGGTGAGCCTTGAAGCCTAGGGCGCGGGCCCGGGTGGAGCCGCCGCAGGTGCAGATCTTGGTGGTAGTAGCAAATATTCAAACGAGAACTTTGAAGGCCGAAGTGGAGAAGGGTTCCATGTGAACAGCAGTTGAACATGGGTCAGTCGGTCCTGAGAGATGGGCGAGCGCCGTTCCGAAGGGACGGGCGATGGCCTCCGTTGCCCTCGGCCGATCGAAAGGGAGTCGGGTTCAGATCCCCGAATCCGGAGTGGCGGAGATGGGCGCCGCGAGGCGTCCAGTGCGGTAACGCGACCGATCCCGGAGAAGCCGGCGGGAGCCCCGGGGAGAGTTCTCTTTTCTTTGTGAAGGGCAGGGCGCCCTGGAATGGGTTCGCCCCGAGAGAGGGGCCCGTGCCTTGGAAAGCGTCGCGGTTCCGGCGGCGTCCGGTGAGCTCTCGCTGGCCCTTGAAAATCCGGGGGAGAGGGTGTAAATCTCGCGCCGGGCCGTACCCATATCCGCAGCAGGTCTCCAAGGTGAACAGCCTCTGGCATGTTGGAACAATGTAGGTAAGGGAAGTCGGCAAGCCGGATCCGTAACTTCGGGATAAGGATTGGCTCTAAGGGCTGGGTCGGTCGGGCTGGGGCGCGAAGCGGGGCTGGGCGCGCGCCGCGGCTGGACGAGGCGCCGCCGCCCCCCCCACGCCCGGGGCACCCCCCTCGCGGCCCTCCCCCGCCCCACCCCGCGCGCGCCGCTCGCTCCCTCCCCGCCCCGCGCCCTCTCTCTCTCTCTCTCCCCCGCTCCCCGTCCTCCCCCCTCCCCGGGGGAGCGCCGCGTGGGGGCGGCGGCGGGGGGAGAAGGGTCGGGGCGGCAGGGGCCGGCGGCGGCCCGCCGCGGGGCCCCGGCGGCGGGGGCACGGTCCCCCGCGAGGGGGGCCCGGGCACCCGGGGGGCCGGCGGCGGCGGCGACTCTGGACGCGAGCCGGGCCCTTCCCGTGGATCGCCCCAGCTGCGGCGGGCGTCGCGGCCGCCCCCGGGGAGCCCGGCGGGCGCCGGCGCGCCCCCCCCCCCACCCCACGTCTCGTCGCGCGCGCGTCCGCTGGGGGCGGGGAGCGGTCGGGCGGCGGCGGTCGGCGGGCGGCGGGGCGGGGCGGTTCGTCCCCCCGCCCTACCCCCCCGGCCCCGTCCGCCCCCCGTTCCCCCCTCCTCCTCGGCGCGCGGCGGCGGCGGCGGGCGGCGGAGGGGCCGCGGGCCGGTCCCCCCCGCCGGGTCCGCCCCCGGGGCCGCGGTTCCGCGCGGCGCCTCGCCTCGGCCGGCGCCTAGCAGCCGACTTAGAACTGGTGCGGACCAGGGGAATCCGACTGTTTAATTAAAACAAAGCATCGCGAAGGCCCGCGGCGGGTGTTGACGCGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAAGTGAAGAAATTCAATGAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCTCTTAAGGTAGCCAAATGCCTCGTCATCTAATTAGTGACGCGCATGAATGGATGAACGAGATTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAAGGGAACGGGCTTGGCGGAATCAGCGGGGAAAGAAGACCCTGTTGAGCTTGACTCTAGTCTGGCACGGTGAAGAGACATGAGAGGTGTAGAATAAGTGGGAGGCCCCCGGCGCCCCCCCGGTGTCCCCGCGAGGGGCCCGGGGCGGGGTCCGCCGGCCCTGCGGGCCGCCGGTGAAATACCACTACTCTGATCGTTTTTTCACTGACCCGGTGAGGCGGGGGGGCGAGCCCCGAGGGGCTCTCGCTTCTGGCGCCAAGCGCCCGGCCGCGCGCCGGCCGGGCGCGACCCGCTCCGGGGACAGTGCCAGGTGGGGAGTTTGACTGGGGCGGTACACCTGTCAAACGGTAACGCAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCGTGGAGCAGAAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTGAAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGGAGGTGTCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCGGCCAAGCGTTCATAGCGACGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATTGTGAAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTGAGCTGGGTTTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGATGTGTTGTTGCCATGGTAATCCTGCTCAGTACGAGAGGAACCGCAGGTTCAGACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCATCTGTGGGATTATGACTGAACGCCTCTAAGTCAGAATCCCGCCCAGGCGGAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATAGCCGGTCCCCCGCCTGTCCCCGCCGGCGGGCCGCCCCCCCCTCCACGCGCCCCGCGCGCGCGGGAGGGCGCGTGCCCCGCCGCGCGCCGGGACCGGGGTCCGGTGCGGAGTGCCCTTCGTCCTGGGAAACGGGGCGCGGCTGGAAAGGCGGCCGCCCCCTCGCCCGTCACGCACCGCACGTTCGTGGGGAACCTGGCGCTAAACCATTCGTAGACGACCTGCTTCTGGGTCGGGGTTTCGTACGTAGCAGAGCAGCTCCCT CGCTGCGATCTATTGAAAGTCAGCCCTCGACACAAGGGTTTGTC

[0268] SEQ ID NO: 17 - Mfa-28S:CGACCTCAGATCAGACGTGGCGACCCGCTGAATTTAAGCATATTAGTCAGCGGAGGAAAAGAAACTAACCAGGATTCCCTCAGTAACGGCGAGTGAACAGGGAAGAGCCCAGCGCCGAATCCCCGCCCCGCGGTGGGGCGCGGGAAATGTGGCGTACGGAAGACCCACTCCCCGGCGCCGCTCGTGGGGGGCCCAAGTCCTTCTGATCGAGGCCCAGCCCGTGGACGGTGTGAGGCCGGTAGCGGCCCCCGGCGCGCCGGGCCCGGGTCTTCCCGGAGTCGGGTTGCTTGGGAATGCAGCCCAAAGCGGGTGGTAAACTCCATCTAAGGCTAAATACCGGCACGAGACCGATAGTCAACAAGTACCGTAAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGGGCGTGAAACCGTTAAGAGGTAAACGGGTGGGGTCCGCGCAGTCCGCCCGGAGGATTCAACCCGGCGGCGGGTCCGGCCGTGTCGGCGGCCCGGCGGATCTTTCCCGCCCCCCGTTCCTCCCGACCCCTCCACCCGCCCTCCCTCCCCCGCCGCCCCTCCTCCTCCTCCCCGGAGGGGGCGGGCTCCGGCGGGTGCGGGGGTGGGCGGGCGGGGCCGGGGGTGGGGTCGGCGGGGGACCGTCCCCCGACCGGCGACCGGCCGCCGCCGGGCGCATTTCCACCGCGGCGGTGCGCCGCGACCGGCTCCGGGACGGCTGGGAAGGCCTGGCGGGGAAGGTGGCTCGGGGGGCCCCGTCCCGCCCCGTCTTCCCCCCGCCCGCGTCCTCCCCCGGGAGGGCGCGGGTCGGGGTGGCGGCGGCGGTGGCGGCGGGACCACCCCCCGAGTGTTACAGCCCCCCGGCAGCAGCACTCGCCGAATCCCGGGGCCGAGGGAGCGAGACCCGTCGCCGCGCTCTCCCCCCTCCCGGCGCCCACCCCCGCGGGGGCCCCCCGCGAGGGGGTCCCCCCCGCGGGGGCGCGCCGGCGTTCCTCGTGGGGGGCCGGGCCACCCCTCCCACGGCGCGACCGCTCTCCCACCCCCTCCCCGCACCCCCGGCGACGGGGGCCCGCGCGGGTGGGGGCGGGGCGGACTGTCCCCAGTGCGCCCCGGGCGGGTCGCGCCGTCGGGCCCGGGGGGGTTCTCTCGGGGCCACGCGCGCGTCCCTCGAAGAGGGGGACGGCGGAGCGAGCGCACGGGGTCGGCGGCGATGTCGGCTACCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACACGTGCGCGAGTCAGGGGCTCGCACGAAAGCCGCCGTGGCGCAATGAAGGTGAAGGCCGGCGCGCTCGCCGGCCGAGGTGGGATCCCGAGGCCTCTCCAGTCCGCCGAGGGCGCACCACCGGCCCGTCTCGCCCGCCGCGCCGGGGAGGTGGAGCACGAGCGCACGTGTTAGGACCCGAAAGATGGTGAACTATGCCTGGGCAGGGCGAAGCCAGAGGAAACTCTGGTGGAGGTCCGTAGCGGTCCTGACGTGCAAATCGGTCGTCCGACCTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCCTCCGAAGTTTCCCTCAGGATAGCTGGCGCTCTCGCAAACCCAACCTCCCACGCAGTTTTATCCGGTAAAGCGAATGATTAGAGGTCTTGGGGCCGAAACGATCTCAACCTATTCTCAAACTTTAAATGGGTAAGAAGCCCGGCTCGCTGGCGTGGAGCCGGGCGTGGAATGCGAGTGCCTAGTGGGCCACTTTTGGTAAGCAGAACTGGCGCTGCGGGATGAACCGAACGCCGGGTTAAGGCGCCCGATGCCGACGCTCATCAGACCCCAGAAAAGGTGTTGGTTGATATAGACAGCAGGACGGTGGCCATGGAAGTCGGAATCCGCTAAGGAGTGTGTAACAACTCACCTGCCGAATCAACTAGCCCTGAAAATGGATGGCGCTGGAGCGTCGGGCCCATACCCGGCCGTCGCCGGCAGTCGAGAGTGGACGGGAGCGGCGGGGGTCGGCGCGCGTGGGGGTGCAGCGTGCGTGGGGGGGGTCTCCCCTCCTCCTCCTCCCCCCCCGCCCGCCCCCGGAGCCCCGCGGACGCTACGCCGCGACGAGTAGGAGGGCCGCTGCGGTGAGCCTTGAAGCCTAGGGCGTGGGCCCGGGTGGAGCCGCCGCAGGTGCAGATCTTGGTGGTAGTAGCAAATATTCAAACGAGAACTTTGAAGGCCGAAGTGGAGAAGGGTTCCATGTGAACAGCAGTTGAACATGGGTCAGTCGGTCCTGAGAGATGGGCGAGCGCCGTTCCGAAGGGACGGGCGATGGCCTCCGTTGCCCTCAGCCGATCGAAAGGGAGTCGGGTTCAGATCCCCGAATCCGGAGTGGCGGAGATGGGCGCCGCGAGGCGTCCAGTGCGGTAACGCGACCGATCCCGGAGAAGCCGGCGGGAGCCCCGGGGAGAGTTCTCTTTTCTTTGTGAAGGGCAGGGCGCCCTGGAATGGGTTCGCCCCGAGAGAGGGGCCCGTGCC TTGGAAAGCGTCGCGGTTCCGGCGGCGTCCGGTGAGCTCTCGCTGGCCCTTGAAAATCCGGG GGAGAGGGTGTAAATCTCGCGCCGGGCCGTACCCATATCCGCAGCAGGTCTCCAAGGTGAAC AGCCTCTGGCATGTTGGAACAATGTAGGTAAGGGAAGTCGGCAAGCCGGATCCGTAACTTCG GGATAAGGATTGGCTCTAAGGGCTGGGTCGGTCGGGCTGGGGCGCGAAGCGGGGCTGGGCGC GCGCCGCGGCTGGACGAGGCGCCGCCGCCCCCCCCACGCCCGGGGCACCCCCCTCGCGGCCC TCCCCCGCCCCACCCCGCGCGCCTCTCGCTCCCTCCCCCGCGCCCTCTCTCCCCCTCCCCTC CCCGGGGGTGCGGGGGGAAGGGTCGGGCGGAGGGGCGGCGGCGGCCGCGGGGCCCCGGTGGC GGGGGCACGGTCCCCCGCGGGGGGGGCCCGGGCACCCGGGGGGCCGGCGGCGGCGGCGACTCTGGACGCGAGCCGGGCCCTTCCCGTGGATCGCCCCAGCTGCGGCGGGCGTCGCGGCCGCCCC CGGGGAGCCCGGCGGGCGCCGGCGCGCCCCGCTCGCTCCGCCGTCGCGCGCGTCCGCGGGGG CGGGGAGCGGTCGGGCGGCGGCGTCGGTGGGCGGCGGGCGGGGGTTCGTCCCCCCGCCTCCC CCCCGGCCCGTCCGCCCCCCGTTCCCCCCCCTCCTCGCCGCGCGGCGGCGGCGGCGGCGGGC CGCGGGCCGGTCCCCCCCGCCGGGTCCGCCCCCGGGGCCGCGGTTCCGCGCGGCGCCTCGCC TCGGCCGGCGCCTAGCAGCCGACTTAGAACTGGTGCGGACCAGGGGAATCCGACTGTTTAAT TAAAACAAAGCATCGCGAAGGCCCGCGGCGGGTGTTGACGCGATGTGATTTCTGCCCAGTGC TCTGAATGTCAAAGTGAAGAAATTCAATGAAGCGCGGGTAAACGGCGGGAGTAACTATGACT CTCTTAAGGTAGCCAAATGCCTCGTCATCTAATTAGTGACGCGCATGAATGGATGAACGAGA TTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAAGGGAACGGGCTTGGCGGAAT CAGCGGGGAAAGAAGACCCTGTTGAGCTTGACTCTAGTCTGGCACGGTGAAGAGACATGAGA GGTGTAGAATAAGTGGGAGGCCCCCGGCGCCCCTCCGTCCCCGCGAGGGGGCGGGGCGGGGT CCGCCGGCCTTGCGGGCCGCCGGTGAAATACCACTACTCTGATCGTTTTTTCACTGACCCGG TGAGGCGGGGGGGCGAGCCCCGAGGGGCTCTCGCTTCTGGCGCCAAGCGCCCGGCCGCGCGC CGGCCGGGCGCGACCCGCTCCGGGGACAGTGCCAGGTGGGGAGTTTGACTGGGGCGGTACAC CTGTCAAACGGTAACGCAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCGTGGA GCAGAAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTGAAAGCGGG GCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGGAGGTGTCAGAAAAGTTACCACAGG GATAACTGGCTTGTGGCGGCCAAGCGTTCATAGCGACGTCGCTTTTTGATCCTTCGATGTCG GCTCTTCCTATCATTGTGAAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGG AACGTGAGCTGGGTTTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGATGTGTTG TTGCCATGGTAATCCTGCTCAGTACGAGAGGAACCGCAGGTTCAGACATTTGGTGTATGTGC TTGGCTGAGGAGCCAATGGGGCGAAGCTACCATCTGTGGGATTATGACTGAACGCCTCTAAG TCAGAATCCCGCCCAGGCGGAACGATACGGCAGCGCCGCGGAGCCTCGGTTGGCCTCGGATA GCCGGTCCCCCGCCTGTCCCCGCCGGCGGGCCGCCTCGCCCCGCGCGGGGCGTGCCCCGCCG CGCGCCGGGACCGGGGTCCGGTGCGGAGTGCCCTTCGTCCTGGGAAACGGGGTGCGGCCGGA AAGGCGGCCGCCCCCTCGCCCGTCACGCAACGCACGTTCGTGGGGAACCTGGCGCTAAACCA TTCGTAGACGACCTGCTTCTGGGTCGGGGTTTCGTACGTAGCAGAGCAGCTCCCTCGCTGCG ATCTATTGAAAGTCAGCCCTCGACACAAGGGTTTGT

