Dimethyl Sulfate Research Articles

Comparative Analysis of MicroRNA Expression in Three Paulownia Species with Phytoplasma Infection

Paulownia witches’ broom (PaWB), caused by phytoplasma, is an important disease of Paulownia. To further identify the key miRNAs associated with the formation of PaWB symptoms, miRNA and degradome sequencing were performed to explore important miRNAs–target regulation in healthy and diseased Paulownia tomentosa, Paulownia fortunei, and P. tomentosa × P. fortunei seedlings, and the corresponding diseased seedlings treated with 75 mg L−1 dimethyl sulfate. A total of 212, 111, and 197 differentially expressed miRNAs (DEMs) were obtained in P. tomentosa, P. fortunei, and P. tomentosa × P. fortunei, respectively. Degradome sequencing detected 559, 251, and 568 target genes of the DEMs in P. tomentosa, P. fortunei, and P. tomentosa × P. fortunei, respectively. The expression patterns of selected miRNAs and the target genes were verified be qRT-PCR. Through analysis of the expression level of the DEMs in this study, combined with the results in our previous studies, as well as with those reported in other phytoplasma-infected plants, we concluded that miR156 is an important miRNA related to witches’ broom. According to the functions of the target genes of DEMs, we constructed a co-regulatory network of the DEMs-target genes interaction. These results will help to advance the understanding of the mechanism of PaWB.

Адаптивний потенціал генофонду салату посівного листкового, створеного методом індукованого мутагенезу на основі вітчизняного сорту Вельможа

Адаптивний потенціал генофонду салату посівного листкового, створеного методом індукованого мутагенезу на основі вітчизняного сорту Вельможа

Photochemical degradation of trypan blue.

PurposeTo investigate the photochemical degradation of trypan blue (TB) and to identify decomposition products.MethodsDefined solution samples of TB and a mixture with lutein/zeaxanthin were exposed to blue light. Thermal degradation processes were ruled out using controls not subjected to irradiation. All samples were analyzed using optical microscopy, UV/Vis spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance (NMR) spectrometry. Degradation kinetics were determined based on changes in absorbance; intermediates were identified by analyzing mass differences of characteristic fragment ion peaks within the fragmentation patterns, and assignments were verified by NMR.ResultsTB demonstrated a photochemical degradation, which can be triggered by lutein/zeaxanthin. Intermediates vary depending on the presence of lutein/zeaxanthin. The self-sensitized photodegradation of TB occurs under generation of dimethyl sulfate and presumed formation of phenol. In contrast, within the presence of lutein/zeaxanthin the decomposition of TB indicates the formation of methoxyamine and sulfonyl arin. Thermal degradation processes were not observed.ConclusionsTB demonstrated a photodegradation that may be triggered by lutein/zeaxanthin and results in the formation of cytotoxic decomposition products. Our findings contribute to understand degradation mechanisms of TB and may elucidate previous clinical and experimental observations of cellular toxicity after TB application.

Optimization of the method for the determination of diethyl sulfate at workplaces

Diethyl sulfate (DES) is a substance classified to the group of carcinogens. The value of maximum admissible concentration for this substance in workplace air is not specified in Poland. Due to the use of DES in domestic companies there is a need to develop a sensitive method for the determination of diethyl sulfate in the work environment. Studies were performed using gas chromatography (GC) technique. An Agilent Technologies chromatograph, series 7890A, with a mass selective detector (5975C, Agilent Technologies, USA) was used in the experiment. Separation was performed on a capillary column with Rtx-5MS (30 m × 0.25 mm × 0.25 μm) (Restek, USA). The possibility of using sorbent tubes filled with activated carbon (100 mg/50 mg), silica gel (100 mg/50 mg) and Porapak Q (150 mg/75 mg) for absorption of diethyl sulphate was investigated. The method of sampling air containing diethyl sulfate was developed. Among the sorbents to absorb DES Porapak Q was chosen. Determination of the adsorbed vapor includes desorption of DES, using dichloromethane/methanol mixture (95:5, v/v) and chromatographic analysis of so obtained solution. Method is linear (r = 0.999) within the investigated working range of 0.27- -5.42 μg/ml, which is an equivalent to air concentrations 0.0075-0.15 mg/m3 for a 36 l air sample. The analytical method described in this paper allows for selective determination of diethyl sulfate in the workplace air in the presence of dimethyl sulfate, ethanol, dichloromethane, triethylamine, 2-(diethylamino)ethanol, and triethylenetetramine. The method meets the criteria for performing procedures aimed at measuring chemical agents, listed in EN 482. Med Pr 2018;69(3):291-300.

