Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects  In HPLC, a liquid mobile phase (MP) carries a mixture of compounds through a column packed with particles. As a pump forces the MP through the column, the components of the mixture interact with the stationary phase - the particle’s surface - to different degrees and are separated in the process. HPLC’s first application was the resolution of nucleic acids exploiting IEX normal mode3, even though this type of chromatography was later abandoned. Since then a steady improvement in instrumentation and column packings have yielded methods for analysis and purification of both synthetic materials as well as compounds from biological fluids7. HPLC remains the most widely used analytical technology especially for purity determination and batch-to-batch comparison of compounds poised for pharmaceutical use8. Most chromatography suppliers claim applications for oligonucleotide analysis, but in reality good analytical methods exist for up to 25nt10 and become less optimal as the length increases6.The discovery of Chromatography, i.e. separation of a mixture into its components, approximately 120 years ago is credited to Mikhail Tsvet, a Russian-Italian botanist. A major revolutionary step in chromatography was the advent of high-performance liquid chromatography (HPLC) instruments invented 50 years ago16. Protection of 2′-OH and deprotection at the end of the synthesis may not be accomplished at 100% yield, so the overall efficiency of RNA synthesis is compromised compared to DNA. Coupling efficiency in oligonucleotide manufacturing refers to the success rate of a synthesizer adding a new base to a growing nucleic acid chain. This measure is especially important for long oligos, because as the length of an oligo increases, small differences in coupling efficiency have dramatic effects on the yield of the full-length product. Here are two examples to illustrate the compounding effect of less than 100% efficiency in the synthesis of a 100nt vs the synthesis of a 50nt. With an average 99.0% efficiency at each step, the calculated fraction of the 100nt in the final product is 0.366, whereas the calculated fraction of the 50nt is 0.605, almost double. With an average 98.0% efficiency at each step (just 1.0% less), the corresponding calculated fraction for a 100nt is 0.13 and for a 50nt is 0.36. The “theoretical” 13% yield to make a 100nt with an 98.0% average step efficiency is consistent with literature claiming 5.5% yield, after purification, for the optimized synthesis of a 110nt RNA oligo17. With synthetic oligos the highest HPLC peak is very likely to correspond to the desired oligo. Identification of the final product is conducted using IP-RP HPLC analysis directly followed by mass spectrometric (MS) detection18. It should be noted that product identification by mass determination does not require resolution, and therefore identification is independent of relative abundance. However the effectiveness of purification/isolation of the desired product correlates with the product’s abundance in the crude mixture6.The synthesis as well as the purification of oligoribonucleotides are intrinsically less efficient compared to the synthesis and purification of their deoxy counterparts, primarily due to the presence of the 2′-OH that needs to be protected during synthesis20. Such self-structure could be due to short intramolecular double stranded regions between distant sequences, to regional stem-loop folding, to Hoogsteen base-pairs in G-rich sequences, and/or to a wide array of base-stacked conformations. Self-structure leads to the presence of several conformers of comparable stability. Conformers may bear scientific interest regarding in-vitro and in-vivo activity, but they are an additional obstacle to good separation of the mixture into the desired oligo and its impurities. Hence analysis of longer RNAs necessitates denaturing conditions. Such conditions are (i) aqueous MP at pH 12 (see later), (ii) high temperature, and (iii) the presence of an organic cosolvent, such as methanol (CH3OH) or acetonitrile (CH3CN), in the MP. Additional additives are formamide and urea, favored by molecular biologists, but not by HPLC analysts. All these additives/conditions act by disrupting base-pairing and base-stacking interactions, and therefore practically linearize the nucleic acid. Linear, non-structured, polymers may elute as sharp peaks by HPLC, and thus resolve from closely related impurities.In addition to the synthetic efficiency issue, the longer oligoribonucleotides, let us say longer than the 50nt, exhibit self-structure within the otherwise linear polymer23 . In the 1970s-80s the primary mode of chromatography was normal phase24, eventually completely replaced by reversed-phase. In the 1970s Orgel and coworkers discovered the non-enzymatic, template-directed synthesis of oligoribonucleotides25. Using phosphoimidazolide activated ribomononucleotides, as building blocks, and homo- or hetero-polymeric nucleic acids, as templates, they demonstrated formation of the complementary strand as a series of oligos up to the 40nt26. In order to investigate length and linkage, they took Kel-F packing, coated it non-covalently with Adogen, a mixture of tetraalkylammonium compounds, and packed it in HPLC columns (named RPC5)28. This was the first packing known to resolve oligos up to the 40nt based on length and internucleotide bond linkage 26. For the next two decades oligonucleotide analysis was accomplished using packing material from Orgel’s Laboratory, and a MP made out of aqueous 10 mM NaOH  and NaClO4 for salt gradient elution31. The RPC5 packing was not user-friendly, and ultimately replaced by commercially available ones, that barely claim to match its resolving power.Credit for the development of column packings and methods to resolve oligonucleotide mixtures, in this author’s opinion, should be given to the late Dr. Leslie E. Orgel of the Salk Institute and his coworkers for the research they spearheaded in support of the “RNA world” hypothesis32. Since nucleic acids are easily retained on packings covalently modified with tri- or tetra-alkylammonium substituents, IEX methods became popular. In IEX increased salt concentration in the MP promotes elution of oligo of length N ahead of oligo with length N + 1, simply because oligo N is retained less strongly than oligo N + 1. The addition of a volatile ion-pairing agent, such as triethylamine acetate buffer (TEAA), in the MP of a reversed-phase packing facilitates both elution from the column and good separation18. The latter, IP-RP, quickly became a favorite, as it allows for leading the LC flow directly onto a mass detector, and enables MS determination for every HPLC peak. Other modes, like size-exclusion, affinity, and hydrophobic interaction have also been used in special cases. State-of-the-art in RNA oligo separations is currently up to the 60nt using capillary gel electrophoresis (CGE)33, or HPLC in conjunction with IP-RP36 or with IEX modes38. Limited number of companies and research groups17 claim synthesis, analysis, identification, and purification of RNA oligos at the 100nt level. Transfer RNAs (tRNAs) are about 76nt long and known to be the most self-structured RNAs, typically found in a stable cloverleaf conformation39. Research in proteomics has led to large-scale, cell-free use of in-vitro transcribed (IVT) tRNAs as carriers of both native and non-native amino acids to synthesize novel proteins40. While analysis/purification of native as well as IVT tRNA is laborious and presents challenges, the IEX HPLC method developed here may provide purity evaluation and serve as a purification method (see Results).Several HPLC interaction modes have been used successfully for the separation of an oligonucleotide mixture. While initially the mode of choice was normal phase IEX, in 1987 we introduced reversed-phase mode for separation of oligos with N < 517. In this report we illustrate that IP-RP may be less optimal for purity evaluation of sgRNA compared to conventional IEX. We then develop a fully denaturing basic IEX HPLC method for purity evaluation of sgRNA, and demonstrate that such method is also suitable for tRNA and mRNA. Most importantly, we design and illustrate protocols to use with any suitable IEX column and any RNA sample to assess on-the-column and off-the-column stability.Until the discovery and commercialization of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a technology with therapeutic potential for human diseases, HPLC analytical methods for RNA oligos up the 60nt were adequate for most applications. CRISPR is a family of DNA sequences in bacteria that contain sequences from viruses that have attacked the bacterium. These sequences play a key role in a bacterial defense system, and form the basis of a genome editing technology known as CRISPR/Cas9 that allows for genome modification (addition or deletion) within any organism, including humans. A number of companies are currently actively promoting CRISPR technologies for several diseases, and plan to submit to the FDA Investigational Drug Applications (IND) for Cas9 protein, messenger RNA (mRNA) responsible for Cas9 protein synthesis, and sgRNA with sequence complementary to the gene that is to be cut out or replaced. sgRNAs are typically 100nt long and analytical methods for RNAs of such length are sparse41. Earlier method development in our own Laboratory comparing a number of HPLC columns from different suppliers showed base-line resolution among a series of 20nt long oligodeoxyadenylates where the 11th base was replaced by a different nucleotide than dA42. Literature illustrating N + 1 resolution of synthetic 60nt oligos41 led us to the conjecture that DNAPacIEX may exhibit the highest possible separation for oligos with N > 60. Some of the advantages of the DNAPacIEX are: usability in a pH range of 2.5 < pH < 12.5, in a temperature range up to 85 °C, and the compatibility with MeOH or CH3CN41. There is actually a newer version of the DNAPAC PA200 with a 4 μ bead size, and consequently of better resolution, but it exerts higher back pressure (>450bars) compared to what our HPLC instrument can handle, and therefore was not tested here. As IP-RP HPLC column we selected one from the same supplier, DNAPAC RP (abbreviated DNAPacRP)34, with the reasoning that any IP-RP HPLC column produced by this specific supplier would have been compared side-by-side with the DNAPacIEX before entering the market place, and therefore judged to be, at least, as resolving as the DNAPacIEX. Indeed literature for DNAPacRP illustrates separation of synthetic 60nt RNA oligos with N + 1 resolution34. As test articles we used two 32nt RNA oligos differing by 4 nucleotides, a canonical 100nt RNA and the 94nt truncated RNA with the same sequence as the 100nt, but missing 6nt at the 3′end, and a heavily methylated 100nt RNA of the identical sequence as the canonical 100nt, but with 46 2′OMe groups. In addition we tested tRNA (E. Coli) and two mRNAs, EGFP with 996nt and Cas9 with 4500nt. The 100nt RNAs serve as representative examples for sgRNA. Suppliers and RNA sequences can be found in the Materials and Methods section.Method development efforts centered around the DNAPAC PA200, 8 μ bead size, (abbreviated DNAPacIEX) form ThermoFisher Scientific (earlier Dionex) as IEX HPLC column4) for elution, led to the observation of two well resolved peaks. These observations in conjunction with the experiments using IP-RP (see next section and Supplementary Information) led