[0269] SEQ ID NO: 18 - Mmu-28S:CGCGACCTCAGATCAGACGTGGCGACCCGCTGAATTTAAGCATATTAGTCAGCGGAGGAAAA GAAACTAACCAGGATTCCCTCAGTAACGGCGAGTGAACAGGGAAGAGCCCAGCGCCGAATCC CCGCCGCGCGTCGCGGCGTGGGAAATGTGGCGTACGGAAGACCCACTCCCCGGCGCCGCTCG TGGGGGGCCCAAGTCCTTCTGATCGAGGCCCAGCCCGTGGACGGTGTGAGGCCGGTAGCGGC CCCCGGCGCGCCGGGCTCGGGTCTTCCCGGAGTCGGGTTGCTTGGGAATGCAGCCCAAAGCG GGTGGTAAACTCCATCTAAGGCTAAATACCGGCACGAGACCGATAGTCAACAAGTACCGTAA GGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGGGCGTGAAACCGTTAAGAGGTAA ACGGGTGGGGTCCGCGCAGTCCGCCCGGAGGATTCAACCCGGCGGCGCGCGTCCGGCCGTGCCGGTGGTCCCGGCGGATCTTTCCCGCTCCCCGTTCCTCCCGACCCCTCCACCCGCGCGTCGT TCCCCTCTTCCTCCCCGCGTCCGGCGCTCCGGCGGCGGGCGCGGGGGGTGGTGTGGTGGTGGCGCGCGGGCGGGGCCGGGGGTGGGGTCGGCGGGGGACCGCCCCCCGGCCGGCGACCGGCCGCCGCCGGGCGCACTTCCACCGTGGCGGTGCGCCGCGACCGGCTCCGGGACGGCCGGGAAGGCCCGGTGGGGAAGGTGGCTCGGGGGGGGCGGCGCGTCTCAGGGCGCGCCGAACCACCTCACCCCGAGTGTTACAGCCCTCCGGCCGCAGCTTTCGCCGAATCCCGGGGCCGAGGAAGCCAGATACCCGTCGCCGCGCTCTCCCTCTCCCCCCGTCCGCCTCCCGGGCGGGCGTGGGGGTGGGGGCCGGGCCGCCCCTCCCACGGCGCGACCGCTCTCCCACCCCCCTCCGTCGCCTCTCTCGGGGCCCGGTGGGGGGCGGGGCGGACTGTCCCCAGTGCGCCCCGGGCGTCGTCGCGCCGTCGGGTCCCGGGGGGACCGTCGGTCACGCGTCTCCCGACGAAGCCGAGCGCACGGGGTCGGCGGCGATGTCGGCTACCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACGCGTGCGCGAGTCAGGGGCTCGTCCGAAAGCCGCCGTGGCGCAATGAAGGTGAAGGGCCCCGCCCGGGGGCCCGAGGTGGGATCCCGAGGCCTCTCCAGTCCGCCGAGGGCGCACCACCGGCCCGTCTCGCCCGCCGCGCCGGGGAGGTGGAGCACGAGCGTACGCGTTAGGACCCGAAAGATGGTGAACTATGCCTGGGCAGGGCGAAGCCAGAGGAAACTCTGGTGGAGGTCCGTAGCGGTCCTGACGTGCAAATCGGTCGTCCGACCTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCCTCCGAAGTTTCCCTCAGGATAGCTGGCGCTCTCGCTCCCGACGTACGCAGTTTTATCCGGTAAAGCGAATGATTAGAGGTCTTGGGGCCGAAACGATCTCAACCTATTCTCAAACTTTAAATGGGTAAGAAGCCCGGCTCGCTGGCGTGGAGCCGGGCGTGGAATGCGAGTGCCTAGTGGGCCACTTTTGGTAAGCAGAACTGGCGCTGCGGGATGAACCGAACGCCGGGTTAAGGCGCCCGATGCCGACGCTCATCAGACCCCAGAAAAGGTGTTGGTTGATATAGACAGCAGGACGGTGGCCATGGAAGTCGGAATCCGCTAAGGAGTGTGTAACAACTCACCTGCCGAATCAACTAGCCCTGAAAATGGATGGCGCTGGAGCGTCGGGCCCATACCCGGCCGTCGCCGGCAGTCGGAACGGAACGGGACGGGAGCGGCCGCGGGTGCGCGTCTCTCGGGGTCGGGGGTGCGTGGCGGGGGCCCGTCCCCCGCCTCCCCTCCGCGCGCCGGGTTTCGCCCCCGCGGCGTCGGGCCCCGCGGACGCTACGCCGCGACGAGTAGGAGGGCCGCTGCGGTGAGCCTTGAAGCCTAGGGCGCGGGCCCGGGTGGAGCCGCCGCAGGTGCAGATCTTGGTGGTAGTAGCAAATATTCAAACGAGAACTTTGAAGGCCGAAGTGGAGAAGGGTTCCATGTGAACAGCAGTTGAACATGGGTCAGTCGGTCCTGAGAGATGGGCGAGTGCCGTTCCGAAGGGACGGGCGATGGCCTCCGTTGCCCTCGGCCGATCGAAAGGGAGTCGGGTTCAGATCCCCGAATCCGGAGTGGCGGAGATGGGCGCCGCGAGGCGTCCAGTGCGGTAACGCGACCGATCCCGGAGAAGCCGGCGGGAGCCCCGGGGAGAGTTCTCTTTTCTTTGTGAAGGGCAGGGCGCCCTGGAATGGGTTCGCCCCGAGAGAGGGGCCCGTGCCTTGGAAAGCGTCGCGGTTCCGGCGGCGTCCGGTGAGCTCTCGCTGGCCCTTGAAAATCCGGGGGAGAGGGTGTAAATCTCGCGCCGGGCCGTACCCATATCCGCAGCAGGTCTCCAAGGTGAACAGCCTCTGGCATGTTGGAACAATGTAGGTAAGGGAAGTCGGCAAGCCGGATCCGTAACTTCGGGATAAGGATTGGCTCTAAGGGCTGGGTCGGTCGGGCTGGGGCGCGAAGCGGGGCTGGGCGCGCGCCGCGGCTGGACGAGGCGCCGCCGCCCTCTCCCACGTCCGGGGAGACCCCCCGTCCTTTCCGCCCGGGCCCGCCCTCCCCTCTTCCCCGCGGGGCCCCGTCGTCCCCCGCGTCGTCGCCACCTCTCTTCCCCCCTCCTTCTTCCCGTCGGGGGGCGGGTCGGGGGTCGGCGCGCGGCGCGGGCTCCGGGGCGGCGGGTCCAACCCCGCGGGGGTTCCGGAGCGGGAGGAACCAGCGGGCCCCCGGTGGGGCGGGGGGCCCGGACACTCGGGGGGGCCGGCGGCGGCGGCGACTCTGGACGCGAGCCGGGCCCTTCCCGTGGATCGCCTCAGCTGCGGCGGGCGTCGCGGCCGCTCCCGGGGAGCCCGGCGGGTGCCGGCGCGGGTCCCCTCCCCGCGGGGCCTCGCTCCACCCCCCCATCGCCTCTCCCGAGGTGCGTGGCGGGGGCGGGCGGGCGTCCCGCGCGTGTGGGGGGAACCTCCGCGTCGGTGTTCCCCCGCCGGGTCCGCCCCCCGGGCCGCGGTTTTCCGCGCGGCGCCTCGCCTCGGCCGGCGCCTAGCAGCCGACTTAGAACTGGTGCGGACCAGGGGAATCCGACTGTTTAATTAAAACAAAGCATCGCGAAGGCCCGCGGCGGGTGTTGACGCGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAAGTGAAGAAATTCAATGAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCTCTTAAGGTAGCCAAATGCCTCGTCATCTAATTAGTGACGCGCATGAATGGATGAACGAGATTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAAGGGAACGGGCTTGGCGGAATCAGCGGGGAAAGAAGACCCTGTTGAGCTTGACTCTAGTCTGGCACGGTGAAGAGACATGAGAGGTGTAGAATAAGTGGGAGGCCCCCGGCGCCCGGCCCCGTCCTCGCGTCGGGGTCGGGGCACGCCGGCCTCGCGGGCCGCCGGTGAAATACCACTACTCTCATCGTTTTTTCACTGACCCGGTGAGGCGGGGGGGCGAGCCCCGAGGGGCTCTCGCTTCTGGCGCCAAGCGTCCGTCCCGCGCGTGCGGGCGGGCGCGACCCGCTCCGGGGACAGTGCCAGGTGGGGAGTTTGACTGGGGCGGTACACCTGTCAAACGGTAACGCAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCGTGGAGCAGAAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTGAAAGCGGGGCCTCACGATCCTTCTGACCTTTTGGGTTTTAAGCAGGAGGTGTCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCGGCCAAGCGTTCATAGCGACGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATTGTGAAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTGAGCTGGGTTTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGATGTGTTGTTGCCATGGTAATCCTGCTCAGTACGAGAGGAACCGCAGGTTCAGACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCATCTGTGGGATTATGACTGAACGCCTCTAAGTCAGAATCCCGCCCAGGCGGAACGATACGGCAGCGCCGAAGGAGCCTCGGTTGGCCCCGGATAGCCGGGTCCCCGTCCGTCCCCGCTCGGCGGGGTCCCCGCGTCGTCCCCGCGGCGGCGCGGGGTCTCCCCCCGCCGGGCGTCGGGACCGGGGTCCGGTGCGGAGAGCCGTTCGTCTTGGGAAACGGGGTGCGGCCGGAAAGGGGGCCGCCCTCTCGCCCGTCACGTTGAACGCACGTTCGTGTGGAACCTGGCGCTAAACCATTCGTAGACGACCTGCTTCTGGGTCGGGGTTTCGTACGTAGCAGAGCAGCTCCCTCGCTGCGATCTATTGAAAGTCAGCCCTCGACACAAGGGTTTGTCTCTGCGGGCTTTC

