Direct and Indirect Photochemical Reactions in Viral RNA Measured

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Direct and indirect photochemical reactions in viral RNA measured with RT-qPCR and mass spectrometry Zhong Qiao, and Krista R. Wigginton Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04281 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 12, 2016

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Environmental Science & Technology

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Direct and indirect photochemical reactions in viral

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RNA measured with RT-qPCR and mass

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spectrometry

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Zhong Qiao,† and Krista R. Wigginton†* †

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Department of Civil and Environmental Engineering,

University of Michigan, Ann Arbor, Michigan 48109, USA

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_____________________________________ *corresponding author: [email protected] Tel. (734) 763-9661 Fax. (734) 764-4292 Word Counts 207 words in Abstract + 4,399 words in Text + 1,800 words in Figures + 600 words in Tables = Total 7,006

1 ACS Paragon Plus Environment


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Abstract: RNA carries the genetic instructions for many viruses to replicate in their host cells.

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The photochemical reactions that take place in RNA and affect viral infectivity in natural and

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engineered environments, however, remain poorly understood. We exposed RNA oligomer

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segments from the genome of bacteriophage MS2 to UV254, simulated sunlight, and singlet

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oxygen (1O2), and analyzed the oligomer reaction kinetics with RT-qPCR and quantitative

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MALDI-TOF mass spectrometry (MS). Following UV254 exposure, quantitative MALDI-TOF-

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MS detected significantly more RNA modifications than RT-qPCR, suggesting that certain

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chemical modifications in the RNA were not detected by the reverse transcriptase enzyme. In

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contrast, MALDI-TOF-MS tracked as much 1O2-induced RNA damage as RT-qPCR. After 5

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hours of simulated sunlight exposure (5100 J/m2 UVB and 1.2 × 105 J/m2 UVA), neither

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MALDI-TOF-MS nor RT-qPCR detected significant decreases in the oligomer concentrations.

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High-resolution ESI-Orbitrap MS analyses identified pyrimidine photohydrates as the major

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UV254 products, which likely contributed to the discrepancy between the MS- and RT-qPCR-

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based results. Reactions between RNA oligomers and 1O2 resulted in an unidentified major

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product with a mass change of +6 Da. These results shed light on the photochemical reactions

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that take place in RNA and suggest that the analytical techniques used to detect RNA reactivity

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could bias the observed reaction kinetics.

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Introduction

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Many illnesses are transmitted by enteric viruses,1 often by exposure to drinking or

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recreational waters that have not been appropriately treated. Disinfection is the main line of

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defense for inactivating viruses in water. Understanding virus disinfection mechanisms helps

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improve treatment technologies and also predict the fate of non-culturable or newly emerged

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viruses during disinfection processes. Most enteric viruses are composed of a small RNA or

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DNA genome that is protected by a protein capsid. The specific chemical reactions that take

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place in viral proteins during disinfection and the biological significance of those reactions have

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been the focus of recent studies,2-5 but our understanding of the specific reactions that take place

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in viral nucleic acids is more limited.

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Disinfection treatments that harness photochemistry, including ultraviolet (UV) germicidal

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irradiation and solar water disinfection (SODIS), primarily target the viral genome.6,7

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Photochemical reactions in viral genomes can take place via direct or indirect photolysis

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pathways.8 In the direct mechanism, viral nucleic acids absorb UV light and then react to form

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photoproducts.9 In indirect pathways, exogenous sensitizers outside of the organism (e.g., NOM)

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absorb light and then react with the nucleic acids or react with other constituents in the water to

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form reactive species (e.g., singlet oxygen (1O2), hydroxyl radical (OH•)) that subsequently react

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with the nucleic acids.10 Alternatively, endogenous molecules within the virus particle can also

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act as sensitizers.3

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Most of the past research on nucleic acid photochemistry has focused on DNA. The major

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DNA modifications induced by UV radiation include cis-syn cyclobutane pyrimidine dimers

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(CPD), (6-4) photoproducts, and pyrimidine hydrates, with other modifications occurring at



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lower levels.11 Oxidants that form from indirect photolysis pathways, like 1O2, preferentially

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react with guanine bases in DNA,12 although all four bases are susceptible to oxidative damage.13

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The oxidation product 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) is often used as a

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marker of oxidative damage in deoxynucleosides.14-17

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Studying the mechanistic fate of viral RNA through photochemical treatment processes

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requires both microbiological and analytical methods. Infective viruses can be enumerated before

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and after disinfecting treatments, as long as the viruses of interest are culturable.18-20 A number

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of important waterborne viruses, however, are not culturable or are difficult to culture with

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available cell lines (e.g., human norovirus, hepatitis A virus). Quantitative RT-PCR (RT-qPCR)

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is widely used to enumerate viral genomes when viruses are not readily cultureable7,21,22 and to

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study the kinetics of reactions that take place in viral genomes.2,23 When used to track genome

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inactivation, there is a common assumption that RT-qPCR tracks all of the modifications in

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RNA, but the validity of this assumption has not been readily examined.7,24 Reverse transcriptase

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has a high error rate;25 for example, it makes inaccurate base modifications while transcribing

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RNA into DNA. It is therefore possible that RT-qPCR fails to recognize all RNA modifications

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that take place during direct and indirect photolysis reactions, some of which may be important

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for virus infectivity.