to the conclusion that there are, at least, two stable conformers associated with this specific sequence, one of them being the random coil, the only one stable at the highest temperature.HPLC analyses of the two 32nt RNAs  using DNAPacIEX in MP with pH in the range of 7 < pH < 12 exhibit sharp peaks and comparable impurity profiles, simply because these two RNAs are short and devoid of secondary structure. On the contrary, a 100nt RNA with 46 2′-OMe groups, abbreviated RNA(2′OMe), see sequence in Materials and Methods, exploited here as an example of a sgRNA, exhibited a complex HPLC profile upon analysis in a pH 7 aqueous buffered MP, reminiscent of size exclusion chromatography of a protein mixture and discordant with the profile of a synthetic oligo. Similarly, analysis of the RNA(2′OMe) by Capillary Zone Electrophoresis (CZE) at pH 9.3 in borate buffer exhibits a very broad peak. CZE does not have the capability to resolve a mixture of similar oligos, and should produce a single sharp peak. A single sharp peak by CZE was only observed after incubating the sample at 95 °C for 5 min, known to disrupt secondary structure, cooling it down to room temperature (RT) instantaneously, and analyzing it within minutes. We hypothesized that the secondary structure was primarily the result of the heavy methylation, so we tested the unmethylated 100nt RNA(2′OH), same sequence as the RNA(2′OMe), only to observe a comparable HPLC profile  100nt and the truncated 94nt resolve in a mixture by 0.35 min by IEX and not by IP-RP analysis strongly supports the proposition to use IEX for long RNA oligos. Practically speaking, the conditions at which increased temperature and/or a certain concentration of an organic cosolvent will yield complete RNA linearization may not be predictable, or even experimentally easy to determine. Hence high temperature and the presence of a cosolvent may not confidently lead to the best chromatography for a long synthetic RNA oligo. It would be reasonable to simply use IEX MP at pH 12 with suitable HPLC columns, but the stability of the RNA phosphodiester bond in base is a major concern.The relative instability of RNA compared to DNA is well known. To assess whether or not pH 12 eluent in RNA analysis is a usable medium or not, we address this issue in three ways: First we consulted with the literature, second we measured average internucleotide bond stability with a 32nt RNA oligo in pH 12, and third we conducted on-the HPLC column stability in pH 12 for a number of RNAs, spanning the range of 32nt to 4500nt. The following results clearly support pH 12 chromatography for purity evaluation of RNAs.45, leading to a cyclic intermediate that gets easily cleaved in base to form a mixture of 2′- and 3′-monophosphates. Li and Breaker44 conducted their studies using deoxyoligos with a single RNA linkage in the middle and reported at 23 °C, pH 12 with 10 mM KOH a rate of degradation k = 6 × 10−5 min−1. Kaukinen et al. used 2′OMe modified RNA oligos with a single 2′OH base in the middle45, conducted their studies at higher temperatures and reported that stability varies depending on the flanking bases45. Accounting for the higher temperature some of the degradation rates reported by Kaukinen et al.45 agree with Li and Breaker44. Using k = 6 × 10−5 min−1 as a representative degradation rate we calculate that at pH 12 and at 23 °C each RNA linkage will degrade at about 6 × 10−4, during the approximate 10 min of HPLC analysis until the RNA of interest elutes from the column. One way to look at this result is to think that an RNA internucleotide bond has a chance of 0.0006 to degrade during analysis, or if an RNA molecule is 10,000nt long, then, on average, it will be cut at 6 random bonds. An obvious way to suppress this degradation is to conduct HPLC analysis at a lower temperature. Hence we implemented 10 °C as the HPLC column compartment temperature, and expect about a factor of 2 less degradation compared to 23 °C.Detailed studies concluded that the mechanism of the 3′-5′ internucleotide bond cleavage in RNA is specific base catalyzed by the deprotonated ribosyl 2′OH−5 per min per bond. Hence 5.5 × 10−5 per min as the average degradation rate of an internucleotide bond at 15 °C in pH 12 and in the presence of 35% MPB or the equivalent of 0.53 M NaCl. This result is in excellent agreement with the reported 6.0 × 10−5 per min at 23 °C44, and suggests that if one were to conduct this experiment at 10 °C, instead of 15 °C, the observed rate would be somewhat lower than 5.5 × 10−5 per min (upper limit). Extrapolation of the two experimental values to zero ionic strength  provides degradation rate 2.1 × 10−5 per min per bond at 15 °C; see later for experimental confirmation of this estimate.RNA degradation at pH 12 was revisited using the 32nt RNA oligo with better than 85% purity, which allows for monitoring the actual 32nt RNA peak free from impurities. This oligo was incubated in pH 12.0 with 10 mM NaOH and increasing ionic strength by preparing mock solutions with the HPLC eluents at 20% MPB and 50% MPB (MPB is 1.5 M NaCl in 10 mM NaOH). The samples were incubated at 15 °C in the instrument’s autosampler and analyzed automatically every 20 minutes, using a pH 10 sodium carbonate eluent with 10 °C column temperature. Sodium carbonate buffer at pH 10 is an excellent chromatography eluent for short oligos, like the 32nt RNAs, and exhibits a 100-fold less degradation compared to a pH 12 solution. Hence degradation in a pH 10 chromatography is negligible. The data are included in Table We also designed a protocol to test degradation of any RNA at pH 12.0 in 10 mM NaOH, and assess directly extent of the on-the-column degradation; this protocol could be used with any IEX column qualified for pH 12 MP. The on-the-column stability method involves delaying the onset of the salt gradient, while keeping the gradient steepness the same, and results, in the absence of degradation, to an identical chromatographic profile, but shifted in time. Any detectable degradation due to additional pH 12 exposure, will appear as a decrease of the main peak and as an increase in degradants, i.e. small peaks eluting ahead of the main peak. The advantage of running the on-the-column stability is that it can be done automatically for any RNA sample. A visual comparison of the main peak height and the absence of degradants in the HPLC region in the 3 to 4 minutes elution area ahead of the main peak provide unequivocal evidence for the usability of the IEX pH 12 method. On-the-column stability can be conducted only in the absence of salt. Using a buffered eluent, even at concentrations as low as 20 mM buffer, broadens the HPLC peak and alters slightly the profile, due to the added salt that the sample is exposed to, and therefore prevents quantitative comparison.−5 per min (lower limit) per bond at 10 °C. Please note that the two experimentally obtained degradation rates in the absence of salt are in good agreement, i.e. 0.00047 per min at 10 °C , choice of buffer, the presence/absence of a cosolvent, initial MP composition, and gradient steepness. HPLC methods for purity analysis should run about 30 min or less, including equilibration to initial conditions.RNA2′OH), 100nt and 94nt, and 100nt RNA(2′OMe)  were analyzed by both IP-RP and IEX methods. The HPLC profiles in neutral pH  Figs  and S3 aIt is desirable to scale up the best analytical method and use it to do fraction collection in an effort to obtain purer RNA, and this is even more important now for sgRNAs targeting FDA approval. Because we do not have a fraction collection module on our HPLC instrument, we did it manually collecting approximately 0.25 mL in a preweighted test tube right after the detector. The exact weight of the fraction was obtained and the corresponding HCl to neutralize it was added, followed by addition of TRIS.HCl buffer to yield an approximate 25 mM pH 8 buffered solution that was then subject to salt removal by TrimGen purification . The process lasted no more than 20 min per sample, and was implemented with both the 100nt RNA(2′OH) and the truncated 94nt RNA(2′OH). Both materials were obtained in good purity as seen in Figs E. Coli, a mixture of over 40 components, was conducted by both IP-RP . For a typical 10 min analysis time, this rate translates to 5.5 × 10−4 degradation per bond, which is negligible degradation for any oligo. It could be a measurable quantity in the case of a pure oligo, a resolving HPLC column, and a high sample load. For example, a pure 100nt oligo would exhibit a single bond cleavage in only 5.5% of the molecules which is barely outside experimental error. Moreover if the 100nt oligo is composed of, let us say, 50% 2′-OMe substitutions, then degradation should measure only 2.75%. Such low level of degradation in pH 12 may be considered negligible, and the HPLC method scaled up and used for isolation/purification, as long as fraction collection is conducted using a suitably neutralizing and buffered solution for collection. If IEX HPLC indicates that the specific sgRNA is not of acceptable purity, and if manufacturing process development does not lead to improvements, then synthesis of two shorter fragments and chemical ligation47 may be a better solution to obtain quality material.Why is degradation and/or shift of material sometimes detectable and sometimes not? There are a number of parameters critical for degradation and its detection: These parameters are (i) the degradation rate under the specific analytical conditions, (ii) the length of the RNA, (iii) the sample load, and (iv) the resolution of the HPLC column for the tested material. In addition there is a statistical factor that will be discussed later. Both literature values and our own experiments suggest an upper limit degradation rate of 5.5 × 10Degradation becomes detectable with a 1000nt long RNA, such as the mRNA EGFP, primarily because 55% of the molecules will exhibit on average a single bond cleavage. Consequently, if the molecule was a pure 1000nt long RNA and if the HPLC method could resolve a 999nt long from a 1000nt long molecule, then degradation would be measurable. The reality, as it is seen in Fig. −5 per min for RNA internucleotide bond degradation in 10 mM NaOH at 15 °C in the presence of 0.5 M NaCl, and a lower limit of 1.5 × 10−5 per min per bond in 10 mM NaOH at 10 °C. These values translate to practically negligible degradation of an RNA oligo during HPLC analysis time. With long RNAs, such as mRNAs, degradation appears as a small shift of the material from longer to shorter lengths, but does not alter the overall HPLC profile and does not produce detectable short oligo degradants. This experimentally shown phenomenon is consistent with the statistical notion that the longer the RNA, the fewer the short sequences produced by degradation. The relative stability of RNA oligos in basic IEX HPLC can be exploited for purity/impurity analysis, batch-to-batch comparison, as well as for fraction collection as long as care has been taken that the isolated fractions are stored in a neutral medium. With mRNAs pH 12 IEX HPLC is a suitable method for identifying low molecular weight process development contaminants, as well as for quality control as shown by the reproducibility of the HPLC profile. Since RNAs are moving in the realm of pharmaceuticals, high purity materials are required for testing in in-vitro and in-vivo assays and in order to minimize the sources for off-target effects48. Purity analysis of such RNAs should be conducted using HPLC methods with