[0270] SEQ ID NO: 19 - Ssc-28S:TGCCGCGCCCCGCCCCGCCCGCCCGTCCCCGTGCCCGCCGGCTCGTGCTTCCCCGTGCCGCCGCCCGTCTGTGTTCCCTTCTGCCTCCCACCCCCGGCGCCTCCCCCCGCTTGAGCGGGTGGGGGCGGGGTGGGGTGGGGTGGGGGAGCCAGGCGGGTGGAGGCTGGACCGGGGGTCGGTCCTCGCGCGTGGCTCTCGTACCGTGTCCCCTTCCCACCTGGCGGGTGCCCTGTTCCCCGCGCGTGGGCGAGCGAGCGAGCGAGCGGACGAGCGAGGGGGAGCCGATCGGGCTGTCCGCGCCCGTGTCCCATCTGTCCCGTCCCGCCCGCCCGCCCGCTTCGCCCTCCGCCCCTCCGAGACGCGACCTCAGATCAGACGTGGCGACCCGCTGAATTTAAGCATATTAGTCAGCGGAGGAAAAGAAACTAACCAGGATTCCCTCAGTAACGGCGAGTGAACAGGGAAGAGCCCAGCGCCGAATCCCCGCCCCGCGGTGGGGCGCGGGACATGTGGCGTACGGAAGACCCACTCCCCGGCGCCGCTCGTGGGGGGCCCAAGTCCTTCTGATCGAGGCCCAGCCCGTGGACGGTGTGAGGCCGGTAGCGGCCCCCGGCGCGCCGGGCCCGGGTCTTCCCGGAGTCGGGTTGCTTGGGAATGCAGCCCAAAGCGGGTGGTAAACTCCATCTAAGGCTAAATACCGGCACGAGACCGATAGTCAACAAGTACCGTAAGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGGGCGTGAAACCGTTAAGAGGTAAACGGGTGGGGTCCGCGCAGTCCGCCCGGAGGATTCAACCCGGCGGCGGGTCCGGCCGTGCCGGCGGCCCGGCGGATCTTTCCCGCTCCCCGTGCCTCCCGGCCCCTCCACCCGCCCTCCGTCCGCGCCCCCTCGCCGCTTCGGCGGCGGGGGGTGTCGCGGGGGTGGGCGGGCGGGGCCGGGGGTGGGGCCGGCGGGGGACCGCCCCCCGGTCGGCGTCCACGGCCGCCGCCGGGCGCATTTCCACCGCGGCGGTGCGCCGCGACCGGCTCCGGGACGGCTGGGAAGGCCGGTGGGGAAGGTGGCTCGGGGGGCCCCGTCGTCCTCTCCCTCCCTCCTCTCCGGAGGGGGAGGGGGGGGCCCGCGGCGGGCCCACCGCCCCGAGTGTTACAGCCCCCCGGCAGCAGCGCTCGCCGAATCCCGGGGCCGAGGGAGTGAGACCTGTCGCCGCGCTCTCCCCCCTCCCGGCGCTCCCCCCCGCGGGGGCGCTTCCCCGCCAGGGGGGGTGTCCCTCGCGGGGGCGCGCCGGGTTTTTCTTGGTGGGGGCCGGGCCGCCCCTCCCACGGCGCGACCGCTCTCCCACCCCGGCCTCGCCTCCTCCTCCCCCCCTTCCCGGGGTGTGGGGAGGGCCACGGTGCGGGTCGGGGCGGGGCGGACTGTCCTCAGTGCGCCCCGGGCGGGTCGCGCCGTCGGGCCCGGGGGGTTGGTTTTTTTGGTCCTCGGCCACGCCGTCACGAGCGAAGCGAGCGCACGGGGTCAGCGGCGATGTCGGCCACCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACACGTGCGCGAGTCAGGGGCTCGCACGAAAGCCGCCGTGGCGCAATGAAGGTGAAGGCCGCCGCTCGCCGGCGGCCGAGGTGGGATCCCGAGGCCTCTCCGAGTCCGCCGAGGGCGCACCACCGGCCCGTCTCGCCCGCCGCGCCGGGGAGGTGGAGCATGAGCGCACGTGTTAGGACCCGAAAGATGGTGAACTATGCCTGGGCAGGGCGAAGCCAGAGGAAACTCTGGTGGAGGTCCGTAGCGGTCCTGACGTGCAAATCGGTCGTCCGACCTGGGTATAGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTCCCTCCGAAGTTTCCCTCAGGATAGCTGGCGCTCTCGCAGGAAACAGTTTTATCCGGTAAAGCGAATGATTAGAGGTCTTGGGGCCGAAACGATCTCAACCTATTCTCAAACTTTAAATGGGTAAGAAGCCCGGCTCGCTGGCGTGGAGCCGGGCGTGGAATGCGAGTGCCTAGTGGGCCACTTTTGGTAAGCAGAACTGGCGCTGCGGGATGAACCGAACGCCGGGTTAAGGCGCCCGATGCCGACGCTCATCAGACCCCAGAAAAGGTGTTGGTTGATATAGACAGCAGGACGGTGGCCATGGAAGTCGGAATCCGCTAAGGAGTGTGTAACAACTCACCTGCCGAATCAACTAGCCCTGAAAATGGATGGCGCTGGAGCGTCGGGCCCATACCCGGCCGTCGCCGGCAGTCGGAGCAGCGCGCGAGAGGGACGGGAGCGGGGCGCGCGGTCGCCCGGGGACGCCGGGGTCGCGGGGGGTGGGGGTCTGGGGGGTGCGTCGCGGGGGTGGCGGCGGGGGGCCTCTCCCGCCCGTCGCCCGCGCGCGCGCGCGCCCCTCTCTCCCTCCCCCCCCTCCCCGGCCCCCCGTGGCGCCGCCGCCGGAAACCCCCCACCGCGGACGCTACGCCGCGACGAGTAGGAGGGCCGCTGCGGTGAGCCTTGAAGCCTAGGGCGTGGGCCCGGGTGGAGCCGCCGCAGGTGCAGATCTTGGTGGTAGTAGCAAATATTCAAACGAGAACTTTGAAGGCCGAAGTGGAGAAGGGTTCCATGTGAACAGCAGTTGAACATGGGTCAGTCGGTCCTGAGAGATGGGCGAGTGCCGTTCCGAAGGGACGGGCGATGGCCTCCGTTGCCCTCAGCCGATCGAAAGGGAGTCGGGTTCAGATCCCCGAATCCGGAGTGGCGGAGATGGGCGCCGCGAGGCGTCCAGTGCGGTAACGCAACCGATCCCGGAGAAGCCGGCGGGAGCCCCGGGGAGAGTTCTCTTTTCTTTGTGAAGGGCAGGGCGCCCTGGAATGGGTTCGCCCCGAGAGAGGGGCCCGTGCCTTGGAAAGCGTCGCGGTTCCGGCGGCGTCCGGTGAGCTCTCGCTGGCCCTTGAAAATCCGGGGGAGAGGGTGTAAATCTCGCGCCGGGCCGTACCCATATCCGCAGCAGGTCTCCAAGGTGAACAGCCTCTGGCATGTTGGAACAATGTAGGTAAGGGAAGTCGGCAAGCCGGATCCGTAACTTCGGGATAAGGATTGGCTCTAAGGGCTGGGTCGGTCGGGCTGGGGCGCGAAGCGGGGCTGGGCGCGCGCCGCGGCTGGACGAGGCGCCGCCGCCACCCCCACGCCCGGGGCACCCCCGGCCGGGCCCGCCCCCGCGGTCCTCCTCCGCCCCACCCCGCGCGGCTCCCTCCACCCGCCCCCCCCTTCGCCGGTCTCCTCCCGCCCCCCCGCCTCCCACCTCCGCGGGGGGCGGGTGGGGGGGCGGCGGGACGGGTGTCGGGGGGAAGGGCGGGAGCGGCCGGGGCCCCGGTGGCGGGGGGGGTCCCCCGCGGGGTCCCGCGGGCCCACGGGGGCCCGGGCACCCGGGGGGCCGGCGGCGGCGGCGACTCTGGACGCGAGCCGGGCCCTTCCCGTGGATCGCCCCAGCTGCGGCGGGCGTCGCGGCCGCCCTCGGGGAGCCCGGCGGGCGCCGGCGCGCCCCCGGCCGCGCGCGCGCGCGCGCGTGTGCGCGGGAGGGGTGCGGTGCGTGCGTGTCGCGCCGCGCCACCACCCCGCCTCCTCTCGCCGCCGCGCCGCGCGCGTGCGCGTCGGGTCCCGGCGCGCCGGTCCCCCCCGCCGGGTGCGCCCCCGGGGCCGCGGTTCCGCGCGGCGCCTCGCCTCGGCCGGCGCCTAGCAGCCGACTTAGAACTGGTGCGGACCAGGGGAATCCGACTGTTTAATTAAAACAAAGCATCGCGAAGGCCCGCGGCGGGTGTTGACGCGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAAGTGAAGAAATTCAATGAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCTCTTAAGGTAGCCAAATGCCTCGTCATCTAATTAGTGACGCGCATGAATGGATGAACGAGATTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAAGGGAACGGGCTTGGCGGAATCAGCGGGGAAAGAAGACCCTGTTGAGCTTGACTCTAGTCTGGCACGGTGAAGAGACATGAGAGGTGTAGAATAAGTGGGAGGCCCCCGGCGTCCCGTCCCGTGGTCTCCCCGCGAGGGGCGGCGCGGGGCGGGGGTCCGCCGGCCTTGCGGGCCGCCGGTGAAATACCACTACTCTGATCGTTTTTTCACTGACCCGGTGAGGCGGGGGGGCGAGCCCCGAGGGGCTCTCGCTTCTGGCGCCAAGCGCCCGGCCGCGCGCCGGCCGGGCGCGACCCGCTCCGGGGACAGTGCCAGGTGGGGAGTTTGACTGGGGCGGTACACCTGTCAAACGGTAACGCAGGTGTCCTAAGGCGAGCTCAGGGAGGACAGAAACCTCCCGTGGAGCAGAAGGGCAAAAGCTCGCTTGATCTTGATTTTCAGTACGAATACAGACCGTGAAAGCGGGGCCTCACGATCCTTCTGACCTTTGGGGTTTTAAGCAGGAGGTGTCAGAAAAGTTACCACAGGGATAACTGGCTTGTGGCGGCCAAGCGTTCATAGCGACGTCGCTTTTTGATCCTTCGATGTCGGCTCTTCCTATCATTGTGAAGCAGAATTCACCAAGCGTTGGATTGTTCACCCACTAATAGGGAACGTGAGCTGGGTTTAGACCGTCGTGAGACAGGTTAGTTTTACCCTACTGATGATGTGTTGTTGCCATGGTAATCCTGCTCAGTACGAGAGGAACCGCAGGTTCAGACATTTGGTGTATGTGCTTGGCTGAGGAGCCAATGGGGCGAAGCTACCATCTGTGGGATTATGACTGAACGCCTCTAAGTCAGAATCCCGCCCAGGCGGAACGATACGGCAGCGCCGCGGGAGCCTCGGTTGGCCTCGGATAGCCGGTCCCCCGCCGTCCCCGCCGGCGGGCCGCCGCGCGCGCCCGCGTGCGGCGTGTCCCGTCGCGCGTCGGGACCGGGGTCCGGTGCGGAGAGCCCTTCGTCCTGGGAAACGGGGCGCGGCCGGAAAGCGCGGCCGCCCCCTCGCCCGTCACGCACCGCACGTTCGTGGGGAACCTGGTGCTAAACCATTCGTAGACGACCTGCTTCTGGGTCGGGGTTTCGTACGTAGCAGAGCAGCTCCCTCGCTGCGATCTATTGAAAGTCAGCCCTCGACACAAGGGTTTGTCGCTCCCGGCGCCGACGCCGChemical synthesis