Synthesis of tetradentate metal Schiff base complexes towards the synthesis of novel antimicrobials

Schiff bases are molecules that carry a characteristic imine bond that are derived from carbonyl compounds and are widely studied due to the versatility of their steric and electronic properties. Schiff bases are significant because of their biologically active properties that can improve and develop agrochemicals, anti‐inflammatories, antivirals, and even radiopharmaceuticals for cancer targeting. This study is focused on synthesizing a family of novel metal Schiff base complexes that have potential to act as antimicrobials over a wide range of pathogenic species.In a previous study, a family of metal Schiff base complexes with an NN'NN coordination sphere was synthesized. This study found that antimicrobial activity using Schiff base complexes with chromium, iron, or manganese as their center showed greater inhibition of Aspergillus sp. growth than a variety of known antimicrobial drugs (Kumar et al., 2012). In an attempt to expand the antimicrobial potential of these compounds, we changed the structure of these Schiff base ligands to utilize an NN'OS coordination sphere with the same set of metal centers. We hypothesized that the chemical activity of the sulfur atom would provide an improved range of antimicrobial function.Our synthesis of NN'OS coordination sphere‐based antimicrobials began with the compound cyclopentanone. Cyclopentanone was condensed with carbon disulfide, recrystallized, protonated, and methylated with dimethyl sulfate. The resultant methylated product was aminated with two different diamines to form a set of novel primary amines. These primary amines were reacted with salicylaldehydes with different substitutions to form a family of Schiff base ligands. The last step in the synthesis process was to add a transition metal to the Schiff base ligands (chromium, iron, or manganese) through a reflux reaction. After synthesis, each metal Schiff base complex was characterized through 1H and 13C nuclear magnetic resonance, infrared, and gas chromatography‐mass spectroscopy.With synthesis complete, current work focuses on the examination of these compounds as effective antimicrobials. The compounds are being tested for these properties against Aspergillus furmigatus as a representative of a broad range of Aspergillus species. Characterized Schiff base complexes are being compared for their level of antimicrobial activity against that shown by Miconazole, an established antimicrobial used to treat an array of fungal infections. The results of these tests may establish Schiff base ligands with NN'OS coordination spheres as a novel effective means of treating an array of dangerous microbial infections.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identifying the RNA Kinetic Traps in Ribosome Assembly

The ribosome is the cellular machine responsible for protein production in every living cell. While the final structure of the mature ribosome is known from x-ray crystallography studies, the assembly process remains largely unknown. To fulfill the cells’ demand for proteins, ribosome assembly in vivo occurs fast and efficiently. Consequently, intermediates of the assembly process are short lived and do not accumulate in large amounts for investigation. To increase the accumulation of in vivo intermediates of large subunit ribosome assembly in Escherichia coli, we take advantage of the helicase inactive DbpA construct R331A. When R331A DbpA is expressed in cells, three large subunit particles, with sedimentation coefficients of 27S, 35S, and 45S, accumulate. The particles rearrange to form the native 50S large subunit via parallel pathways. We interrogated the RNA structure of the 45S particle using dimethyl sulfate and SHAPE chemical probing reagents combined with Next Generation Sequencing. The structural probing experiments revealed perturbed secondary structures of the P-loop and the helices of the peptidyltransferase center. Additionally, it revealed an incorrect orientation of helix 38, the finger of the A-site, and several critical, broken tertiary RNA contacts between distant regions of the 23S rRNA. The data suggests the peptidyltransferase center's maturation is one of the final events during assembly. This severe misfolding explains the slow rearrangement of the 45S particle to the native 50S subunit. Next, we will characterize the RNA structure of the 27S and 35S particles. The structural probing of the 27S and 35S particles will provide insights into the early steps of ribosome assembly, which remain a complete mystery.