documented resolving power.Well resolving HPLC methods must be developed and implemented to facilitate process improvements in synthetic or transcription protocols of man-made RNAs. IEX chromatography at pH 12 was extensively used for RNA analysis during the period of 1970 to 1990, and the current study confirms its usability and superiority compared to IP-RP. Our study provided an experimental approach of how-to select chromatography mode and HPLC column for a specific application. It also established that the most efficacious denaturing medium for HPLC analysis of RNA is aqueous pH 12 at 10 °C, and showcased a protocol for testing on-the-column stability with any RNA material, with no special requirement for the IEX HPLC column, besides its compatibility with pH 12. We measured an upper limit of 5.5 × 10AGC CAG AGA GAG CCA GAG AGC CUU CA bold indicates the four different bases, purines in Oligo2 replacing four Cs in Oligo1.Custom-made RNA 32nt oligos and mRNAs (EGFP CleanCap™ and Cas9 CleanCap™) were purchased from TriLink Biotechnologies. Oligo purity was determined by us. Oligo1, sequence: AGA GAG CCC CAG AGA GCC CCA GAG AGC CUU CA. Oligo2, sequence: AGA GAG in vivo degradation and this is why we studied it here.Custom-made 100nt and 94nt Oligos were purchased from Dharmacon. RNA(2′OH) 100nt sequence: 5′-UUA CAG CCA CGU CUA CAG CAG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU U-3′. RNA(2′OH) 94nt sequence: same as RNA(2′OH) 100nt but missing the 6 nucleotides at the 3′end. Please note that out of the six, five are pyrimidines and found by us to be important for secondary structure  buffer 2 M was purchased from Life Technologies. TRIS.HCl 1.0 M buffer pH 7.0 was purchased from Sigma and same buffer pH 8.0 Ultrapure from Invitrogen. NaCl crystal ACS min 99.0%, and sodium perchlorate monohydrate ACS, 85 to 90%, from Alfa Aesar. Sodium hydroxide solution and Hydrochloric acid solution both at 1.0 N, both BioReagent purchased from Sigma. Sodium phosphate dibasic anhydrous AR granular purchased from Mallinckrodt . Sodium carbonate and sodium bicarbonate, both BioXtra, from Sigma Aldrich. Distilled water from ArrowHead or Alhambra was used for preparation of HPLC MP.Spin columns (TC-100 FC from TrimGen Corporation) were used to remove salt and buffer according to the manufacturer’s instructions; practically 100% recovery of RNA is achieved with minor volume/concentration changes.pH measurements were conducted with an Orion 3 pH meter equipped with a Gel pH electrode 9146BN from Thermo Scientific after 3-point calibration with standards LSS purchased from Grainger.As the primary analytical tool, we used an Agilent 1100/1200 LC HPLC equipped with a binary pump, Diode Array Detector (DAD), a 1290 Infinity Autosampler/Thermostat, and Chemstation software Rev.B.04.01 SP1 for data acquisition and processing. Both the autosampler and the column compartment have individual temperature control. Typically the autosampler was kept at 15 °C, unless otherwise noted.HPLC columns, DNAPac PA200 from ThermoFisher Scientific (Dionex), were used with configurations 2 × 250 mm or 4 × 250 mm, typically at about 0.5 mL or 0.9 mL flow per min, respectively. A second HPLC column used was from the same supplier, DNAPac RP in the configuration of 2.1 × 100 mm and used with the flow of about 0.35 mL. The first type of column is used for IEX and the second for IP-RP chromatography. The performance of the instrument and the column was qualified using standards every time ahead as well as after analysis of samples. Mobile phase was prepared and the pH was measured. The same buffer was contained in both MPA and MPB. TEAA buffer was used for IP-RP, typically at 100 mM concentration. As buffers for IEX we used TRIS.HCl at pH 7.0 and at pH 8.0, sodium carbonate at pH 10.0 and at pH 11.0, sodium phosphate at pH 12.0; all these buffers were used at 25 mM concentration in both MPA and MPB. 10 mM NaOH was used as a pH 12.0 eluent; no buffer necessary. The pH values were acceptable in the range pH ± 0.3, and typically not adjusted. The HPLC methods reported here can be used for RNA of any length, and are not optimized for individual RNAs, unless noted otherwise.42. CGE method was conducted with product μPAGE-3 (PN 191–3211), a 75 μx75 cm capillary preloaded with 3%T, 3%C polyacrylamide gel and with buffer, TRIS-borate/urea pH 8.3 and poly(A) standard supplied by Agilent and following supplier’s protocol for analysis.Selected analyses were conducted with an Agilent G1600 Capillary Electrophoresis (CE) instrument equipped with DAD and Chemstation software Rev.B.04.03 for data acquisition and processing; the CE was used in conjunction with a circulating bath to control the autosampler’s temperature, typically kept at 15 °C. The capillary’s temperature was controlled by the instrument’s software. Typical capillary zone electrophoresis (CZE) analyses were conducted with an untreated fused silica capillary (50 μm × 40 cm) with an extended light path purchased from Agilent Technologies in pH 9.3 50 mM sodium tetraborate buffer as detailed elsewhereBoth CE and HPLC peaks were detected and identified using the DAD in the UV–vis region 200–450 nm and the electropherograms and chromatograms were recorded among other wavelengths at 260 nm; the latter are reported here.HPLC methods for purity evaluation of man-made single-stranded RNAs


Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein–protein interactions can guide substrate recognition for RNA editors.Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs  Diverse enzymes catalyze modification of nucleotides in all types of nucleic acids present in the cell. While transfer RNAs (tRNAs) are the most extensively modified molecules in the cell , recent in vivo.Loss of the enzymes responsible for A-to-I editing, adenosine deaminases that act on RNA (ADARs), results in lethality in mice and behavioral phenotypes in worm and fly model systems . Consistin vitro  assay for ADR-1 and biochemical editing assays, we determined that ADR-1 primarily binds mRNA through its first dsRBD, and binds ADR-2 with its second dsRBD, and both ADR-1 dsRBDs are required to promote editing by ADR-2. ADR-2 RIP coupled to high-throughput sequencing indicated that ADR-1 was required for ADR-2 to stably associate with 983 of 1235 transcripts in WT worms. Together, these findings indicate that ADR-1 acts as an endogenous co-factor to direct ADR-2 to specific substrates in vivo, and opens the possibility that ADAR-editing enzymes in humans could interact with other RNA-binding proteins, including deaminase-deficient ADAR family members, to recognize different target mRNAs in vivo.To gain insight into the molecular mechanisms of ADAR substrate recognition, we compared e viable , thus prin worms ,36. Althin worms . To undeEscherichia coli OP50  and 79 ng/μl of 1 kb DNA ladder (NEB). Transgenic strains were maintained by passaging worms expressing the Green Fluorescent Protein (GFP) marker. As described previously  and 3′ UTR (1560 nt downstream of the stop codon) and three copies of the FLAG epitope (DYKDHD) immediately after the start codon. Mutations to the dsRBDs of adr-1 were generated by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. The following transgenic worm strains were utilized in this study and previously published: BB21 adr-1(tm668) + blmEx1 (adr-1(tm668) + blmEx2 (adr-1(tm668) + blmEx11 and BB21 adr-1(tm668) + blmEx12.Worms were maintained under standard laboratory conditions on Nematode growth media (NGM) plates seeded with oli OP50 . Transgeoli OP50 . The injeviously , the tra3′ UTR)]  and BB213′ UTR)] . Newly gSf9 and Hi5 insect cell cultures were maintained as described in the Invitrogen instruction manual, ‘Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques’ and , respectlam-2 (WormBase ID: WBGene00016913), pop-1 (WormBase ID: WBGene00004077), C35E7.6 (WormBase ID: WBGene00016458) and clec-41 (WormBase ID: WBGene00007153) cDNA abundance in both total RNA (input) and immunoprecipitated RNA from the indicated strains. qPCR reactions were performed on 1/10 of the total reverse transcription volume for each sample, with two technical replicates for each qPCR reaction. The total volume of the qPCR reaction was 10 μl, used the SybrFast Master Mix  and was performed on an Eppendorf Realplex Mastercycler using the PCR program . All qPCR quantification for each biological replicate included eight wells of the amplicon of interest (standards), which were 10-fold serial diluted and used to generate a standard curve of cycle threshold versus the relative concentration of amplicon. The standard curves were plotted with a logarithmic scale in regard to concentration and fit with a linear line. The fit of the lines (R2) ranged from 0.98 to 1.00, and all data points fell within the standard curve. All amplicons spanned an exon–exon junction  and adr-1(-) worms as described above. Isolated RNA was subjected to ribosomal RNA depletion using Ribo-Zero gold kits (Human/Mouse/Rat: Illumina Ref: MRZ116C) according to the manufacturer’s instructions . The rRNA-depleted samples were used as the starting material to generate sequencing libraries using the KAPA stranded RNA-seq library preparation Kit (Illumina Ref: KK8400). Briefly, RNA samples were fragmented into 200–300 bp strands by high temperature (94°C for 6 min) and used to make first and second strands of cDNA. Adapters (KAPA S1 adapter kit Ref: 08005770001) were ligated to the cDNA and the libraries were amplified using minimal PCR cycles. Libraries were sequenced on an Illumina NextSeq500 instrument at the Indiana University Center for Genomics and Bioinformatics. Approximately 30–50 million 75 bp single-end sequence reads were obtained for each library.RNA was isolated from input lysates and immunoprecipitates from WT, . FeatureCounts (v1.5.2) was used to count mapped reads to Wormbase (WS262) gene annotations using the [-s 2] flag for reversely stranded reads. Genes with read counts of zero across all samples were removed as indicating non-expressed transcripts. To determine enrichment of transcripts in the IP, RNA-sequencing datasets for two biological replicates of both immunoprecipitate and input samples for each strain were analyzed. Raw read counts were input into DESeq2 (v1.18.1) to test for ratio of ratios, in this case ((IPA/InputA)/(IPB/InputB)), using a likelihood ratio test. To determine high confidence editing sites in the input RNA-Seq datasets from WT, adr-2(-) and adr-1(-), the aligned reads for the two replicates of each sample were merged to increase coverage at edited sites. These sites were then identified using SAILOR as previously described  between experimental (WT or adr-1(-)) and control (adr-2(-)) were considered to be enriched in the ADR-2 IPs. A list of C. elegans edited transcripts from several published RNA-seq datasets  using the following STAR (v2.5.2b) parameters: escribed . Sites wdatasets  was overadr-1 (WormBase ID: WP: CE32459) was PCR amplified and cloned into the pFastBacHTB vector downstream of a 6-histidine tag and a TEV protease recognition site. Mutations to the individual dsRBDs of ADR-1 were generated using site-directed mutagenesis (KKxxK into EAxxA) and the double dsRBD mutant was generated by subcloning the dsRBD2 mutations into the dsRBD1 mutant plasmid. The coding region of adr-2 (WormBase ID: WP: CE25120) was PCR amplified and cloned into the pLK2-10His-3C vector  and Insect Cell Culture Techniques’ as well as the methods described in , respectWT ADR-1 and ADR-1 dsRBD mutants were expressed in Sf9 