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The goal of this study was to characterize RNA reactions during direct and indirect photolysis.

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In particular, we studied direct photolysis by UV254 and sunlight radiation, and indirect

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photolysis with 1O2. We focused on 1O2 due to the fact that it is a principal oxidant involved with

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virus inactivation in waters containing NOM.26 Using quantitative Matrix Assisted Laser

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Desorption Ionization time-of-flight mass spectrometry (MALDI-TOF-MS), high-resolution

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Electrospray ionization (ESI) Orbitrap mass spectrometry, and RT-qPCR, we characterized the


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photolysis reaction kinetics and products in two RNA oligomers from the genome of

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bacteriophage MS2.

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Experimental Methods

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Chemicals and reagents

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Furfuryl alcohol (98%), THAP (2’-4’-6’ Trihydroxyacetophenone monohydrate), dibasic

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ammonium citrate (HOC(CO2H)(CH2CO2NH4)2) and Rose Bengal (dye content 95%) were

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purchased from Sigma-Aldrich (St. Louis, MO). HPLC grade acetonitrile (ACN) was purchased

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from Acros Organics (New Jersey, USA). UltraPure DNase/RNase-Free distilled water was

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purchased from ThermoFisher Scientific, (Grand Island, NY).

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UV254, solar spectrum, and 1O2 reaction protocols

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Two RNA oligonucleotides were designed with sequences from selected regions of the

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bacteriophage MS2 viral genome (Table 1). Oligomer A was rich in pyrimidine bases, including

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several pairs of neighboring pyrimidines, and poor in guanines. Oligomer B was poor in

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pyrimidines and rich in guanines. The size of the synthetic RNA oligomers (24-mer) was small

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enough for quantitative RNA mass spectrometry measurements and large enough for RT-qPCR

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measurements. The photolysis experiments were conducted in DNA/RNAse-free water and run

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in triplicate.

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In the UV254 irradiation treatments, 20 µL RNA solution in DNase/RNase-free distilled water

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(4 µM, pH 6.2) was added to the wells of a 96-well plate (Eppendorf, Hauppauge, NY). The

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plate was placed approximately 25 cm below four 15 W germicidal low-pressure mercury vapor

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lamps (model G15T8, Philips, Andover, MA) inside a collimated beam unit. Based on chemical

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actinometry measurements,27 the UV irradiance was 0.17 mW/cm2 at 254 nm. The RNA



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oligomer samples were irradiated for up to 20 minutes, or a dose of 204 mJ/cm2. Shielding

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calculations indicated that 99% of the incident light was transmitted through the sample, thus

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shielding corrections were not deemed necessary.

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For solar spectrum irradiation and 1O2 experiments, samples of oligomers (1 mL, 1.2 µM)

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were pipetted into 5 mm diameter quartz NMR tubes (Wilmand, Vineland, NJ). The tubes were

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placed in a test chamber of a Suntest XLS+ solar simulator (Atlas Material Testing Technology,

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Mt Prospect, IL). The solar simulator spectrum (300 to 800 nm) was monitored with a built-in

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photo-diode detector, with measured irradiances equal to 34 W/m2 and 1.4 W/m2 for the UVA

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(320-400 nm) and UVB (280-320 nm) ranges, respectively. This is equivalent to approximately

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2.4× and 3.4× the intensity of midday sun in Ann Arbor, MI during the summer (Figure S1). The

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temperature in the test chamber was maintained at 25 ºC by an air- and water-cooling system.

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For the 1O2 experiments, Rose Bengal was added to the tubes to a concentration of 1.5 mg/L

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(1.5 µM). To maintain a constant 1O2 concentration, Rose Bengal was replenished in the

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experimental solution to the initial concentration of 1.5 mg/L every 20 minutes. This approach

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resulted in a relatively constant 1O2 concentration of 9 x 10-11 M throughout the experiment, as

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measured with the 1O2 probe compound furfuryl alcohol (Figure S2).26 Control experiments

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conducted either in the dark (i.e. Dark Control) or without Rose Bengal (i.e. No Rose Bengal

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Control) were included in each set of 1O2 experiments.

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In both the 1O2 experiments and direct photolysis experiments, aliquots of the experimental

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solutions were collected from the reaction tubes in the simulator chamber periodically and stored

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refrigerated in the dark. The 1O2 experiments were conducted for two hours and the direct

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photolysis experiments were conducted for five hours. Samples were analyzed immediately after

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the completion of the experiments by RT-qPCR and mass spectrometry.


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Stem-loop primer based RT-qPCR Assay

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The stem-loop quantitative RT-qPCR method applied here was originally developed to

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quantify MicroRNAs (miRNAs) and therefore works well for RNA oligomers that are 18-25

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bases long.28 In brief, stem-loop RNA primers were designed for the two 24-mer RNA targets

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(Table 1). The RNA oligomer standards for RT-qPCR calibration curves were prepared at

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concentrations between 1.3 × 10-3 and 8.0 × 10-2 pmole/µL (7.5 × 108 and 4.8 × 1010 copies/µL).