Synthesis and Antimicrobial Activity of Sulfur Derivatives of Quinolinium Salts.

A novel method for cleavage of the dithiine ring in 5,12-(dimethyl)-thioqinantrenium bis-chloride 1 “via” reaction with sodium hydrosulfide leads to 1-methyl-3-mercaptoquinoline-4(1H)-thione 2. Further transformation of thiol and thione functions of compound 2 leads to a series of sulfide and disulfide derivatives of quinolinium salts 4 and 6. 1-Methyl-4-chloro-3-benzylthioquinoline chloride 8 was obtained by N-alkylating 4-chloro-3-benzylthioquinoline using dimethyl sulfate. Antimicrobial activity of the obtained compounds was investigated using six Gram-positive and six Gram-negative bacterial strains, as well as Candida albicans yeast. Greater activity was demonstrated towards Gram-positive strains. MIC values for compounds and with benzylthio 4d and benzoylthio 4f substituents in 3-quinoline position were found to be in the 0.5–1 μg/mL range, at a level similar to that of ciprofloxacin (reference). Compounds 4d and 4f also demonstrated interesting antifungal properties (MIC = 1).

Novel cationic surfactants based on waste frying oil for cleaning water surface from petroleum films: Synthesis, antimicrobial and surface properties

A novel series of environmentally friendly cationic surfactants were synthesized by reaction of mixed fatty acids, extracted from waste frying oil (WFO) as a low-cost raw material, and N,N-Dimethyl-1,3-propanediamine with high yield products. The synthesized fatty amides were reacted with different alkyl halides and dimethyl sulphate to form the desired cationic surfactants. The acquired compounds were elucidated via different spectroscopic tools as IR, 1H and 13C NMR spectra. Surface properties including critical micelle concentration, effectiveness, maximum surface excess, minimum surface area…. etc. was investigated. Also, thermodynamic parameters including free energy of adsorption (ΔGadso) and micellization (ΔGmico) were calculated. The petroleum capacities of synthesized compounds were evaluated in the diluted and solid form with waters varying in salinity. The results showed that the synthesized compounds have good surface properties, high tendency to be adsorbed at the surface than micellized and high ability to collect and disperse the petroleum film from water surface (the effect ranging from 95–97%). Moreover, the antimicrobial activities of synthesized cationic surfactants against various microbes were investigated.

Suppression of Dendritic Lithium Growth by in Situ Formation of a Chemically Stable and Mechanically Strong Solid Electrolyte Interphase.

The growth and proliferation of Li dendrites during repeated Li cycling has long been a crucial issue that hinders the development of secondary Li-metal batteries. Building a stable and robust solid state electrolyte interphase (SEI) on the Li-anode surface is regarded as a promising strategy to overcome the dendrite issues. In this work, we report a simple strategy to engineer the interface chemistry of Li-metal anodes by using tiny amounts of dimethyl sulfate (DMS, C2H6SO4) as the SEI-forming additive. With the preferential reduction of DMS, an SEI layer composed of Li2S/Li2O forms on the Li surface. This inorganic SEI layer features high structural modulus and low interfacial resistant, enabling a dense and dendrite-free Li deposition as evidenced by scanning electron microscopy, atomic force microscopy, and in situ optical images. In addition, this SEI layer can prevent the deposited Li from direct contact with corrosive electrolytes, thus rendering an improved cycling stability of Li anodes with an average Coulombic efficiency of 97% for up to 150 cycles. When the DMS additive is introduced into a Li/NCM full cell, the cycle life of Li-metal batteries can be also improved significantly. This work demonstrates a feasible route to suppress Li dendrite growth by designing appropriate film-forming additives to regulate the interfacial properties of the SEI layer, and also the sulfonyl-based derivatives revealed in this work represent a large variety of new film-forming molecules, providing a broad selectivity for constructing high efficiency and cycle-stable Li anodes to address the intrinsic problems of rechargeable Li-metal batteries.