cells. Baculovirus-infected cells were collected by centrifugation 72 h after infection. Cells were resuspended in Lysis Buffer  and sonicated for 20 s at an amplitude 20 for three times, resting on ice for 1 min between each sonication (Misonix-ultrasonic liquid processor). Following ultracentrifugation at 179,200 g for 30 min at 4°C, the supernatant was added to Ni-NTA (Qiagen) resin and incubated for 1 h. Resin was washed with buffer  and His-ADR-1 was eluted with buffer .WT ADR-2 and ADR-2 mutants were expressed in Hi5 cells. Baculovirus-infected cells were collected by centrifugation 72 h after infection. Cells were resuspended in Amylose wash buffer , sonicated and centrifuged as described for recombinant ADR-1. The supernatant was incubated with Amylose resin (NEB) for 1 h and then washed with 5–10 column volumes of wash buffer. ADR-2 was cleaved from the amylose-bound MBP tag by 3C protease. Cleaved ADR-2 was diluted and then incubated with Heparin resin  and eluted using a high-salt buffer .Both ADR-1 and ADR-2 were dialyzed into storage buffer . Protein purity and concentration were determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining, alongside known concentrations of bovine serum albumin (BSA-Sigma) standards. Proteins were concentrated using Amicon Ultra-4 centrifugal filters (30 K), aliquoted and stored at −80°C.C. elegans lam-2 3′ UTR that contain 25 sites that undergo A-to-I editing in vivo, 8 of which require ADR-1 for maximal editing  was used to remove unincorporated nucleotides. RNA concentration was calculated based on the radioactive ATP incorporated in each strand (measured by a scintillation counter). Equal molar ratios of each strand were combined and incubated in 1× annealing buffer ) and then subjected to high heat (90°C) for 2 min in a water bath and cooled gradually to room temperature. The annealed dsRNA was subjected to Native PAGE at a constant power of 3 W for ∼3 h. The dsRNA band was excised from the Native gel, and the RNA was allowed to passively diffuse into 350 μl of 1× electrophoretic mobility shift assay (EMSA) buffer overnight. The concentration of the dsRNA was measured using a Beckman scintillation counter model LS600SC.The 200 bp long dsRNA was prepared by amplifying regions of the  editing . PCR temlam-2 3′-UTR region (32P γ-ATP using Polynucleotide kinase (Polynucleotide kinase-NEB). Free nucleotides were removed using microspin columns (Biorad). Annealing of ssRNAs, gel extraction of dsRNA and quantification were performed as described above.The 46 bp long dsRNA was made by annealing two complementary synthetic ssRNAs from R region . These s2, 0.5 mM dithiothreitol [DTT]) on ice for 30 min. Reactions were stopped by adding 5× loading buffer  and 75% of the reaction was loaded into a Native 6% (29:1 acrylamide/bisacrylamide (Biorad)) gel. Electrophoresis was performed at 4°C at 200 V for 2–2.5 h with 0.5× Tris-borate-EDTA (TBE) running buffer. Gels were dried, exposed to phosphorimager screens overnight and autoradiographed (Typhoon FLA 9500). ImageQuant TL (1D v8.1) was used to quantify the bound and free RNA. RNA-binding isotherms were obtained using GraphPad Prism 7.0a with a curve fit of specific binding with Hill slope.Gel mobility shift assays were performed as previously described . Briefly2HPO4, 0.02 M KH2PO4, 0.009 M NH4Cl, 0.02 M NaCl) and two volumes of TGKED buffer  containing Complete protease inhibitor (Roche) were added to the worm pellet. The worm suspension was sonicated at an amplitude of 20 (power ∼4) for 10 s four times and at an amplitude of 35 four times (Misonix-ultrasonic liquid processor), resting the tube on ice for 1 min between each sonication. Lysates were centrifuged at 4°C at 16 000 g for 45 min and quantified using a Bradford assay. Extracts were aliquoted, flash frozen and stored at −80°C.Worm extracts were made as previously described . Briefly2, 5% glycerol, 1 mM DTT and 0.4 U/μl RNasin (Promega)) with a final concentration of 1 nM radiolabeled lam-2 3′ UTR (long) and 100–150 μg worm extracts. Reactions were incubated at 20°C for 2 h and stopped by the addition of a 10:1 Trizol:chloroform mixture. RNA was isolated from the aqueous layer, ethanol precipitated and subjected to cleavage with P1 nuclease  at 37°C for 30 min. Cleaved nucleotides were extracted using a 25:24:1 phenol/chloroform/isoamyl mixture and then lyophilized using a speed vacuum. Mononucleotides were resuspended in water and spotted on a thin-layer chromatography (TLC) plate  that was run with solvent (197.5 ml saturated (NH4)2SO4, 47.5 ml 0.1 M NaOAc, pH 6.0, 5 ml isopropanol) for 2 h, exposed to phosphorimager overnight and autoradiographed (Typhoon FLA 9500).Deamination reactions (100 μl) were performed in assay buffer , except worms were not subjected to ultraviolet (UV) crosslinking. The immunoprecipitates were washed with RIP wash buffer, resuspended in 2× SDS buffer and subjected to SDS-PAGE. For the in vitro pulldown assay, Dynabeads  were incubated with ADR-2 antibody . RIP wash buffer with freshly added detergents  was used to wash the antibody-bound beads before the addition of ADR-2 to the final concentration of 5 nM. After 2 h of incubation at 4°C, unbound ADR-2 was removed with wash buffer. BSA was added to the beads at a final concentration of 0.4 μg/μl to reduce non-specific binding and incubated for 1 h. The beads were then incubated with ADR-1 proteins  in EMSA buffer  for 1 h with or without 46 bp dsRNA . The beads were washed with RIP wash buffer, resuspended in 30 μl of 2× SDS buffer and subjected to SDS-PAGE. Immunoblotting analysis for the in vivo IP utilized FLAG and ADR-2 (PA6496) antibodies, while the in vitro pulldowns utilized ADR-2 antibody (IU529) and a custom ADR-1 antibody previously described  was ∼76.3 ± 2.1 nM . However, each ADR-1 dsRBD mutant still exhibited ∼50–80-fold stronger affinity toward dsRNA than that of ADR-2, which contains a single dsRBD. Together, these data indicate that the dsRBD of C. elegans ADR-2 has a lower affinity for dsRNA in contrast to all previously studied ADARs, including C. elegans ADR-1.To date, most ADAR proteins have been shown to bind tightly to dsRNA, with apparent affinities in the subnanomolar to 10 nM range . To testpurified . Bindingpurified . The binM Figure  and H. TM Figure  and F. T results , an ADR-o Figure  and H. Eo Figure  and H  worms. Equal expression and IP of WT ADR-1 and mutants was confirmed by immunoblot  strain  strain. Interestingly, and in contrast to the in vitro RNA-binding data, the individual dsRBD mutants of ADR-1 bound differentially to mRNAs in vivo  and inosine (edited) monophosphate. Extracts were prepared from worms lacking adr-1(-) and transgenic worms expressing WT ADR-1 and both ADR-1 individual dsRBD mutants. Equal concentrations of the lysates were used in the assay and immunoblot indicated equivalent amounts of the ADR-2-editing enzyme in each lysate (adr-1(-) worms  worms  lysate  lysate . Togethein vivo, we sought to determine whether dsRBD2 of ADR-1 might be required for a direct protein–protein interaction with ADR-2. In all kingdoms of life, it has been shown that dsRBPs can interact directly with other dsRBPs via protein–protein interactions between dsRBDs  worms, indicating an in vivo interaction . In contrast, the addition of ADR-2 did not have any significant change in binding of the ADR-1 dsRBD2 mutant  lysates were compared. Importantly, ADR-2 was efficiently immunoprecipitated from both WT and adr-1(-) lysates, but not from lysates of adr-2(-) worms  immunoprecipitates, indicating that ADR-2 stably associates with the lam-2 mRNA in WT worms  worm lysates, ADR-2 failed to enrich lam-2, suggesting that, in vivo, ADR-2 is unable to interact with the lam-2 mRNA in the absence of ADR-1  worms by 37- and 7-fold, respectively, but both showed dramatically lower enrichment from adr-1(-) worm lysates  lysates exhibited similar enrichment for the C35E7.6 mRNA, suggesting that stable association of ADR-2 with C35E7.6 mRNA does not require ADR-1  lysates, as well as the negative control immunoprecipitates from adr-2(-) lysates, were subjected to high-throughput RNA sequencing (RIP-seq). In addition, high-throughput sequencing was performed on RNA isolated from each worm strain before immunoprecipitation, which serves as a normalization for the transcript expression in the worm strains. To identify RNAs that immunoprecipitated with ADR-2, differential enrichment of transcripts in the WT immunoprecipitates  compared to the immunoprecipitates from adr-2(-) worms  worms) was performed for two biological replicates using DESeq2 (bona fide ADR-2 target RNAs (adr-1(-) worms was analyzed and identified 423 transcripts significantly enriched in the ADR-2 immunoprecipitates  worms (adr-1(-), but not WT worms, suggesting that in the absence of adr-1, ADR-2 may associate with some unique target RNAs  worms . These ds Figure .de novo editing site identification and quantification using our recently developed software, SAILOR (adr-1(-) worms. Within 23 ADR-2-bound transcripts, 260 editing sites had significant read coverage in the adr-1(-) RNA-seq to quantify editing using SAILOR (adr-1(-) worms . This heterodimeric complex binds cellular RNAs via the first dsRBD of ADR-1, which promotes stable association of ADR-2 with substrates to allow for efficient editing. Our proposed molecular mechanism, in which a co-factor not only recognizes, but also tethers an ADAR enzyme to its substrate, has not been demonstrated in any other organism. Human and m dimers ,51,54,55m dimers  and/or mm dimers  for ADAR unknown . A parti Figures . To the C. elegans ADR-1 both physically interacts with ADR-2 and promotes stable binding of the complex to RNA to allow ADR-2 to properly edit. However, previous studies have shown that ADR-1 is not required for ADR-2 to edit all adenosines , suggesting the weak affinity of ADR-2 might only be physiological relevant in cells with high expression of ADR-2. In contrast, the higher affinity to dsRNA for ADR-2 in complex with ADR-1 exhibits less cooperative binding to dsRNA (Hill coefficient of ∼1.2), suggesting that the interaction of ADR-2 with ADR-1 in order to bind substrates may be most important in cells with low expression of ADR-2. Further in vitro studies of the dynamics of ADR-2 interaction with different dsRNAs may provide insight into how ADR-2 binds and edits certain transcripts in the absence of ADR-1.The use of a co-factor to promote RNA binding of a modification enzyme to substrates is widespread outside of the ADAR family. For example, the human and fly RNA-binding proteins, RBM15 and RBM15B, bind the RNA methyltransferase machinery and recruit it to transcripts for methylation of specific adenosines ,59. In aenosines ,36,61 anenosines . These dr dsRBDs . Mutatior dsRBDs . Future C. elegans ADR-2 is also able to function as an A-to-I editing enzyme independent of complex formation with ADR-1. Future work aimed at dissecting the cellular pathways that control dynamics of complex formation between ADR-1 and ADR-2 will be critical for understanding the different modes of substrate recognition by ADAR enzymes.Many types of DNA and RNA modifications are catalyzed by a complex of two proteins, where one subunit catalyzes the modification reaction and the partner subunit is not enzymatically active . Our stuin vivo. In this regard, it was recently reported that binding of human ADAR2 to a specific transcript (CTN-RNA) is enhanced by ADAR1 binding to the same transcript, and vice versa .Supplementary DataClick here for additional data file.