[0271] A 4 mL screw-cap vial was charged with the pseudouridine (0.1 mmol, 1.0 equiv.). The Triethylammonium bicarbonate buffer (TEAB, 1.0 M, 200 pL, pH = 8.5) was then added in one portion at room temperature, followed by the addition of methyl 2-bromoacrylate solution (2.0 M, 100 pL, 2.0 equiv.). The reaction vial was sealed again and incubated at 70 °C for 2 hours. The crude reaction mixture was then concentrated under reduced pressure (in vacuo) and purified by preparative thin layer chromatography (5:2:0.1 DCM: MeOH: NEts) on silica gel to obtain the product as a white powder in 95% isolated yield (30 mg). (Table 1)Reaction Conditions and buffer preparation

[0272] To develop a selective chemical probe for pseudouridine modification, a panel of a, P-unsaturated compounds was screened for reactivity with pseudouridine under mild conditions. The tested compounds included acrylonitrile derivatives with substituents such as chloride (-C1), bromide (-Br), ester (-COOR), and others to enhance reactivity (see FIG. IB for a list of compounds 1-12). Initial screening was conducted using single nucleosides (A, U, G, C, T, m6,6A) to precisely measure reaction kinetics.General procedure

[0273] A single nucleoside mixture solution was prepared with a molar ratio of A: U: G: C: : m6,6A = 1 : 1 : 1 : 1 : 1 : 1 using 100 mM stock solutions of each nucleoside (A, U, G, C, T, m6’6A). A 1.0 pL aliquot of the nucleoside mixture was combined with 19 pL of nuclease-free water, followed by the addition of 25.0 pL TEAB buffer (1.0 M triethylammonium bicarbonate) and 1.0 pL of a 2.0 M compound solution in DMSO. The mixture was incubated at room temperature for 10 minutes. The crude reaction mixture was then diluted to 100 pL with nuclease-free water and filtered through a 0.22 pm filter (Millipore, SLGVR04NL) before being directly analyzed using an Agilent 6130 ESI / CI LC-MS system. For each injection, 10 pL of the sample was injected, and nucleosides were separated by reverse-phase UHPLC using a C18 column (Agilent, 959963-092) followed by mass spectrometry (MS) detection. Nucleosides were quantified using a UV detector with m6,6A as the internal standard. The final reaction ratios of nucleosides were determined using calibration curves obtained from nucleoside standards analyzed under the same conditions. For pH testing, reactions were performed in sodium phosphate buffer across a pH range from 1.0 to 13.0. For temperatureoptimization, reactions were conducted at temperatures ranging from 10 °C to 95 °C. For reaction time variation, incubation times were tested between 5 and 60 minutes. For buffer testing, reactions were performed in TEAB, phosphate, sodium borate (Na3BO3), and triethylammonium acetate (TEAA), all adjusted to pH 7.5 (see Table 1).Table 1 - BuffersReaction of model RNA oligonucleotides with BS and MALDI-TOF MS

[0274] To 8 pL of nuclease-free water, 1 pL of synthetic RNA oligo AGXGA (X= C, U, or T, 100 ng pL1) was added, followed by mixing well via pipetting. The reaction mixture was then supplemented with 10.0 pL of TEAB buffer (1.0 M triethylammonium bicarbonate) and 1.0 pL of a 2.0 M compound solution in DMSO. The mixture was incubated in PCR instrument at 70 °C for 2 min, followed by 37 °C for 3 hours. After cooling to room temperature, 2 pL of the mixture was added to 40 pL resin (Bio-Rad) and left to stand at room temperature for 30 min. The supernatant was then used directly for matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) analysis. For MALDI-TOF, the matrix solution was prepared by mixing 2',4',6'-trihydroxyacetophenone (10 mg mF1in 50% CH3CN / H2O) and ammonium citrate (50 mg ml1in H2O) at an 8: 1 (v / v) ratio. A 1.0 pL aliquot of the purified reaction product was combined with 1.0 pL of the matrix, spotted onto the MALDI sample plate, allowed to dry, and analyzed using a Bruker Ultraflextreme MALDI-TOF / TOF mass spectrometer. The MALDI-TOF MS recorded the signals using negative reflector mode.RNA preparation

[0275] RNA oligo with NN-T-NN motifTable 2. Synthesis by IDT and purified by agarose gel.

[0276] RNA Sources of cell lines samples. Human embryonic kidney cells (HEK293T) were used for method development and validation. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA integrity was verified by agarose gel electrophoresis, and concentrations were quantified with a NanoDrop spectrophotometer. Small RNA: RNAs <200 nucleotides were enriched using the mirVana miRNA Isolation Kit (Ambion). Poly(A)+ RNA: Messenger RNA was isolated using the Poly(A)Purist MAG Kit (Ambion).

[0277] RNA Sources of mammalian tissues. Two tissue samples, brain and testis from 4 species human (Homo sapiens), monkey (Macaca fascicularis), mouse (Mus musculus), and pig (Sus scrofa) were included in this study. Total RNA samples of mammalian species were purchased from Zyagen (see Table 3). Small RNA fraction was selected using RNA Clean and Concentrator kit (Zymo Research).Table 3 - RNA samples of mammalian speciesCatalog NO. Species TissuesPUM-seq Library Construction