Transalkylation of alkyl aryl sulfides with alkylating agents

The reaction of methyl iodide with tert-butylphenylsulfide in DMF leads to a transalkylation that produces methylphenylsulfide. This transalkylation reaction was further studied by 1H NMR spectroscopy. The polarity of the solvent, the electron density on the sulfur atom, and the strength of the alkylating agent (MeI, EtI, BuI, dimethyl sulfate, or dimethyl carbonate) played important roles in the reaction. The suggested mechanism of the reaction involves the formation of a dialkyl aryl sulfonium salt that subsequently eliminates the radical. This mechanism was supported by the observation of higher conversion rates for compounds with more branched alkyl groups on the sulfur atom, which may lead to the formation of more stable radicals.

Harvesting of Microalgae Biomass with Polyethylenimine-Based Sorbents

The purpose of this work was to investigate the sorbents on the basis of polyethylenimine (PEI) intended for collecting biomass of microalgae (MA). For this purpose, a series of porous and insoluble polymeric materials were synthesized by cross-linking of PEI with epichlorohydrine. The analysis of kinetics and efficiency of immobilization assessed for the model culture Chlorella vulgaris, revealed that already within 3 h of incubation, 39–75% of MA cells attached to the surface of tested sorbents. It was shown that on the initial stage of immobilization the sorption activity of polymeric materials depended on the “PEI:crosslinker” ratio. One of the tested sorbents was additionally quartenized by alkylation with dimethyl sulphate resulting in sharp increase of its sorption activity. The estimation of the MA desorption from polymeric surface showed that most Ch. vulgaris cells were practically irreversibly immobilized on all tested sorbents based on the PEI cross-linked with epichlorohydrine.

Convenient iron-catalyzed reductive aminations without hydrogen for selective synthesis of N-methylamines

N-Methylated amines play an important role in regulating the biological and pharmaceutical properties of all kinds of life science molecules. In general, this class of compounds is synthesized via reductive amination reactions using high pressure of molecular hydrogen. Thus, on laboratory scale especially in drug discovery, activated (toxic) methyl compounds such as methyl iodide and dimethyl sulfate are still employed, which also generate significant amounts of waste. Therefore, the development of more convenient and operationally simple processes for the synthesis of advanced N-methylamines is highly desired. Herein, we report the synthesis of functionalized and structurally diverse N-methylamines directly from nitroarenes and paraformaldehyde, in which the latter acts as both methylation and reducing agent in the presence of reusable iron oxide catalyst. The general applicability of this protocol is demonstrated by the synthesis of >50 important N-methylamines including highly selective reductive N-methylations of life science molecules and actual drugs, namely hordenine, venlafaxine, imipramine and amitriptyline.

Potential Impurities of the Anti-epileptic Drug, Clobazam: Synthesis and Characterization