Current technologies for targeted characterization and manipulation of viral RNA primarily involve amplification or ultracentrifugation with isopycnic gradients of viral particles to decrease host RNA background. The former strategy is non-compatible for characterizing properties innate to RNA strands such as secondary structure, RNA–RNA interactions, and also for nanopore direct RNA sequencing involving the sequencing of native RNA strands. The latter strategy, ultracentrifugation, causes loss in genomic information due to its inability to retrieve unassembled viral RNA. To address this, we developed a novel application of current nucleic acid hybridization technologies for direct characterization of RNA. In particular, we modified a current enrichment protocol to capture whole viral native RNA genomes for downstream RNA assays to circumvent the abovementioned problems. This technique involves hybridization of biotinylated baits at 500 nucleotides (nt) intervals, stringent washes and release of free native RNA strands using DNase I treatment, with a turnaround time of about 6 h 15 min. RT-qPCR was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. We report that this protocol was able to successfully purify viral RNA by 561- to 791-fold. We also report that application of this protocol to direct RNA sequencing yielded a reduction in human host RNA background by 1580-fold, a 99.91% recovery of viral genome with at least 15× coverage, and a mean coverage across the genome of 120×. This report is, to the best of our knowledge, the first description of a capture-based purification method for assays that involve direct manipulation or characterisation of native RNA. This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes.The online version of this article (10.1186/s13568-019-0772-y) contains supplementary material, which is available to authorized users. Viruses with RNA genomes are the cause of many infectious diseases with serious consequences to human health and mortality such as flaviviruses, HIV-1, SARS-coronavirus, HTLV-1 and influenza virus  and amplified via polymerase chain reaction (PCR) to suit the sensitivities of the downstream assays  was used as the primary proof of concept that capture-based purification indeed removes host background. Subsequently, capture-based purification was applied to direct RNA sequencing as proof of concept that capture-based purification can be coupled with downstream RNA assays. The application of capture-based purification to direct RNA sequencing as an alternative to current strategies will also be discussed.® 150 cm2 cell culture flasks (Sigma Aldrich). Prior to infection, spent media was discarded and Huh-7 cell monolayer was washed with 10 mL 1× PBS twice. Viral inoculum was provided by Low et al. (dengue virus strain D1/SG/05K2402DK1/2005-1-I (DENV1) was diluted with 2 mL pure DMEM media and mixed via pipetting up and down 5 times. Four milliliters of diluted virus was pipetted into each flask and incubated at 37 °C for 1 h. Flasks were rocked every 15 min. Subsequently, inoculum was removed via pipette and 30 mL of pure DMEM media was pipetted to each flask was incubated at 37 °C for 30 h.Viral inoculum and Huh-7 hepatocellular carcinoma cells (Japanese Tissue Repository) were cultured in DMEM media  up to 70–90% confluency in Corningw et al. . A multig, 4 °C for 15 min. Upper aqueous phase in each tube was carefully removed and pipetted to a new 1.5 mL Eppendorf tube (approximately 500 µL). Two microliters of Glycoblue Coprecipitant (Invitrogen) and 500 µL of isopropanol (Sigma Aldrich) was added to each new tube and stored in − 80 °C overnight (or longer). Prior to the capture step, tubes were centrifuged at 12,000g, 4 °C for 15 min and supernatant was discarded. Pellet was re-suspended in 500 µL 75% ethanol diluted with nuclease-free water. Tubes were centrifuged again at 7500g, 4 °C for 3 min and supernatant was discarded. Pellet was air dried. For the cell lysate RNA, total RNA concentration was determined via Nanodrop (Thermo Fisher Scientific) and re-precipitated in ethanol to obtain 30 µg total RNA pellets. Viral supernatant RNA either underwent capture or direct RNA sequencing library preparation without re-precipitation.Extractions were separately performed on viral supernatant and cell lysates. Media of infected flasks  was transferred to a 50 mL Falcon tube. Subsequently the cell monolayer of each flask was treated with 8 mL of Trizol LS Reagent (Invitrogen) and 2.25 mL of 1× PBS (Thermo Fisher Scientific) followed by incubation at 37 °C for 10 min. One milliliter aliquots of this cell lysate mixture were then pipetted to 1.5 mL Eppendorf tubes. 250 µL aliquots of viral supernatant were also pipetted into individual tubes and treated with 750 µL of Trizol LS Regeant. 200 µL of chloroform was then added to individual aliquots of viral supernatant and cell lysate before incubation at room temperature for 10 min. Tubes were shaken vigorously by hand for 30 s, and subsequently inverted by hand every 3 min. Each tube was centrifuged at 12,000https://umasangumathi.github.io/BaitMaker/ . The bait stock in our experiments was previously used for hybridization-based target enrichment in a published study by The Singapore Zika Study Group , optimized for our purposes of RNA capture. SeqCap Hybridization and Wash Kit (Roche) was used in place of Integrated DNA Technologies reagents. Figure RNA Pellet from the extraction step was re-eluted with reagents shown in Additional file 100 µL of Dynabeads M-270 Streptavidin beads (Thermo Fisher Scientific) was pipetted to a 1.5 mL Eppendorf tube and equilibrated at room temperature for 30 min. The tube was put on a magnetic rack (Invitrogen) until eluate was clear and supernatant was discarded. 200 µL 1× Bead Wash Buffer was added and mixture was vortexed for 10 s before placing it on a magnetic rack and discarding the supernatant for a total of two washes. Hybridization mixture was immediately added to the beads and incubated at 65 °C for 45 min.All wash buffers were diluted to 1× prior to washing. Wash Buffer I and Stringent Wash Buffer was pre-heated for 1 h to 65 °C. 100 µL of Wash buffer I was added to the hybridization-bead mixture, agitated by hand and transferred to a new 1.5 mL Eppendorf tube. The new tube is placed on a magnetic rack. Once eluate is clear, supernatant was quickly discarded, taking caution not to remove any beads. 200 µL of Stringent Wash Buffer was added to the pellet and incubated at 65 °C for 5 min, before discarding the supernatant as per the method above, for a total of two washes. Subsequent washes were performed as per the method above without the incubation step in the following sequence: Wash Buffer I, Wash Buffer II, and Wash Buffer III.Viral RNA was released from the beads via DNase I treatment. Beads were re-suspended in 20 µL of nuclease-free water, incubated at 95 °C for 3 min then cooled to 37 °C. Subsequently, 2.7 µL of 10× reaction buffer and 4 µL of TURBO DNase (Thermo Fisher Scientific) was added to the reaction and mixed by pipetting up and down 5 times, followed by further incubation at 37 °C for 40 min. Subsequently, the tube was placed on a magnetic rack to allow the beads to aggregate. The supernatant was then transferred to a clean 1.5 mL Eppendorf tube for a 2.2× Agencourt RNAClean XP beads clean-up (Beckman Coulter). Briefly, 58.7 µL of beads were added to the supernatant and incubated for 8 min at room temperature, and then 2 min on a magnetic rack. The beads were washed twice with 75% Ethanol and residual ethanol was air-dried. The beads were then re-eluted in 30 µL of nuclease-free water.The proof of concept for our capture-based purification method was to demonstrate that host RNA background could be removed from target viral RNA. Absolute quantification of host and viral RNA was done using RT-qPCR. Two 1.5 mL Eppendorf tubes containing 30 µg of total RNA from cell lysate were prepared to represent pre and post-capture groups. 