[0278] PUM-seq libraries were prepared to detect pseudouridine modifications via chemically induced T-to-C mutations during reverse transcription. The process included RNA fragmentation, 3 ’-adapter ligation, chemical treatment, reverse transcription, and library amplification. RNA was fragmented using RNA Fragmentation Reagents (E6150S, NEB) at 94 °C for 3 minutes, followed by purification with the RNA Clean and Concentrator kit (Zymo Research). Fractionated RNA was 3 '-end repaired using T4 polynucleotide kinase (PNK, M0201S, NEB). RNA was combined with 2 pL of 10x T4 PNK Reaction Buffer (B0201S, NEB) and 3 pL of T4 PNK, diluted to a final volume of 20 pL, and incubated at 37 °C for 60 minutes. The reaction was then purified using the RNA Clean and Concentrator kit (Zymo Research) and eluted with 10 pL of RNase-free water. For 3'-adapter ligation, 10 pL of 3'- repaired RNA fragments were incubated with 1 pL of 20 pM RNA 3 '-Adapter (SEQ ID NO: 4) at 70 °C for 2 minutes and immediately placed on ice. The reaction was supplemented with 2.5 pL of 10* T4 RNA Ligase Reaction Buffer (M0373L, NEB), 7.5 pL of PEG8000 (50%), 1 pL of SUPERaserin RNase Inhibitor, and 1 pL of T4 RNA Ligase 2 truncated KQ (M0373L, NEB). The mixture was incubated at 25 °C for 2 hours, followed by an overnight incubation at 16 °C for 12 hours. The reaction was diluted to a final volume of 47 pL with nuclease-free water, and excess adapters were removed by adding 2 pL of 5 '-deadenylase (M0331S, NEB) and incubating at 30 °C for 30 minutes, followed by the addition of 1 pL RecJf (M0264L, NEB) for single-stranded DNA digestion at 37 °C for 30 minutes. The 3 '-end-ligated RNA was purified using the RNA Clean and Concentrator kit (Zymo Research) and eluted with 10 pL of RNase-free water. A 2.0 pl aliquot of the purified RNA was saved for ‘Input’ libraryconstruction, 8.0 pl was subjected to PUM-seq optimized condition, as the ‘Treated’ sample. Ligated RNA was incubated with methyl 2-bromoacrylate (150-200 mM) in TEAB buffer (pH 8.0) at 37 °C for 3 hours to selectively label T sites. An alternative quick protocol used 70 °C for 30 minutes. Both "Input" and "Treated" samples were mixed with 1 pL of 2.0 pM RT primer (SEQ ID NO: 5), heated to 65 °C for 2 minutes, and immediately placed on ice. To this, 4.0 pL of 5* RT Buffer, 1.0 pL of dNTP Solution Mix, 1.0 pL of dCTP Solution, 1.0 pL of RNaseOUT Recombinant Ribonuclease Inhibitor (10777019, Thermo Scientific), and 1.0 pL of Maxima H Minus Reverse Transcriptase (Invitrogen, EP0752) were added. The mixture was diluted with nuclease-free water up to 20 pL and thoroughly mixed and incubated at 50 °C for 1 hour, followed by the addition of 1 pL RNase H (M0297L, NEB) and incubation at 37 °C for 20 minutes. The reaction was then heated to 70°C for 5 minutes, and the resulting cDNA was purified using the DNA Clean and Concentrator kit (DCC, Zymo Research). The reaction buffer was supplemented with 10x dCTP to minimize background mutations. The purified cDNA was mixed with 2.0 pL of 10 pM cDNA adapter (SEQ ID NO: 6), heated at 70 °C for 2 minutes, and immediately placed on ice. To this mixture, 5 pL of 10x T4 RNA Ligase Reaction Buffer, 0.5 pL of 25 mM ATP, 25 pL of PEG8000 (50%), 1.25 pL of 40 mM CO(NH3)6C13solution, 3.75 pL of dimethyl sulfoxide (DMSO), and I pL of T4 RNA Ligase I (high concentration, M0437M, NEB) were added. The reaction was thoroughly mixed and incubated at 25 °C for 8 hours, followed by purification using the DNA Clean and Concentrator kit (Zymo Research), eluting with 20 pL of RNase-free water. The eluted cDNA (20 pL) was stored at - 80 °C, with 8 pL used for PCR amplification. Libraries were amplified via PCR with indexed primers and purified using AMPure XP beads (Beckman Coulter). Libraries were sequenced on the Illumina NovaSeq 6000 platform.Sequencing data analysis and *P sites detection

[0279] Sequencing adapters (P5: ACACGACGCTCTTCCGATCT (SEQ ID NO: 5) and P7: AGATCGGAAGAGCACACGTC (SEQ ID NO: 20) were removed using Cutadapt. The first 10 nucleotides (NNNNNNNNNN) at the beginning of Read 2 (R2) were extracted as unique molecular identifiers (UMIs) for each read pair, and the first two nucleotides of Read 1 (Rl) and the first nucleotide of the trimmed R2 were removed to mask putative random tails generated during reverse transcription (RT) and end-repair steps. Cleaned reads were aligned to reference genomes from Ensembl (human: GRCh38, monkey: Macaca_fascicularis_6.0, mouse: GRCm39, pig: Sscrofal l.l) using HISAT2-3n, with Read 1 and Read 2 swapped before mapping due to the ligation-based library preparation method, which produces a reversestrand sequencing readout for RNA. The alignment was performed using the parametersbase-change T,C -directional-mapping — no-softclip”. Mutation extraction was conducted using custom scripts with bcftools mpileup, and the mutation ratio was calculated as C / (C + T).

[0280] For pseudouridine site identification, positions with mutation rates exceeding 5% in untreated samples were filtered out to exclude potential SNPs or somatic mutations. A statistical model was applied to estimate the reliability of each detected site by leveraging the background noise ratio of the sample. Background noise was modeled for each six-letter motif (NNUNNN), based on the mutation ratio distribution of the bottom 95% quantile of all uridine (U) sites in the treated sample. The average background noise for each motif was calculated, and if the ratio was zero, a small prior value (0.0005) was assigned. The p-value for each site was computed by fitting three parameters: (1) the number of mutation events, (2) the total coverage, and (3) the corresponding background noise of the motif at the given site. Putative T sites were selected using a p-value cutoff of 10'4, and to further improve reliability, an additional filtering step was applied, requiring sites to have at least 20* coverage and a mutation ratio greater than 10%.Statistics and reproducibility

[0281] For PUM-seq libraries, two or three biologically independent replicates were used in each experiment with cultured cells. Immunoblots are representative images from at least three rounds of independent experiments. Data is presented as the mean ± s.d., with two-tailed Student’ s / -tests on the statistical significance of differences between groups. All statistical analysis and data graphing were done using Python. No statistical methods were applied to preevaluate sample size. No data was excluded from analysis. Samples in this study were not randomized. Blinding was not used for this study because cell culture, sample preparation, reagents and experimental settings were kept consistent for each experiment.Example 7. Overview

[0282] PUM-seq is a technology that resolves the long-standing challenge of mapping pseudouridine (T) with high efficiency and minimal RNA damage. Built on a novel tandem chemical reaction that proceeds under exceptionally mild conditions (e.g., 37°C), PUM-seq provides near-quantitative, mutation-based detection of T. This approach overcomes the key limitations of previous methods: it successfully resolves T in challenging uridine-rich contexts where nearly half of all mRNA modifications reside; it preserves RNA integrity, yielding longer, more mappable sequencing libraries; and its exceptionally low background rate (0.25%) provides the statistical power to reliably quantify low-stoichiometry sites for the first time.

[0283] This technological leap enabled the generation of the first multi-species and tissuelevel landscapes for multiple non-coding RNA families, revealing that the evolution of pseudouridylation is defined by a combination of deep conservation and dynamic, lineagespecific regulation. The deeply conserved core includes high-stoichiometry T sites that are under strong purifying selection, acting as essential architectural cornerstones in rRNA, tRNA, and snRNA. These sites are not randomly placed; they cluster in functionally critical domains, such as the ribosome’s peptidyl transferase center, the hinge region of tRNA’s L-shaped structure, and the snRNA-pre-mRNA interaction interface of the spliceosome. The strict conservation of not just their position but also their consistent high stoichiometry supports a functional “dosage” requirement, where even minor deviations are evolutionarily detrimental to the cell's core machinery. In stark contrast to this static foundation, a dynamic layer of modifications that appears to be a hotbed of adaptive evolution was uncovered. The gain of lineage-specific sites, such as the primate-specific modification at 18S:210, represents a distinct molecular event on a specific evolutionary branch, raising the question of whether such changes subtly altered ribosome function to adapt to the unique proteomic features of that lineage. Perhaps most strikingly, the discovery that multiple, independently-evolved mammalian sites show enrichment in the brain suggests a convergent evolutionary trend linking novel T modifications to the development of the nervous system. This work lays the groundwork for a new field of evolutionary epitranscriptomics, prompting key concepts: mechanisms that these brain-enriched T sites regulate neuronal function, translation of synaptic proteins and / or stabilization of key neural RNAs; linking the appearance of these novel modifications in the evolutionary record to major advances in cognitive complexity.

[0284] In summary, PUM-seq represents a significant methodological advance that empowers the scientific community to investigate pseudouridylation with unprecedented accuracy and depth. By providing a reliable tool for quantitative, cross-conditional analysis, this work opens the door to systematically exploring how T stoichiometry is regulated during development, disease, and environmental stress. The preservation of long RNA fragments makes PUM-seq uniquely suited for future integration with single-molecule long-read sequencing platforms, which would enable amplification-free analysis and the study of T phasing. Ultimately, PUM-seq provides the foundation for dissecting the full functional impact of this enigmatic RNA modification, from its ancient role in shaping RNA structure to its dynamic contributions to organismal complexity and adaptation.* * *