Background: Clobazam (CLB) has been used as an anxiolytic, anticonvulsant and antiepileptic drug under the trade names, Frisium, Urbanol, Onfi and Tapclob. Impurity profiling in the pharmaceutical industries is a central task in the manufacturing of drug product. The quantification of impurities in active pharmaceutical ingredients (APIs) is also receiving considerable importance from the regulatory authorities in the point of stringent quality requirement. Thus, the provision of substantial quantity of impurities as standards, which are accomplished either by isolation from crude reaction mass or the synthesis of material with proposed structure using new synthetic route, to analytical method development and validation is a good demand. Therefore, easily accessible synthetic routes to the preparation of impurities with the lowest cost are sought after. To date, no synthetic specifics were found on the synthesis and characterization of CLB Imp-B, -C, -D and –F, and the synthesis of CLB Imp-E. Therefore, the comprehensive study was undertaken on the synthesis and characterization of CLB related impurities and this study might be useful to prepare CLB impurities in the required quantity with purity to use as a reference standard of analytical method development in pharmaceutical industry. Methods: The synthetic routes were developed for the synthesis of CLB impurities such CLB Imp-B to Imp-F and isolated them using purification techniques with purity in the required quantity. The commercially available key starting materials, reagents and solvents were used in the course of synthesis of impurities. The analytical techniques such as NMR, IR and mass were used to characterize the structure of synthesized CLB related impurities. Results: The easy accessible synthetic methods were developed for the synthesis of clobazam related impurities, CLB Imp-B to F in good to excellent yields with purity as well as described the cause to formation of these impurities. The KSM related impurity, 2-chloro-1-nitrobenzene and the active methylene hydrogens in clobazam are caused to the formation of CLB Imp-B and CLB Imp-C&-D, respectively. CLB Imp-E and –F are formed due to the degradation of drug molecule, CLB in the presence of base (NaOH). CLB Imp-B was prepared from 2-chloro-1-nitrobenzene by following similar process of CLB reported in the synthetic specific. CLB Imp-F was prepared from 5-chloro-N1 -phenylbenzene-1,2-diamine by coupling with mono-methyl malonate followed by N-methylation using dimethyl sulfate. The rest of all impurities were planned to prepare by taking CLB as starting material. CLB Imp-C and –D were synthesized by treating CLB with methyl iodide in the presence of inorganic base. The mixture of aqueous NaOH and CLB was agitated vigorously at 75-80°C to obtain CLB Imp-E. All the synthesized compounds were purified using purification techniques such as crystallization and column chromatography, and well characterized by IR, ESI-mass, 1H NMR, 13C NMR and DEPT analyses. Conclusion: The immaculate discussion about synthetic approaches and structural elucidation using analytical applications such as IR, NMR (1H, 13C and DEPT) of CLB Imp-B to F, which are listed in the European pharmacopoeia monograph of clobazam, has been described. All the impurities were obtained with stringent purity (>98%) in the described synthetic routes and the same impurities have been used in analytical method development in our pharmaceutical industry. Also, the plausible mechanistic path ways to the formation of impurities, keeping in view the regulatory importance of clobazam, were discussed in detail. Keywords: APIs, clobazam, characterization, EP impurities, synthesis, NMR.

Modification and comparison of three Gracilaria spp. agarose with methylation for promotion of its gelling properties

In order to improve the gelling properties of agarose, we modified it by methylation. The agarose was prepared from Gracilaria asiatica, G. bailinae, and G. lemaneiformis with alkaline, treated with diatomaceous earth and activated carbon, and anhydrous alcohol precipitation. The methylation reaction process of agarose was performed with dimethyl sulfate while the chemical structure of low-gelling temperature of agarose was also studied by 13C-NMR and FT-IR spectra. Results showed that the quality of agarose from G. asiatica is optimal. Its electroendosmosis is 0.116, sulfate content is 0.128%, and its gel strength (1.5%, w/v) is 1024 g cm−2, like those of the Sigma product (A9539). The gelling temperature, melting temperature, and gel strength of the low-gelling temperature agarose is 28.3, 67.0 °C, and 272.5 g cm−2, respectively. FT-IR Spectra and 13C-NMR spectra also showed that agarose was successfully methylated. Overall, this work suggests that low-gelling temperature agarose may have potential uses as an agar embedding material in various applications such as biomedicine, food, microbiology, and pharmaceutical.

Glyoxals as in vivo RNA structural probes of guanine base-pairing.

Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson–Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram-positive and Gram-negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.