15 µL and 10 µL of nuclease-free water was added to the pre and post-capture group respectively. QuantiTect Probe RT-PCR Kit was used according to the manufacturer’s instructions. Primer targets were selected in the nonstructural 1 protein region of DENV1 and human GAPDH  as a reference gene.The volumes of reagents, shown in Additional file n copies/µL . Each dilution was quantified in duplicate. The sequences of the oligoes, primers and probes used are shown in Additional file q values for the serial dilutions were linearly regressed on log2(copies/µL) and Pearson’s product-moment correlation coefficient was calculated. Equation (q > on log2(copies/µL).A standard curve was constructed using a serial dilution of an oligo complementary to the primer target in accordance with MIQE Guidelines  was calculated using the respective equations of the regression lines.The absolute quantity of DENV1 RNA, normalized to human GAPDH reference gene, was then compared between pre and post-capture groups. The ratio of the absolute quantity of DENV1 and human RNA pre and post-capture was compared, and purification factor calculated using Eq. .2\documeIn this study we defined purification factor as a measure of how well host RNA background is removed from viral RNA.E. coli Poly(A) Polymerase (New England Biolabs). The volumes of reagents shown in Additional file Pre and post-capture RNA extracted from supernatant were sequenced on the MinION. Separately, we pooled 6 captures to generate the concentrated post-capture group. Six captures each consisting of 30 µL of purified RNA was added to the same RNAeasy MiniKit (Qiagen) column and final elution volume was 30 µL. Prior to library preparation, all RNA samples were polyadenylated using The reaction was then incubated in a thermocycler with the following program: 37 °C for 30 min then cooled to 4 °C before proceeding to a 2.2× Agencourt RNAClean XP cleanup (Beckman Coulter). The RNA was eluted in 11.5 µL nuclease-free water. Subsequently, library preparation and sequencing was performed according to the protocol for Direct RNA Sequencing Kit (SQK-RNA001) using 9.5 µL of RNA. All ligation reactions were extended to 20 min. The RNA Library was loaded on a R9.4 MinION flow cell and sequencing was ran for 48 h using MinKNOW v1.14.1 (GUI 2.1.14) interface. Live basecalling was turned off.https://github.com/hyeshik/poreplex) which internally calls Albacore v2.3.1 (Oxford Nanopore Technologies). Basecalled read metrics and plots were then generated using NanoPlot v1.0.0 (https://github.com/wdecoster/NanoPlot). Subsequently, mapping and alignment was performed using Graphmap v0.5.2 (https://github.com/isovic/graphmap) with default parameters. Mean coverage and mapping quality was calculated using Qualimap v0.2.0 ,,4), a bihttps://www.illumina.com/Documents/products/technotes/technote_snp_caller_sequencing.pdf), we assumed the criterion that at least 10 reads were correctly called and mapped to a single base (k = 10).Since the common heuristic for variant calling with Illumina reads requires 10× coverage . This suggests that our capture-based purification method is highly successful in the removal of host RNA background. The minimum coverage required for variant calling was, as described above, benchmarked to that of Illumina reads so that the effectiveness of our capture-based purification method could be more accurately evaluated based on the higher error read rates of direct RNA sequencing technology. Even with the higher adjusted heuristic for variant calling of 15×, the 3.89-fold increase in percentage of the genome above 15× coverage suggests that application of our capture-based purification method increases the amount of target genetic information retrieved.We demonstrated with RT-qPCR that our novel RNA capture technique can be used to retrieve and purify viral RNA in its native form for downstream assays. This was corroborated by our direct RNA sequencing results. Indeed, unpurified viral RNA represented by the pre-capture group did not produce sufficient throughput for any meaningful downstream analysis due to the indiscriminate nature of the sequencing of both host and viral RNA. However, after treatment with our capture method, the amount of host RNA background has decreased greatly as seen from the 272-fold increase in mapping rates to target viral RNA. In comparison, even with the incorporation of PCR amplification for shotgun sequencing of n et al.  reportedt et al.  observedOur method fundamentally allows for the capture of unknown viral strains and genotypes due to the flexibility of adding multiple baits complementary to different regions of the viral genome, or even multiple baits specific to different viruses in a single capture. Indeed, Deviatkin et al.  has repoAnother major advantage to our capture-based purification method is that large machinery like an ultracentrifuge is not required. To perform our capture method, only a thermocycler and a heat block is required. This is especially applicable to research environments where an ultracentrifuge is not readily available. More fundamentally, our capture-based purification method allows for the capture of both unassembled and assembled viruses, which any method leveraging on the differential physiochemical properties of encapsulated viral RNAs cannot. This would be important in studies of unassembled RNA segments, which have been demonstrated to be of great biological importance. Indeed, in the case of DENV1, subgenomic flaviviral particles have been shown to underlie mosquito immunity and viral transmission  adapter which would hybridize with poly(A) tails, facilitating adapter ligation  tail since the conventional poly(T) adapter was substituted with a custom sequencing adapter specific to the target. However, one drawback of this strategy is that foreknowledge of the 3′ end of RNA targets is required. This entails that sequencing of unknown, novel, or mutant viruses would be difficult. Another limitation is that this strategy may be ineffective in cases where the RNA sample is fragmented since the 3′ end of each fragment of viral RNA is distinct.The custom adapter strategy employed by Keller et al.  greatly Degenerate adapters may be used to circumvent the first limitation, as with the degenerate adapters at the + 4 position used by Keller et al. . HoweverIn this study, we demonstrated the second and third strategies, which make use of the conventional poly(T) adapter. This was done using DENV1, a positive single stranded RNA virus lacking a poly(A) tail. The protocol used to generate the pre-capture group sequencing results employ the second strategy while that for the post-capture and concentrated post-capture results employ the third. The polyadenylation of RNA and subsequent use of the conventional poly(T) adapter has several advantages. First, the polyadenylation of RNA prior to library preparation and sequencing allows for sequencing of RNA viruses with or without an inherent poly(A) tail. Second, usage of the conventional poly(T) adapter removes the need for any foreknowledge of the target sequence. Third, all RNA fragments would be ligated with adapters since the adapters are not sequence specific, circumventing the problem of fragmented RNA. However, the main disadvantage strategies involving polyadenylation of RNA is that sequencing specificity is greatly reduced as host mRNA transcripts include a poly(A) tail and will also be sequenced. Wongsurawat et al.  independThe issue of low sequencing specificity is, however, less pronounced in the third strategy. Comparing the second strategy (pre-capture) and third strategy (post-capture), there is a 50-fold increase in percentage of reads mapping to DENV1. Furthermore, a comparison of pre-capture and concentrated post-capture results yields a 123-fold increase in mapping rates to DENV1. This suggests that while simply polyadenylating RNA samples leads to low sequencing specificity, applying our capture-based purification prior allows us to reap the advantages of using the conventional poly(T) adapter whilst having a significantly higher sequence specificity. Additionally, coupling capture-based purification with polyadenylation yields a higher target mapping rate of 31.31% with regard to the 3.98% observed when the fourth strategy was employed (Wongsurawat et al. In this study, we demonstrated a novel capture-based method for the purification of viral RNA from host RNA. In addition, we described a successful attempt of applying our capture method to the direct RNA sequencing of viral culture, using a DENV1 model, exemplifying how our capture method could significantly remove host background in downstream assays requiring viral RNA in its native form. Further improvements to the capture yield are underway and could potentially expand its application to clinical samples where starting RNA material is low. The fundamental advantages of using our capture-based purification method makes it a superior alternative to conventional viral purification methods and addresses limitations of current strategies for direct RNA sequencing, potentially paving a new path for the characterization and direct RNA sequencing of viral RNA.Additional file 1: Table S1. Reagents and volumes for hybridization step. Table S2. Reagents and volumes for RT-qPCR. Table S3. RT-qPCR program set on Applied Biosystems ViiA 7 Real Time-PCR System. Table S4. Primers, probes and standards used with the respective modifications for RT-qPCR. Table S5. Reagents and volumes used for polyadenylation of RNA samples. Figure S1. DENV1 and GAPDH standard curves used for calculation of primer efficiency. Figure S2. Coverage plot against nucleotide position on DENV1 reference for pre-capture MinION sequencing run. Figure S3. Coverage plot against nucleotide position on DENV1 reference for post-capture MinION sequencing run. Figure S4. Coverage plot against nucleotide position on DENV1 reference for concentrated post-capture MinION sequencing run.