[0285] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.REFERENCESThe following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Davis, F.F., and Allen, F.W. (1957). RIBONUCLEIC ACIDS FROM YEAST WHICH CONTAIN A FIFTH NUCLEOTIDE. Journal of Biological Chemistry 227, 907-915. https: / / doi.org / 10.1016 / 80021-9258(18)70770-9.2. Carlile, T.M., Rojas-Duran, M.F., Zinshteyn, B., Shin, H., Bartoli, K.M., and Gilbert, W.V. (2014). Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515, 143-146. https: / / doi.org / 10.1038 / naturel3802.3. Schwartz, S., Bernstein, D.A., Mumbach, M.R., Jovanovic, M., Herbst, R.H., Leon- Ricardo, B.X., Engreitz, J.M., Guttman, M., Satija, R., Lander, E.S., et al. (2014). Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA. Cell 159, 148-162. https: / / doi.Org / 10.1016 / j.cell.2014.08.028.4. Dai, Q., Zhang, L.-S., Sun, H.-L., Pajdzik, K., Yang, L., Ye, C., Ju, C.-W., Liu, S., Wang, Y., Zheng, Z., et al. (2022). Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution. Nat Biotechnol 41, 344-354. https: / / doi.org / 10.1038 / s41587-022-01505-w.5. Hamma, T., and Ferre-D’Amare, A.R. (2006). Pseudouridine Synthases. Chemistry & Biology 13, 1125-1135. https: / / doi.Org / 10.1016 / j.chembiol.2006.09.009.6. Borchardt, E.K., Martinez, N.M., and Gilbert, W.V. (2020). Regulation and Function of RNA Pseudouridylation in Human Cells. Annu. Rev. Genet. 54, 309-336. http s : / / doi . org / 10.1146 / annurev-genet- 112618-043830.7. Edelheit, S., Schwartz, S., Mumbach, M.R., Wurtzel, O., and Sorek, R. (2013).Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet 9, el003602. http s : / / doi . org / 10.1371 / j ournal . pgen .1003602.8. Carlile, T.M., Rojas-Duran, M.F., and Gilbert, W.V. (2015). Pseudo-Seq. Methods Enzymol 560, 219-245. https: / / doi.org / 10.1016 / bs.mie.2015.03.011.9. Li, X., Zhu, P., Ma, S., Song, J., Bai, J., Sun, F., and Yi, C. (2015). Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nat Chem Biol 11, 592- 597. https: / / doi.org / 10.1038 / nchembio.1836.10. Zhang, M., Jiang, Z., Ma, Y., Liu, W., Zhuang, Y., Lu, B., Li, K., Peng, J., and Yi, C. (2023). Quantitative profiling of pseudouridylation landscape in the human transcriptome. Nat Chem Biol 19, 1185-1195. https: / / doi.org / 10.1038 / s41589-023-01304-7.11. Burrows, C.J., and Fleming, A.M. (2023). Bisulfite and Nanopore Sequencing forPseudouridine in RNA. Acc. Chem. Res. 56, 2740-2751. https: / / doi.org / 10.1021 / acs.accounts.3c00458.12. Khoddami, V., Yerra, A., Mosbruger, T.L., Fleming, A.M., Burrows, C.J., and Cairns, B.R. (2019). Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution. Proceedings of the National Academy of Sciences 116, 6784-6789. https: / / doi.org / 10.1073 / pnas.1817334116.13. Fleming, A.M., Alenko, A., Kitt, J.P., Orendt, A.M., Flynn, P.F., Harris, J.M., and Burrows, C.J. (2019). Structural Elucidation of Bisulfite Adducts to Pseudouridine That Result in Deletion Signatures during Reverse Transcription of RNA. J. Am. Chem. Soc. 141, 16450- 16460. https: / / doi.org / 10.1021 / jacs.9b08630.14. Fleming, A.M., Xiao, S., and Burrows, C.J. (2022). Pseudouridine and Nl- Methylpseudouridine Display pH-Independent Reaction Rates with Bisulfite Yielding Ribose Adducts. Org. Lett. 24, 6182-6185. https: / / doi.org / 10.1021 / acs.orglett.2c02427.15. Fleming, A.M., Zhu, J., Done, V.K., and Burrows, C.J. (2023). Advantages and challenges associated with bisulfite-assisted nanopore direct RNA sequencing for modifications. RSC Chem. Biol. 4, 952-964. https: / / doi.org / 10.1039 / D3CB00081H.16. Xu, H., Kong, L., Cheng, J., Al Moussawi, K., Chen, X., Iqbal, A., Wing, P.A.C., Harris, J.M., Tsukuda, S., Embarc-Buh, A., et al. (2024). Absolute quantitative and baseresolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome. Nat Methods 21, 2024-2033. https: / / doi.org / 10.1038 / s41592-024-02439-8.17. Chambers, R.W., Kurkov, V., and Shapiro, R. (1963). The Chemistry of Pseudouridine.Synthesis of Pseudouridine-5’ -Diphosphate*. Biochemistry 2, 1192-1203. https: / / doi.org / 10.1021 / bi00906a003.18. Chambers, R.W. (1965). The Chemistry of Pseudouridine. IV. Cyanoethylation *. Biochemistry 4, 219-226. https: / / doi.org / 10.1021 / bi00878a007.19. Yoshida, M., and Ukita, T. (1965). Selective Modifications of Inosine and 0-Uridine with Acrylonitrile out of the Other Ribonucleosides. The Journal of Biochemistry 57, 818-821.20. Yoshida, M., and Ukita, T. (1968). Modification of nucleosides and nucleotides: VII. Selective cyanoethylation of inosine and pseudouridine in yeast transfer ribonucleic acid. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis 157, 455-465. https: / / doi.org / 10.1016 / 0005-2787(68)90145-7.21. Takai, K. (2022). The uridine to pseudouridine modification at the wobble position of eukaryotic isoleucine tRNA species is unlikely to induce mistranslation. Nucleosides, Nucleotides & Nucleic Acids 41, 137-153. https: / / doi.org / 10.1080 / 15257770.2021.2011916.22. Babaian, A., Rothe, K., Girodat, D., Minia, I., Djondovic, S., Milek, M., Spencer Miko, S.E., Wieden, H.-J., Landthaler, M., Morin, G.B., et al. (2020). Loss of mlacp3'P Ribosomal RNA Modification Is a Major Feature of Cancer. Cell Reports 31, 107611. https: / / doi.Org / 10.1016 / j.celrep.2020.107611.23. Takai, K., and Yokoyama, S. (2003). SURVEY AND SUMMARY: Roles of 5- substituents of tRNA wobble uridines in the recognition of purine-ending codons. Nucleic Acids Res 31, 6383-6391. https: / / doi.org / 10.1093 / nar / gkg839.24. Critchfield, F.E., Funk, G.L., and Johnson, J.B. (1956). Determination of Alpha, Beta- Unsaturated Compouds by Reaction with Morpholine. Anal. Chem. 28, 76-79. https: / / doi.org / 10.1021 / ac60109a024.25. Martinez, G., Arumugam, J., Jacobs, H.K., and Gopalan, A.S. (2013). 3,2- Hydroxypyridinone (3,2-HOPO) vinyl sulfonamide and acrylamide linkers: aza-Michael addition reactions and the preparation of poly-HOPO chelators. Tetrahedron Letters 54, 630- 634. https: / / doi.Org / 10.1016 / j.tetlet.2012. l l.136.26. Noordzij, G.J., and Wilsens, C.H.R.M. (2019). Cascade aza-Michael Addition- Cyclizations; Toward Renewable and Multifunctional Carboxylic Acids for Melt- Polycondensation. Front. Chem. 7. https: / / doi.org / 10.3389 / fchem.2019.00729.27. Genest, A., Portinha, D., Fleury, E., and Ganachaud, F. (2017). The aza-Michael reaction as an alternative strategy to generate advanced silicon-based (macro)molecules and materials. Progress in Polymer Science 72, 61-110. https : / / doi . org / 10.1016 / j . progpolymsci .2017.02.002.28. Coetzee, J.F., and Padmanabhan, G.R. (1965). Dissociation and Homoconjugation ofCertain Phenols in Acetonitrile. J. Phys. Chem. 69, 3193-3196. https: / / doi.org / 10.1021 / j l00893a516.29. Baddar, F.G., Wahhab, S.A., Awad, B.M., Guindy, N.M., Wahba, M., and Malek, B. A.A. (1970). Phenolic esters. Part I. Rate of hydrolysis of phenyl, p-tolyl, and p-chlorophenyl hydrogen succinate. J. Chem. Soc. B, 739-745. https: / / doi.org / 10.1039 / J29700000739.30. Carey, F.A., and Sundberg, R.J. (2007). Advanced Organic Chemistry: Part A: Structure and Mechanisms (Springer Science & Business Media).31. Boncel, S., and Walczak, K. (2006). The Influence of Base on Regioselectivity of 5- Substituted Uracils Addition to Michael Acceptors. Letters in Organic Chemistry 3, 534-538. https: / / doi.org / 10.2174 / 157017806778341942.32. Boncel, S., Mqczka, M., and Walczak, K.Z. (2010). Michael versus retro-Michael reaction in the regioselective synthesis of N-l and N-3 uracil adducts. Tetrahedron 66, 8450- 8457. https: / / doi.Org / 10.1016 / j.tet.2010.08.059.33. Taoka, M., Nobe, Y., Yamaki, Y., Sato, K., Ishikawa, H., Izumikawa, K., Yamauchi, Y., Hirota, K., Nakayama, H., Takahashi, N., et al. (2018). Landscape of the complete RNA chemical modifications in the human 80S ribosome. Nucleic Acids Research 46, 9289-9298. https: / / doi.org / 10.1093 / nar / gky811.34. Tavakoli, S., Nabizadeh, M., Makhamreh, A., Gamper, H., McCormick, C.A., Rezapour, N.K., Hou, Y.-M., Wanunu, M., and Rouhanifard, S.H. (2023). Semi-quantitative detection of pseudouridine modifications and type I / II hypermodifications in human mRNAsusing direct long-read sequencing. Nat Commun 14, 334. https: / / doi.org / 10.1038 / s41467-023- 35858-w.35. Hughes, D.G., and Maden, B.E. (1978). The pseudouridine contents of the ribosomal ribonucleic acids of three vertebrate species. Numerical correspondence between pseudouridine residues and 2’-O-methyl groups is not always conserved. Biochem J 171, 781— 786. https: / / doi.org / 10.1042 / bj l710781.36. Maden, E.H., and Wakeman, J.A. (1988). Pseudouridine distribution in mammalian 18 S ribosomal RNA. A major cluster in the central region of the molecule. Biochem J 249, 459- 464. https: / / doi.org / 10.1042 / bj2490459.37. Milenkovic, I., Cruciani, S., Llovera, L., Lucas, M.C., Medina, R., Pauli, C., Heid, D., Muley, T., Schneider, M.A., Klotz, L.V., et al. (2025). Epitranscriptomic rRNA fingerprinting reveals tissue-of-origin and tumor-specific signatures. Molecular Cell 85, 177-190.e7. https: / / doi.Org / 10.1016 / j.molcel.2024. l 1.014.38. Talross, G.J.S., Deryusheva, S., and Gall, J.G. (2021). Stable lariats bearing a snoRNA (slb-snoRNA) in eukaryotic cells: A level of regulation for guide RNAs. Proceedings of the National Academy of Sciences 118, e2114156118. https: / / doi.org / 10.1073 / pnas.2114156118.Bykhovskaya Y, Casas K, Mengesha E, Inbal A, Fischel-Ghodsian N. 2004. The American Journal of Human Genetics 74: 1303-8Dai Q, Zhang L-S, Sun H-L, Pajdzik K, Yang L, et al. 2023. Nature Biotechnology 41 :344-54 de Brouwer APM, Abou Jamra R, Kbrtel N, Soyris C, Polla DL, et al. 2018. The American Journal of Human Genetics 103: 1045-52Dudev, T. & Lim, C. Ring Strain Energies from ab Initio Calculations. J. Am. Chem. Soc. 120, 4450-4458 (1998).Frye M, Harada BT, Behm M, He C. 2018. Science 361 : 1346-9Ge R, Ye C, Peng Y, Dai Q, Zhao Y, et al. 2023. Nature Protocols 18:626-57Heiss NS, Knight SW, Vulliamy TJ, Klauck SM, Wiemann S, et al. 1998. Nature Genetics 19:32-8Hu L, Liu S, Peng Y, Ge R, Su R, et al. 2022. Nature Biotechnology 40: 1210-9Knutson SD, Ayele TM, Heemstra JM. 2018. Bioconjugate Chemistry 29:2899-903Matta, C. F. & Gillespie, R. J. Understanding and Interpreting Molecular Electron Density Distributions. J. Chem. Educ. 79, 1141 (2002).Rennie, R. A Dictionary of Chemistry. (Oxford university press, Oxford, 2020).Roundtree IA, Evans ME, Pan T, He C. 2017. Cell 169: 1187-200Smith, M. & March, J. March ’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. (J. Wiley &sons, Hoboken (N.J.), 2007).Verma, D. K., Dewangan, Y. & Verma, C. Chapter 1 - Organic reaction mechanism, in Handbook of Organic Name Reactions (eds. Verma, D. K., Dewangan, Y. & Verma, C.) 1- 153 (Elsevier, 2023). doi:10.1016 / B978-0-323-95948-3.00001-5.Weng X, Gong J, Chen Y, Wu T, Wang F, et al. 2020. Nature Chemical Biology 16:489-92Wiberg, K. B. The Concept of Strain in Organic Chemistry. Angewandte Chemie International Edition in English 25, 312-322 (1986).Wu T, Cheng AY, Zhang Y, Xu J, Wu J, et al. 2024. Nature BiotechnologyWu T, Lyu R, You Q, He C. 2020. Nat Methods 17:515-23Xu, H. et al. Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome. Nat Methods 21, 2024-2033 (2024).The reference, Xu, H., Kong, L., Cheng, J. et al. Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome. Nat Methods (2024), is also fully incorporated by reference herein.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a pseudouridine labeling molecule solution in a buffer, wherein the pseudouridine labeling molecule comprises an electron-withdrawing group.

2. The pseudouridine labeling molecule of claim 1, wherein the pseudouridine labeling molecule is of the formula:whereinR1 is the electron-withdrawing group;R2 is hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; andR3 and R4 are each independently H or C1-C3 alkyl.

3. The pseudouridine labeling molecule of claim 1 or 2, wherein the electron-withdrawing group is nitrile, nitro, ester, carboxylic acid, ester, amide, ketone, aldehyde, sulfone, sulfonic acid, thioester, or thioate.