Activation barriers for methylation of DNA bases by dimethyl sulfate

The SN2 transition states of the methylation reaction of DNA bases with dimethyl sulfate were examined employing DFT/ M06-2X/6-31+G∗ and DFT/B3LYP-D3/6-311+G (2df, 2p) levels of theory. Solvation effects were examined using the conductor-like polarizable continuum model (CPCM). Calculation results and feedback from electrostatic potential maps show that in water, charge separation lowers the activation barriers relative to the gas phase for the reactions at N7 of guanine, N3 of adenine and cytosine. Also, the reaction at the O6 site of guanine is governed by steric interference and exhibits a higher activation barrier in water.

DMS-Seq for In Vivo Genome-wide Mapping of Protein-DNA Interactions and Nucleosome Centers

Protein-DNA interactions provide the basis for chromatin structure and gene regulation. Comprehensive identification of protein-occupied sites is thus vital to an in-depth understanding of genome function. Dimethyl sulfate (DMS) is a chemical probe that has long been used to detect footprints of DNA-bound proteins invitro and invivo. Here, we describe a genomic footprinting method, dimethyl sulfate sequencing (DMS-seq), which exploits the cell-permeable nature of DMS to obviate the need for nuclear isolation. This feature makes DMS-seq simple in practice and removes the potential risk of protein re-localization during nuclear isolation. DMS-seq successfully detects transcription factors bound to cis-regulatory elements and non-canonical chromatin particles in nucleosome-free regions. Furthermore, an unexpected preference of DMS confers on DMS-seq a unique potential to directly detect nucleosome centers without using genetic manipulation. We expect that DMS-seq will serve as a characteristic method for genome-wide interrogation of invivo protein-DNA interactions.

Benzothiazole thiourea derivatives as anticancer agents: Design, synthesis, and biological screening

In a systematic effort to identify a potent anticancer agent, we synthesized benzothiazole thiourea derivatives and examined their cytotoxic activity against five different human and animal cancer cell lines. Benzothiazolylthiocarbamides have been prepared in excellent yields by reaction of substituted 2-amino benzothiazoles with carbon disulfide and dimethyl sulfate followed by their ammonolysis. Cytotoxicity of the four compounds were screened for antitumor activity against human breast cancer cells (MCF-7), human cervix epithelial carcinoma (HeLa), human colon cancer cell line (HT-29), human leukemia cell line (K-562), and mouse neuroblastoma cell line (Neuro-2a) using cisplatin as a reference by MTT assay. Our results presented herein provide experimental evidence that benzothiazolylthiocarbamides induce apoptosis in cancer cell lines. According to flow cytometry results, treatment of HT-29 cells with 1-(6-ethoxy-1,3-benzothiazol- 2-yl)thiourea produced a large population of apoptotic cell (79.45%), which was 1.2-fold higher than that produced by cisplatin (65.28%) at the same concentration.

RNA structure inference through chemical mapping after accidental or intentional mutations

Despite the critical roles RNA structures play in regulating gene expression, sequencing-based methods for experimentally determining RNA base pairs have remained inaccurate. Here, we describe a multidimensional chemical-mapping method called "mutate-and-map read out through next-generation sequencing" (M2-seq) that takes advantage of sparsely mutated nucleotides to induce structural perturbations at partner nucleotides and then detects these events through dimethyl sulfate (DMS) probing and mutational profiling. In special cases, fortuitous errors introduced during DNA template preparation and RNA transcription are sufficient to give M2-seq helix signatures; these signals were previously overlooked or mistaken for correlated double-DMS events. When mutations are enhanced through error-prone PCR, in vitro M2-seq experimentally resolves 33 of 68 helices in diverse structured RNAs including ribozyme domains, riboswitch aptamers, and viral RNA domains with a single false positive. These inferences do not require energy minimization algorithms and can be made by either direct visual inspection or by a neural-network-inspired algorithm called M2-net. Measurements on the P4-P6 domain of the Tetrahymena group I ribozyme embedded in Xenopus egg extract demonstrate the ability of M2-seq to detect RNA helices in a complex biological environment.

Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA.

Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given the variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely nonoverlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.