RNA structure and dynamic studies by NMR spectroscopy suffer from chemical shift overlap and line broadening, both of which become worse as RNA size increases. Incorporation of stable isotope labels into RNA has provided several solutions to these limitations. Nevertheless, the only method to circumvent the problem of spectral overlap completely is the solid-phase chemical synthesis of RNA with labeled RNA phosphoramidites. In this review, we summarize the practical aspects of this methodology for NMR spectroscopy studies of RNA. These types of investigations lie at the intersection of chemistry and biophysics and highlight the need for collaborative efforts to tackle the integrative structural biology problems that exist in the RNA world. Finally, examples of RNA structure and dynamic studies using labeled phosphoramidites are highlighted. Ty. Ty40]. tert-butyldimethylsilyl (tBDMS), [(triisopropylsilyl)oxy]methyl (TOM), and 2′-cyanoethoxymethyl (CEM) group are preferred. Despite the stability under acidic conditions and ease of addition and removal, tBDMS suffers from a low coupling efficiency, long coupling time, and migration from the 2′- to 3′-OH during RNA synthesis undec-7-ene (DBU) [2-15N2]-adenosine was prepared in 7 steps with 45% yield starting from an inexpensive pyrimidine, 6-amino-2-thioxo-1,2-dhydro-4(3H)-pyrimidone, and [15N]-NaNO2 and [15N]-NH4Cl as 15N sources for N7 and NH2, respectively [15N]-hypoxanthine was generated in 81% yield and is readily converted to [7-15N]-6-chloropurine for enzymatic transglycosylation with commercially available purine nucleoside phosphorylase (EC 2.4.2.1) and 7-methylguanosine as the ribose source. Selective amination at C6 was achieved with [15N]-NH4Cl. -adenosine was prepared similarly, except during the ring closure of the 4,5-diaminopyrimidine intermediate the 13C label was introduced by [13C]-sodium ethyl xanthate -adenosine or -guanosine can be synthesized starting with -adenosine in 5 steps with 60%–62% yield and 4 steps with 65%–70% yield, respectively. Although this synthetic strategy is well suited for purine nucleoside phosphoramidites, the 2′-O-tBDMS regioselectivity is not applicable to pyrimidines -purines and [3-15N]-pyrimidines by N1/N3 nitration of unlabeled protected nucleoside precursors and subsequent substitution with in situ generated 15NH3 [15N]-adenosine and -guanosine and [3-15N]-uridine and -cytidine 2′-O-TOM phosphoramidites starting from commercially available unlabeled 6N-Ac,5′-O-DMTr,2′-O-TOM-adenosine and 5′-O-DMTr,2′-O-TOM-uridine, respectively, with [15N]-NH4Cl as the 15N source [15N]-adenosine was prepared in 5 steps with 37% yield and an inosine intermediate was used as a starting point to synthesize [1-15N]-guanosine in 6 steps with 24% yield. [3-15N]-uridine was obtained in 3 steps with 49% yield and was converted to [3-15N]-cytidine with 81% yield in two additional steps. The isotope labeled, N-Ac,5′-O-DMTr,2′-O-TOM-nucleosides were then converted into phosphoramidites with 2-cyanoethyl diisopropylphosphoramidochloridite (CEP-Cl) in 1 step with at least 90% yield. The main advantage of this synthesis is that the incorporation of 15N occurs after the low yielding 2′-O-TOM protection.Wenter and Pitsch developed a convenient approach to obtain -purine and [3-15N]-pyrimidine phosphoramidites with either 2′-O-tBDMS or 2′-O-TOM protection [15N]-adenosine phosphoramidite was prepared in 8 steps with 16% yield. During this synthesis, a nitroso-inosine intermediate was used to synthesize the [1-15N]-guanosine phosphoramidite in 11 steps and 9% yield. As far as the pyrimidines were concerned, [3-15N]-uridine was synthesized starting from unlabeled 2′-O-tBDMS-uridine in 6 steps with 22% yield. The [3-15N]-cytidine was obtained from 2′-O-tBDMS-[3-15N]-uridine in 5 steps with 25% yield and -cytidine was synthesized in 6 steps with 16% yield. This protocol circumvents the need for the expensive starting materials used by Wenter and Pitsch but at the expense of lower reaction yields -KCN, C5 from [2-13C]-bromoacetic acid, C4 from [1-13C]-bromoacetic acid, C2 from [13C]-urea, and 15N was inserted at N1 and N3 with [15N]-urea -thiourea and [13C]-formic acid to install 13C at adenine C2 and purine C8, respectively -adenosine and -guanosine and [6-13C-5-2H]-uridine and -cytidine 2′-O-CEM phosphoramidites -adenosine was synthesized in 5 steps with 29% yield. The [8-13C]-guanosine 2′-O-CEM phosphoramidite was synthesized de novo from the labeled nucleobase in 9 steps with 14% yield. Synthesis of [6-13C-5-2H]-uridine 2′-O-CEM phosphoramidite began from the unprotected labeled nucleoside, and proceeded to the phosphoramidite in 5 steps with 13% yield. Finally, [6-13C-5-2H]-cytidine 2′-O-CEM phosphoramidite was synthesized from 3′,5′-O-TIPDS-uridine in 6 steps with 44% yield. The major disadvantage of 2′-O-CEM phosphoramidites is the need to synthesize both unlabeled and stable isotope-labeled material since they are not commercially available. There are also more synthesis steps compared to tBDMS and TOM labeling protocols. Taken together, many stable isotope labeled RNA phosphoramidites have been synthesized with diverse labeling patterns. The next section is devoted to highlighting a few examples of how these labels can be exploited to study RNA structure and dynamics by NMR spectroscopy.Finally, Kremser et al. synthesized isotope labeled 2′-nversion ,48,52. Kamidites . Startin13C/15N-labeled phosphoramidites to monitor both Watson-Crick and non-canonical base pairs in RNA - and -guanosine were position-specifically incorporated into short (8–12 nts) RNA fragments to compare spectral properties of GU to GC base pairs -guanosine and -adenosine were position-specifically incorporated into short (8 nts) RNAs to study face-to-face and sheared GA base pairs -guanosine and -adenosine were incorporated into loop A and B domains (14–24 nts) from the hairpin ribozyme found in the satellite RNA of tobacco ringspot virus [13C isotopes were used as tags to distinguish between the residues in doubly labeled samples since 1JH2C2 or 1JH8C8 splits the 1H resonances for residues that contain 13C directly coupled to 15N. Wenter and Pitsch incorporated [1-15N]-adenosine and -guanosine and [3-15N]-uridine and -cytidine labels into 5 sites of the 32 nt bistable RNA -uridine and [1-15N]-adenosine labels [15N]-adenosine phosphoramidite to verify the formation of a base triplet near the active site of the env22 twister ribozyme [15N]-uridine and [7-15N]-adenosine incorporated at U4 and A49, respectively (15N(3)-U4 and 15N(7)-A49 was lacking in the HNN-COSY spectra. Nevertheless, the use of [7-15N]-adenosine in HNN-COSY experiments is a straightforward way to demonstrate non-canonical base pairing interactions in RNA.Neuner et al. probed the conformational flexibility of the P3 stem of PreQ1 class-II riboswitch interaction with a single-labeled e labels . Base paribozyme . The A49ectively B. Unfort13C]-uridine and -cytidine and [8-13C]-adenosine and -guanosine samples were prepared by solid-phase synthesis. A similar strategy was applied by Weickhmann et al. to solve the structure of the 43 nt SAM/SAH-binding riboswitch bound to SAH [15N2]- uridine, [6-13C]-uridine and [6-13C]-cytidine and -, [8-13C]-adenosine and [8-13C]-guanosine RNA samples were chemically synthesized -pyrimidine and [8-13C]-purine phosphoramidites remove the 13C-13C scalar and dipolar coupling interactions and thus facilitate straightforward probing of functional dynamics in RNA. Strebitzer and Nußbaumer et al. demonstrated the potential of atom-specific 13C-labeling to probe conformational dynamics in the ε-element of the duck Hepatitis B virus RNA (dHBVε) [13C2]-adenosine incorporated at residues A20, A22, and A41. The kinetics of the unfolding process (kex = 1522 ± 128 s-1) were quantified via a 13C-CPMG relaxation dispersion (RD) experiment utilizing the 13C-adenosine labels  between the major and minor RNA states was obtained from the 1H RD profiles of C7, C9, A10, A93, G94, and U95. Moreover, these data agreed with the previously reported 13C RD experiments, validating the 1H RD approach to study milli- to microsecond dynamics in RNA. These approaches are limited to RNAs less than 70 nts and only labeled nucleobase is readily coupled to unlabeled ribose. The introduction of labeled ribose using this method remains a challenge.In addition to NMR structural experiments, isotope labeled phosphoramidites have been used with great success to study RNA dynamics ,59,60,61 (dHBVε) . Wijmeng (dHBVε) . To crose labels A. In addus sites B [61]. T13C2]-adenosine-labeled residue were prepared by solid-phase synthesis and ligated to an unlabeled acceptor fragment with T4 DNA ligase  excited state with a chemical shift matching that of the CCR5:microRNA complex. Chen et al. concluded that the CCR5 samples free and bound conformations in solution and the addition of cognate microRNA stabilizes the bound conformation.Two strategies to overcome the inherent size limitations of solid-phase RNA synthesis involve ligation of small chemically synthesized fragments to a larger in vitro transcribed segment and use of chemo-enzymatic methodology to incorporate both isotope labeled nucleobase and ribose moieties. This approach was recently applied to interrogate the dynamics of 96 nt CCR5 RNA that stimulates -1 programmed ribosomal frameshifting upon binding to a microRNA A 39]. C. C39]. CA ligase B,C. Not O-tBDMS and 2′-O-TOM phosphoramidites, owing to the commercial availability of the unlabeled material. From the developments made by Kreutz and co-workers, Silantes has made the following labeled phosphoramidites commercially available: [3-15N]-, -, and [6-13C-5-2H]-uridine and -cytidine, and [1-15N]-, [8-13C]-adenosine and -guanosine  synthetic approaches to incorporate isotope labels into the ribose and (2) solid-phase synthetic conditions that accommodate RNAs larger than 100 nts.In summary, we present an overview of past and current RNA phosphoramidite isotope labeling methodologies and their applications in RNA structure and dynamic analysis by NMR spectroscopy. The biggest practical considerations in the solid-phase chemical synthesis of labeled phosphoramidites is 2′-OH protection and stable isotope labeling pattern, both of which are specified by the RNA of interest. Most NMR studies discussed herein were with RNAs prepared with 2′-uanosine . The ava