4. The pseudouridine labeling molecule of claim 3, wherein the electron-withdrawing group is substituted.

5. The pseudouridine labeling molecule of claim 4, wherein the electron-withdrawing group is substituted with a linking group.

6. The pseudouridine labeling molecule of claim 5, wherein the linking group is azide, alkyne, thiol, amine, maleimide, N-hydroxysuccinimide ester, tetrazine, or trans-cyclooctene.

7. The pseudouridine labeling molecule of claim 5 or 6, wherein the molecule further comprises a linker between the linking group and the pseudouridine labeling molecule.

8. The pseudouridine labeling molecule of claim 7, wherein the linker is a polyethylene glycols (PEGs), polyglycerols, polysarcosines, polyoxazolines, peptide linkers, or alkyl linkers.

9. The composition of any one of claims 1-8, wherein the pseudouridine labeling molecule is selected from:

10. The composition of any one of claims 1-9, wherein the pseudouridine labeling molecule comprises two electron-withdrawing groups.

11. The composition of claim 10, wherein the two electron-withdrawing groups are each independently nitro, sulfonyl, sulfonate, nitrile, sulfonic acid, aldehyde, ketone, carboxylic acid, ester, acid chloride, or amide.

12. The composition of claim 10, wherein the two electron-withdrawing groups are ester and bromide.

13. The composition of any one of claims 1-12, wherein the pseudouridine labeling molecule is methyl a-bromoacrylate.

14. The composition of any one of claims 1-13, wherein the buffer is a Tris, phosphate, triethylammonium bicarbonate (TEAB), HEPES, NasBCh, sodium cacodylate, triethylammonium acetate (TEAA), and / or ethylenediaminetetraacetic acid (EDTA) buffer.

15. The composition of any one of claims 1-14, wherein the buffer is TEAB.

16. The composition of any one of claims 1-15, wherein the concentration of the buffer is between about 50 mM to about 1000 mM or between about 50 mM to about 500 mM or between about 50 mM to about 250 mM.

17. The composition of one of claims 1-16, wherein the concentration of the buffer is less than about 200 mM.

18. The composition of any one of claims 1-17, wherein the buffer is 150 mM TEAB.

19. The composition of any one for claims 1-18, wherein the buffer has a pH of about 5- 10.

20. The composition of any one of claims 1-19, wherein the buffer has a pH of about 7.75 to about 7.95.

21. The composition of any one of claims 1-20, wherein the buffer comprises an amine.

22. The composition of claim 21, wherein the amine comprises NHA.

23. The composition of any one of claims 1-22, wherein the buffer comprises NH4OH.

24. The composition of any one of claims 21-23, wherein the concentration of the amine is between about 200 - 800 mM.

25. The composition of any one of claims 21-24, wherein the concentration of the amine is about 200 mM.

26. The composition of any one of claims 1-25, the concentration of the pseudouridine labeling molecule in the composition is between about 1 mM - 2000 mM.

27. The composition of any one of claims 1-26, the concentration of the pseudouridine labeling molecule in the composition is about 200 mM.

28. The composition of any one of claims 1-27, wherein the pH of the composition is between about 5.5 and 10.

29. The composition of any one of claims 1-28, wherein the pH of the composition is between about 7 and 10.

30. The composition of any one of claims 1-29, wherein the pH of the composition is about 7.5-8.5.

31. The composition of any one of claims 1-30, wherein the solution further comprises DSMO, ethanol nitrogen, DMS, or the like.

32. The composition of any one of claims 1-31, wherein the composition is stable for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

33. The composition of any one of claims 1-32, further comprising ribonucleic acid (RNA).

34. The composition of claim 33, wherein the RNA comprises at least one pseudouridine modification.

35. The composition of any one of claims 1-34, wherein the ratio of the pseudouridine labeling molecule to pseudouridine is between about 20: 1 to about 0.15: 1, about 10: 1 to about 1.25: 1, or about 5: 1 to about 2.5: 1.

36. The composition of any one of claims 33-35, wherein the RNA is rRNA, tRNA, snRNA,Y-RNA, miRNA, scaRNA, snoRNA and / or mRNA.

37. The composition of claim 36, wherein the RNA is mRNA.

38. The composition of claim 37, wherein the mRNA is polyA enriched mRNA.

39. A method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising use of the composition according to any one of claims 1-38.

40. A method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising incubating acomposition comprising an RNA polynucleotide, a pseudouridine labeling molecule and a buffer, under conditions sufficient to produce a modified pseudouridine that is detectable and / or quantifiable, and a buffer, wherein the pseudouridine labeling molecule comprises an electron-withdrawing group.

41. The method of claim 40, wherein the pseudouridine labeling molecule is of the formula:whereinRi is selected from the group consisting of nitrile, nitro, ester, carboxylic acid, ester, amide, ketone, aldehyde, sulfone, sulfonic acid, thioester, and thioate;R2 is hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; andR3 and R4 are each independently H or C1-C3 alkyl.

42. The method of claim 41, wherein the electron-withdrawing group is substituted.

43. The method of claim 42, wherein the electron-withdrawing group is substituted with a linking group.

44. The method of claim 43, wherein the linking group is azide, alkyne, thiol, amine, maleimide, N-hydroxysuccinimide ester, tetrazine, or trans-cyclooctene.

45. The method of any one of claims 40-44, wherein the molecule further comprises a linker between the linking group and the pseudouridine labeling molecule.

46. The method of claim 45, wherein the linker is polyethylene glycol (PEG), polyglycerol, polysarcosine, polyoxazoline, amino acids, or an alkyl linker.

47. The method of any one of claims 1-46, wherein the pseudouridine labeling molecule reacts with the RNA polynucleotide through an Aza-Michael addition reaction to form an intermediate Aza-Michael addition modified pseudouridine.

48. The method of claim 47, wherein the intermediate Aza-Michael addition product undergoes an intramolecular cyclization reaction to form the modified pseudouridine.

49. The method of claim 48, wherein, when modified pseudouridine comprises a halogen group, the modified pseudouridine further undergoes a halogen hydrolysis to form the modified pseudouridine.

50. The method of any one of claims 39-49, wherein the pseudouridine labeling molecule is selected from:

51. The method of any one of claims 39-50, wherein the labeling molecule comprises two electron-withdrawing groups.

52. The method of claim 51, wherein the two electron-withdrawing groups are each independently, nitro, sulfonyl, sulfonate, nitrile, sulfonic acid, aldehyde, ketone, carboxylic acid, ester, acid chloride, or amide.

53. The method of claim 51 or 52, wherein the wherein the two electron withdrawing groups are ester and bromide.

54. The method of any one of claims 40-53, wherein the wherein the conditions sufficient to produce a modified pseudouridine comprise one or more of: a) the pseudouridine labeling molecule in a concentration at about 1 mM - 2000 mM, b) a pH of equal to or less than 10, c) a temperature of less than 85 °C, and / or d) an incubation time of between about 2 minutes - 4 hours.

55. The method of any one of claims 40-54, wherein the conditions sufficient to produce a modified pseudouridine comprises an incubation time of 37°C for 3 hours or 70°C for 30 minutes.

56. The method of any one of claims 40-55, wherein the pseudouridine labeling molecule is or comprises methyl a-bromoacrylate.

57. The method of any one of claims 40-56, wherein the pseudouridine labeling molecule is not (bromo) acrylonitrile.

58. The method of any one of claims 40-57, wherein the method includes one or more intermediate products.

59. The method of claim 58, wherein the one or more intermediate products is a pseudouridine methyl-propionate ester and / or a pseudouridine bromo delta lactone.

60. The method of claim 58 or 59, wherein the one or more intermediate products is not a pseudouridine propionamide and / or a pseudouridine oxazolidine.

61. The method of any one of claims 40-60, wherein the modified pseudouridine is a sixmembered hydroxylactone.

62. The method of any one of claims 40-61, wherein the modified pseudouridine is not a five-membered oxazolidine.

63. The method of any one of claims 40-62, wherein the modified pseudouridine is a pseudouridine hydroxy lactone.

64. The method of any one of claims 40-63, wherein the modified pseudouridine is not pseudouridine oxazolidine.

65. The method of any one of claims 40-64, wherein the composition comprises a concentration of a pseudouridine labeling molecule between about 50 mM and 500 mM.

66. The method of any one of claims 40-65, wherein the composition comprises a concentration of a pseudouridine labeling molecule of about 200 mM.

67. The method of any one of claims 40-66, wherein a pH of the composition is between about 5.5 and 10.

68. The method of claim 67, wherein the pH of the composition is between about 7 and 9.

69. The method of claim 66 or 67, wherein the pH of the composition is or is about 7.5-8.5.

70. The method of any one of claims 40-69, wherein the temperature is less than about 85 °C.

71. The method of any one of claims 40-70, wherein the temperature is less than about 70 °C.

72. The method of any one of claims 40-71, wherein the temperature is between about SO- 45 °C.

73. The method of any one of claims 40-71, wherein the temperature is about 37 °C.

74. The method of any one of claims 40-73, wherein the incubation time is 30 minutes to 4 hours.

75. The method of any one of claims 40-74, wherein the incubation time is about 3 hours.

76. The method of any one of claims 40-75, further comprising a pre-heating step.

77. The method of claim 76, wherein the pre-heating step is at a higher temperature than the sufficient conditions.

78. The method of claim 76 or 77, wherein the preheating step is less than about 30, 15, 10, 5 or 3 min at a temperature of less than about 70 °C.

79. The method of anyone of claims 40-78, wherein the RNA polynucleotide comprises a low level of damage.

80. The method of anyone of claims 40-79, wherein the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition not comprising the labeling molecule.

81. The method of claim 79 or 80, wherein the damage is assessed as RNA fragmentation.

82. The method of anyone of claims 40-81, wherein the wherein the pseudouridine to modified pseudouridine unconverted rate is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%.

83. The method of anyone of claims 40-82, wherein the pseudouridine to modified pseudouridine unconverted rate is less than about 3%.

84. The method of any one of claims 40-83, wherein greater than 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% of the pseudouridine are converted to modified pseudouridine.

85. The method of anyone of claims 40-84, wherein about 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80- 100% of the pseudouridine are converted to modified pseudouridine.

86. The method of any one of claims 40-85, wherein the RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or 5 ng and 15 ng in the composition.

87. The method of any one of claims 40-86, wherein the RNA is rRNA, tRNA, snRNA, ,Y- RNA, miRNA, scaRNA, snoRNA and / or mRNA.

88. The method of any one of claims 40-87, wherein the RNA is mRNA.

89. The method of claim 88, wherein the mRNA is polyA enriched mRNA.

90. The method of any one of claims 40-89, wherein the modified pseudouridine is detected, quantified, and / or mapped.

91. The method of claim 90, wherein the modified pseudouridine is detected, quantified, and / or mapped, by mass spectrometry.

92. The method of claim 90 or 91, wherein the modified pseudouridine is mixed with dCTP.

93. The method of any one of claims 40-91, wherein RNA with modified pseudouridine is subjected to reverse transcription, cDNA ligation, and / or PCR amplification.

94. The method of any one of claims 40-93, wherein RNA with modified pseudouridine is subjected to reverse transcription, 2nd strand synthesis, dsDNA ligation, and / or PCR amplification.