The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. However, little is known, at such high resolution, how transcription factors directly influence RNA Pol II pausing and directionality. To map the impact of transcription/elongation factors on transcription dynamics genome-wide at base pair resolution, we developed an adapted NET-seq protocol called NET-prism . Application of NET-prism on elongation factors , splicing factors (Sf1), and components of the pre-initiation complex (PIC)  reveals their inherent command on transcription dynamics, with regards to directionality and pausing over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity during gene expression. Transcription is a highly dynamic process that comprises three different stages. Initiation involves RNA Polymerase II recruitment to the promoter followed by release of RNA Pol II towards progressive elongation. Transcriptional termination is promoted when RNA transcripts are processed and RNA Pol II is released from the chromatin template ,2. This S. cerevisiae, such an approach – coined TEF-seq – was developed to interrogate Paf1 – RNA Pol II interaction [Approaches that precisely map the position of RNA Pol II at a high resolution have provided a deeper insight into transcriptional regulatory mechanisms –11. For eraction  and alloHere, we sought to develop a mammalian counterpart to TEF-seq, which includes an immunoprecipitation step of RNA Pol II associated factors, while being efficient enough to capture sufficient amounts of nascent RNA for processing the latter as part of NET-seq type libraries. A detailed protocol is outlined in Similarly to the original yeast NET-seq and TEF-seq protocols ,7, we reWe identified both, positive  and negative (NELF) elongation factors as well as splicing  and TFIID  components as significantly enriched with Pol II under NET-prism conditions  and Supp– NET-seq/prism), indicating that different TFs establish unique patterns of RNA Pol II stalling. Indeed, aligned and averaged NET-prism profiles over the TSS demonstrate additional regulatory complexity during transcriptional initiation and elongation, suggesting that TF binding specificity directly affects RNA Pol II initiation and elongation dynamics. IPs for elongation factors Spt6 and Ssrp1 show strong and broad enrichment of the Pol II complex. These data are in agreement with ChIP-seq densities for both elongation factors [We picked one transcription factor, TFIID (antibody raised against TBP) and two elongation factors, Spt6 and Sssrp1 (subunit of the FACT heterodimer) to validate the NET-prism approach and interrogate the impact of these factors on RNA Pol II activity. We also performed an IP for Mediator (Med14), serving as a negative control, since it did not display a significant association with Pol II under the conditions used to solubilise chromatin ). The da factors . On the Interestingly, as all three factors examined here  also interact with RNA Pol I and III, we asked if nascent transcript would also stem from gene products of these two polymerases. Indeed, all three IPs against these proteins also pull-down nascent transcripts generated by RNA Pol I and Pol III , suggesting that NET-prism might be an ideal tool for the investigation of all three RNA polymerases.Using western blotting, we showed that the conditions of NET-prism allow co-purification of RNA Pol II in the IPs against transcription factors ). HoweveInitially, RNA Pol II was immunoprecipitated using an anti-CTD antibody, followed by competitive elution of RNA Pol II by an excess of CTD peptide (the exact peptide used to generate the α-CTD antibody). The eluent subsequently served as input for the second round of IP using an anti-SSRP1 antibody to capture exclusively SSRP1-bound Pol II complexes. The isolated nascent RNA was subsequently used for library generation. Importantly, comparing single and sequential IP by metagene profiling ) and sin– Total Pol II). Exploration of NET-prism datasets confirmed that only Sf1 exhibited similar pausing at exon boundaries  facilitates the choice of the protein of interest when applying NET-prism. In addition, given the right RNA polymerase inhibitors and antibodies, NET-prism can be extended to specifically interrogate nascent transcription governed by either RNA Pol I or Pol III.We hypothesize that NET-prism will be an ideal tool to investigate transcription/elongation factor interactions with actively travelling RNA polymerase at single nucleotide resolution and with strand specificity. An analogous approach has been previously developed in yeast , where aMoreover, our study yields a global picture of how transcriptional elongation is affected at splicing sites and NET-prism might shed light on an unresolved dogma encompassing splicing catalysis. The idea of transcriptional elongation influencing alternative splicing arises from two unique models; the recruitment model  and the kinetic model (Pol II pausing determines the timing in which splicing sites are presented) ,18. SimiIt is important to recognize though that NET-prism – similarly to ChIP-seq – greatly relies on the quality of the antibody used. Antibody cross-reactivity might result in unspecific binding and thus, generation of artefactual RNA Pol II stalling patterns. Therefore, the choice of a highly specific antibody for the protein of interest is important to achieve unique RNA Pol II footprints.in vivo binding of transcription or splicing factors correlates with transcriptional activity over different cell states and conditions. Therefore, NET-prism will become a valuable tool for unravelling transcriptional and regulatory complexity.Similarly to the human NET-seq , we expe2, on 0.1% gelatin coated plates, in DMEM + GlutaMax™ (Gibco) with 15% fetal bovine serum (Gibco), MEM non- essential amino acids (Gibco), penicillin/streptomycin (Gibco), 550 µM 2-mercaptoethanol (Gibco), and 10 ng/ml of leukaemia inhibitory factor (LIF) (eBioscience).The E14 cell line (mESCs) was cultured at 37°C, 7.5% CO50 μl of Dynabeads G were washed twice in 200 μl of IP buffer (50 mM Tris-HCl (pH 7.0), 50 mM NaCl, 1% NP-40) and ~10 μg of antibody were added. Antibodies used in this study: Spt6 ; Ssrp1 (Biolegends – 10D1); TFIID (Santa Cruz – sc-273); Med14 (Invitrogen – PA5-44864), Total Pol II , Sf1 .8 ES cells were used for each IP. It is important to split cells down to five batches of 2 × 107 each when performing nuclei extraction. All extraction steps are performed on ice to avoid degradation of the nascent RNA. 2 × 107 cells were treated with 200 μl of cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25 µM α-amanitin (Epichem), 10 U RNasin Ribonuclease inhibitor (Promega) and 1× protease inhibitor mix (Thermo)) for 5 min on ice. Lysate was layered on 500 μl of sucrose buffer (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose, 25 µM α-amanitin, 20 U RNasin Ribonuclease inhibitor and 1× protease inhibitor mix) and spun down for 5 min at 16,000g (4ºC). The supernatant was carefully removed and nuclei were resuspended in 100 μl of DNase digestion buffer (1x DNase buffer (NEB), 25 µM α-amanitin, 20 U RNasin Ribonuclease inhibitor and 1× protease inhibitor mix) and further treated with 100 U of DNase I (NEB) for 20 min on ice. It is important for nuclei to be fully resuspended in the DNase digestion buffer. Non-resuspended nuclei are an indication of harsh cytoplasmic lysis conditions – In this case reduce the volume of cytoplasmic lysis buffer.A detailed protocol is available in the Supplementary Information (‘NET-prism protocol’). Briefly, 10Chromatin-solubilised nuclei were spun down at 6,000g (4ºC) for 2 min and the supernatant was carefully removed. Nuclei were further treated with 200 μl of nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 125 mM NaCl, 50 mM urea, 0.2 mM EDTA, 0.625 mM DTT, 25 µM α-amanitin, 20 U RNasin Ribonuclease inhibitor and 1× protease inhibitor mix) for 5 min on ice. Nuclei lysate was spun down at 18,500g (4ºC) for 2 min and supernatants from five different batches were combined. Phosphatase inhibitor mix (x1) (Thermo) was implemented on all the above extraction steps for batches intended for Pol II S2ph and Pol II S5ph immunoprecipitations.8 ES cells usually yields ~200–1000 ng of nascent RNA. Assessment of RNA size is important in order to evaluate the fragmentation time during the library preparation.A detailed protocol is available in the Supplementary Information (‘NET-prism protocol’). Briefly, combined supernatants from the previous step were incubated in a final 1/10 dilution in IP buffer for 2 hours at 4ºC. For the sequential IP, a total Pol II antibody was used for 2 hours, followed by elution twice with 100 μl 2.5 mM CTD peptide  for 30 min. The eluate was further incubated with Ssrp1 antibody-coated beads for an additional 2 hours. Beads were washed 4 times with 1 ml of IP buffer and 700 μl of Qiazol (Qiagen) was directly added to the beads, followed by 140 μl of Chloroform. Samples were spun down and supernatant was ethanol precipitated . Concentration and size of nascent RNA was assessed by Nanodrop and TapeStation 2200, respectively. An IP from 10Two biological replicates were processed for each IP and library preparation. NET-prism libraries were prepared similarly to the human NET-seq protocol  with fewhttps://github.com/BradnerLab/netseq) to align (mm10 genome) and remove PCR duplicates and reads arising from RT bias. Reads mapping exactly to the last nucleotide of each intron and exon (Splicing intermediates) were further removed from the analysis. The final NET-prism BAM files were converted to bigwig (1 bp bin), separated by strand, and normalized to x1 sequencing depth using Deeptools [All the NET-prism fastq files were processed using custom Python scripts (eeptools  (v 2.4) The travelling ratio is calculated via:with Proximal Promoter defined as the Pol II coverage −30 bp and +250 bp around the TSS whereas Gene body region as the Pol II coverage +300 bp downstream of TSS and −200 bp upstream of TES.All ChIP-seq fastq files were aligned to the mm10 genome using Bowtie2 (v 2.2.6) with default parameters . All BAM7 cells were extracted, lysed, and nuclei were treated with DNase I as described above. The supernatant was incubated for 2 hours with a total Pol II antibody (ab817 – Abcam) or IgG  at 4ºC. In total, four samples were prepared for each IP . After thorough washing of beads with IP buffer, samples were incubated overnight at 37°C with Tris pH 8.8 and 300 ng Trypsin Gold (Promega). Peptides were desalted using StageTips [Independent ES cell cultures were grown in 10cm dishes. Per IP, 20 × 10tageTips  and driePeptides were separated on a 25 cm, 75 μm internal diameter PicoFrit analytical column (New Objective) packed with 1.9 μm ReproSil-Pur 120 C18-AQ media (Dr. Maisch) using an EASY-nLC 1200 (Thermo Fisher Scientific). The column was maintained at 50°C. Buffer A and B were 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile. Peptides were separated on a segmented gradient from 6% to 31% buffer B for 45 min and from 31% to 50% buffer B for 5 min at 200 nl/min. Eluting peptides were analyzed on a QExactive HF mass spectrometer (Thermo Fisher Scientific). Peptide precursor m/z measurements were carried out at 60,000 resolution in the 300 to 1800 m/z range. The top ten most intense precursors with charge state from 2 to 7 only were selected for HCD fragmentation using 25% normalized collision energy. The m/z values of the peptide fragments were measured at a resolution of 30,000 using a minimum AGC target of 8e3 and 55 ms maximum injection time. Upon fragmentation, precursors were put on a dynamic exclusion list for 45 sec.The raw data were analyzed with MaxQuant version 1.6.0.13  using thBED files containing typical enhancer and super-enhancer coordinates in mESCs were downloaded from Whyte et al. . Distal NET-seq (GSE90906) , ChIP-seq (Pol II; GSE28247 , Ssrp1;