95. The method of any one of claims 40-94, wherein the buffer is a Tris, phosphate, TEAB, HEPES, NasBCh, sodium cacodylate, TEAA, and / or EDTA buffer,96. The method of any one of claims 40-95, wherein the concentration of the buffer is between about 50 mM to about 1000 mM or between about 50 mM to about 500 mM or between about 50 mM to about 250 mM.

97. The method of any one of claims 40-96, wherein the concentration of the buffer is less than about 200 mM.

98. The method of any one of claims 40-97, wherein the concentration of the buffer is 150 mM TEAB.

99. The method of any one of claims 40-98, wherein the concentration of the buffer has a pH of about 5-10.

100. The method of any one of claims 40-99, wherein the buffer has a pH of about 8.0 to about 9.0.

101. The method of any one of claims 40-95, wherein the buffer comprises an amine.

102. The method of claim 101, wherein the amine comprises NHA.

103. The method of any one of claims 40-102, wherein the buffer comprises NH4OH.

104. The method of any one of claims 101-103, wherein the concentration of the amine is between about 200 - 800 mM.

105. The method of any one of claims 101-104, wherein the concentration of the amine is about 500 mM.

106. A method of detecting a disease or disorder in a subject, wherein the disease or disorder is associated with a pseudouridine RNA modification comprising the method of any one of claims 40-105.

107. The method of claim 106, wherein the subject is a pig, mouse, monkey, or human.

108. A method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, methyl a-bromoacrylate and a buffer under conditions sufficient to produce modified pseudouridine that is detectable and / or quantifiable, wherein the modified pseudouridine is pseudouridine hydroxylactone, and wherein the sufficient conditions comprise: a) the pseudouridine labeling molecule concentration is about 50 mM and 500 mM, b) the pH is equal to or less than 10, c) the temperature is less than 85 °C, and d) the incubation is between 30 min-4 hours.

109. The method of claim 108, wherein the buffer is a Tris, phosphate, TEAB, HEPES, NasBCh, sodium cacodylate, TEAA, and / or EDTA buffer.

110. The method of claim 108 or 109, wherein the concentration of the buffer is between about 50 mM to about 1000 mM or between about 75 mM to about 500 mM or between about 100 mM to about 200 mM.

111. The method of any one of claims 108 -110, wherein the buffer comprises an amine.

112. The method of claim 111, wherein the amine comprises NHA.

113. The method of any one of claims 108-112, wherein the buffer comprises NH4OH.

114. The method of any one of claims 111-113, wherein the concentration of the amine is between about 200 - 800 mM.

115. The method of any one of claims 111-114, wherein the concentration of the amine is about 500 mM.

116. The method of any one of claims 108-114, wherein the composition comprises a concentration of a pseudouridine labeling molecule between about 100 mM and 300 mM.

117. The method of any one of claims 108-116, wherein the composition comprises a concentration of a pseudouridine labeling molecule of about 200 mM.

118. The method of any one of claims 108-117, wherein the pH of the composition is between about 5.5 and 9.

119. The method of any one of claims 108-118, wherein the pH of the composition is between about 8 and 9.

120. The composition of any one of claims 108-119, wherein the pH of the composition is or is about 7.5-8.5.

121. The method of any one of claims 108-120, wherein the temperature is less than about 85 °C.

122. The method of any one of claims 108-121, wherein the temperature is less than about 70 °C.

123. The method of any one of claims 108-122, wherein the temperature is between about 30-45 °C.

124. The method of any one of claims 108-123, wherein the temperature is about 37 °C.

125. The method of any one of claims 108-124, wherein the incubation time is 30 minutes to 4 hours.

126. The method of any one of claims 108-125, wherein the incubation time is 3 hours.

127. The method of any one of claims 108-126, further comprising a pre-heating step.

128. The method of claim 127, wherein the pre-heating step is at a higher temperature than the sufficient conditions.

129. The method of claim 127 or 128, wherein the preheating step is less than 15, 10, or 5 min at a temperature of less than about 70 °C.

130. The method of anyone of claims 108-129, wherein the RNA polynucleotide comprises a low level of damage.

131. The method of anyone of claims 108-130, wherein the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition not comprising the labeling molecule.

132. The method of claim 130 or 131, wherein the damage is assessed as RNA fragmentation.

133. The method of anyone of claims 108-132, wherein the wherein the pseudouridine to modified pseudouridine unconverted rate is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%.

134. The method of anyone of claims 108-133, wherein the pseudouridine to modified pseudouridine unconverted rate is less than about 3%.

135. The method of any one of claims 108-134, wherein greater than 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% of the pseudouridine are converted to modified pseudouridine.

136. The method of anyone of claims 108-135, wherein about 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80- 100% of the pseudouridine are converted to modified pseudouridine.

137. The method of any one of claims 108-136, wherein the RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or 5 ng and 15 ng in the composition.

138. The method of any one of claims 108-137, wherein the RNA is rRNA, tRNA, snRNA, ,Y-RNA, miRNA, scaRNA, snoRNA and / or mRNA.

139. The method of any one of claims 108-138, wherein the RNA is mRNA.

140. The method of claim 139, wherein the mRNA is polyA enriched mRNA.

141. The method of any one of claims 108-140, wherein the modified pseudouridine is detected and / or quantified by mass spectrometry.

142. The method of any one of claims 108-141, wherein RNA with modified pseudouridine is subjected to reverse transcription, cDNA ligation, and / or PCR amplification.

143. The method of any one of claims 108-142, wherein RNA with modified pseudouridine is subjected to reverse transcription, 2ndstrand synthesis, dsDNA ligation, and / or PCR amplification.

144. A method for (1) detecting pseudouridine in an RNA polynucleotide, (2) converting pseudouridine to a modified pseudouridine in an RNA polynucleotide, or (3) facilitating detection of pseudouridine in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, methyl a-bromoacrylate and a buffer under conditions sufficient to produce modified pseudouridine that is detectable and / or quantifiable, wherein the modified pseudouridine is pseudouridine hydroxylactone, and wherein the sufficient conditions comprise: a) the pH is 7-9, b) the temperature is equal or less than 70°C, and c the incubation is between 2-3 hours.

145. The method of claim 144, wherein the buffer is TEAB.

146. The method of claim 144 or 145, wherein the concentration of the buffer is between about 100 mM to about 200 mM.

147. The method of any one of claims 144 -146, wherein the buffer comprises an amine.

148. The method of any one of claims 144-147, wherein the concentration of the amine is between about 100 - 300 mM.

149. The method of any one of claims 144-148, wherein the concentration of the amine is about 200 mM.

150. The method of any one of claims 144-149, wherein the composition comprises a concentration of a pseudouridine labeling molecule between about 100 mM and 250 mM.

151. The method of any one of claims 144-150, wherein the composition comprises a concentration of a pseudouridine labeling molecule of about 150-200 mM.

152. The method of any one of claims 144-151, further comprising a pre-heating step.

153. The method of claim 152, wherein the preheating step is equal or less than 2 min at a temperature of less than about 70 °C.

154. The method of anyone of claims 144-153, wherein the RNA polynucleotide comprises a low level of damage.

155. The method of anyone of claims 144-154, wherein the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition not comprising the pseudouridine labeling molecule.

156. The method of anyone of claims 144-155, wherein the pseudouridine to modified pseudouridine unconverted rate is less than about 3%.

157. The method of any one of claims 144-156, wherein the modified pseudouridine is detected and / or quantified by mass spectrometry.

158. The method of any one of claims 144-157, wherein RNA with modified pseudouridine is subjected to reverse transcription, cDNA ligation, and / or PCR amplification.

159. The method of any one of claims 144-158, wherein RNA with modified pseudouridine is subjected to reverse transcription, 2ndstrand synthesis, dsDNA ligation, and / or PCR amplification.

160. A polynucleotide processing kit comprising the composition of any one of claims 1-38 and optionally instructions for processing a polynucleotide sample.

161. A polynucleotide processing kit comprising: a) a first component comprising a first solution comprising a pseudouridine labeling molecule; b) a second component comprising a buffer; and c) optionally instructions for processing a polynucleotide sample, wherein the pseudouridine labeling molecule comprises an electron-withdrawing group.

162. The method of claim 161, wherein the pseudouridine labeling molecule has comprises the formula:whereinRi is the electron- withdrawing group and is selected from the group consisting of nitrile, nitro, ester, carboxylic acid, ester, amide, ketone, aldehyde, sulfone, sulfonic acid, thioester, and thioate;R2 is hydrogen, halide, ester, nitrile, ester, or substituted or unsubstituted alkyl, ammonium, nitrile, nitro, ester, carboxylic acid, acid chloride, ester, amide, ketone, aldehyde, sulfone, sulfonyl, sulfonic acid, sulfonate, thioester, or thioate; andR3 and R4 are each independently H or C1-C3 alkyl.

163. The polynucleotide processing kit of claim 161 or 162, wherein the electronwithdrawing group is substituted.

164. The polynucleotide processing kit of claim 163, wherein the electron-withdrawing group is substituted with a linking group.

165. The polynucleotide processing kit of claim 164, wherein the linking group is azide, alkyne, thiol, amine, maleimide, N-hydroxysuccinimide ester, tetrazine, or trans-cyclooctene.

166. The polynucleotide processing kit of claim 165, wherein the molecule further comprises a linker between the linking group and the pseudouridine labeling molecule.

167. The polynucleotide processing kit of claim 166, wherein the linker is selected from the group consisting of polyethylene glycols (PEGs), polyglycerols, polysarcosines, polyoxazolines, peptide linkers, and alkyl linkers.

168. The polynucleotide processing kit of any one of claims 161-167, wherein the pseudouridine labeling molecule is selected from:

169. The kit of claim 161 or 168, wherein the buffer is a Tris, phosphate, TEAB, HEPES, NasBCh, sodium cacodylate, TEAA, and / or EDTA buffer.

170. The kit of any one of claims 161-169, wherein the concentration of the buffer is between about 50 mM to about 1000 mM or between about 50 mM to about 500 mM or between about 50 mM to about 250 mM.

171. The kit of any one of claims claim 161-170, wherein the second component comprises one or more amines.

172. The kit of claim 171, wherein the amine comprises NH173. The kit of any one of claims 161-172, wherein the second component buffer comprises NH40H.

174. The kit of any one of claims 161-173, wherein the first and second components are provided separately or as a premixed solution.

175. The kit of any one of claims 161-174, wherein the premixed solution comprises a T labeling molecule concentration between about 1 mM - 2000 mM.

176. The kit of any one of claims 161-175, wherein the first component comprising a first solution comprising a pseudouridine labeling molecule comprises a solvent of DMSO, DMS, ethanol nitrogen, or the like.

177. The kit of claim 176, wherein the solvent is not ethanol alone and / or water alone.

178. The kit of any one of claims 161-177, wherein the instructions comprise instructions for incubating a polynucleotide sample with the first and second components or the premixed solution at a temperature equal to or less than about 80 °C for a time between about 2 minutes - 4 hours.

179. The kit of claim 178, wherein the temperature is between about 30-45 °C.

180. The kit of claim 178 or 179, wherein the temperature is about 37 °C.

181. The kit of any one of claims 161-180, further comprising one or more buffer solutions and / or enzymes.

182. The kit of any one of claims 161-181, wherein the one or more buffers comprise sequencing reagents.

183. The kit of claim 182, wherein the sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

Images