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The RT reaction solutions (15 µL) consisted of 0.15 µL Deoxynucleotides (dNTPs; 100 mM),

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1.00 µL MultiScribe™ Reverse Transcriptase (50 U/µL), 1.50 µL 10X Reverse Transcription

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Buffer, 0.19 µL RNase Inhibitor (20 U/µL; TaqMan® MicroRNA Reverse Transcription Kit,

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ThermoFisher Scientific, Grand Island, NY), 3.0 µL 5X Stem-loop RT primer (Custom

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TaqMan® Small RNA Assay, ThermoFisher Scientific, Grand Island, NY), 4.16 µL nuclease-

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free water, and 5.0 µL of the RNA oligomer stock. RT was performed in a thermal cycler

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(Eppendorf AG 22331 Hamburg, Hauppauge, NY) at 16 ºC for 30 minutes followed by 42 ºC for

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30 minutes. Finally, the preparation was heated at 85 ºC for 5 minutes to denature RNA-DNA

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hybrids and inactivate reverse transcriptase. The resulting cDNA was then amplified by qPCR.

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The 20 µL qPCR reactions included 1.33 µL of the cDNA solution, 1.00 µL of TaqMan®

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Small RNA Assay (20X), 10.00 µL of 2X TaqMan® Universal PCR Master Mix II with UNG

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(ThermoFisher Scientific, Grand Island, NY), and 7.67 µL of nuclease-free water. Amplification

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and detection were performed with a RealPlex2 Mastercycler system (Eppendorf, Hauppauge,

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NY). The amplification procedure included two hold programs, 2 minutes at 50 ºC to activate the

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uracil N’-glycosylase and then 10 minutes at 95 ºC to activate the hot start DNA polymerases,

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followed by 40 cycles consisting of 15 seconds at 95 ºC and 60 seconds at 60 ºC. Real-time



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fluorescence measurements were analyzed with the RealPlex system software. Experimental

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RNA samples and RNA standards were reverse transcribed and amplified in parallel in each

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analysis.

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MALDI-TOF-MS analysis

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The RNA oligomer samples were analyzed with a quantitative MALDI-TOF-MS technique in

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negative-ion mode on a Bruker Autoflex Speed system (Madison, WI). A 20 mg/L solution of

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THAP in 50% ACN/50% H2O with 50 mg/mL ammonium citrate hydrate was used as the

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MALDI matrix. For quantification, a 26-mer internal standard was designed for each viral RNA

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segment by adding one adenine (A) and one uracil (U) to the 24-mer sequence at the 3’ end

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(Table 1).29 Calibration curves for oligomer quantification were prepared by mixing 1 µM 26-

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mer with different 24-mer concentrations, ranging from 0.2 to 2 µM (Figure S3). The resulting

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calibration curve R2 values were always greater than 0.99. Following the UV, solar spectrum,

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and 1O2 experiments, 5 µL aliquots of the treated oligomer solutions were combined with 5 µL

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of the corresponding internal standard solutions with 1 µM as concentration. These mixtures

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were then combined with the matrix solution at a 1:1 ratio and spotted on a polished steel

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MALDI target plate (Bruker, Madison, WI) and allowed to air dry.

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The MALDI mass measurements were calibrated externally with a mixture of five

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oligonucleotides ranging in masses from 1488 to 9137 Da. MALDI spectra were generated in

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linear mode with 12,000 laser shots randomly collected across the sample spot. Samples were

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scanned from 2,000 to 10,000 m/z.

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ESI-Orbitrap Mass Spectrometry


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High-resolution mass analyses were performed with a qExactive ESI-Orbitrap mass

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spectrometer (Thermo Fisher Scientific, MA, USA) coupled with an EQuan Max Plus LC

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system. The samples were separated on a Hypersil GOLD UHPLC column (50 x 2.1 mm, 1.9

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µM particle size, Part No.: 25002-052130, Thermo Fisher Scientific, MA, USA). The mass

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spectrometer was operated in negative-ion mode with 3.8 kV spray voltage, 320 ºC capillary

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temperature and 50 S-lens. The spectrometer was externally calibrated with Pierce ESI Negative

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Ion calibration solution (Prod #: 88324, Thermo Fisher Scientific, MA, USA). For each analysis,

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15 µL of sample was injected with a mobile phase of 2% Hexafluoro-iso-propanol (HFIP) +

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0.4% Triethylamine (TEA) in water and 2% Hexafluoro-iso-propanol (HFIP) + 0.4%

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Triethylamine (TEA) in methanol. The gradient information is provided in Table S1. For product

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detection and identification, RNA oligomer samples were scanned from 400 to 2000 m/z in full-

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scan mode with a resolution power of 70,000. Product fragmentation was performed in Target-

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MS-MS mode with isolation as 4 amu and an HCD level of 20. A scan window of m/z 400 to

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1200 was collected at a resolution power of 17,500. Mass spectra were processed and analyzed

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by Xcalibur Qual Browser software (Thermo Fisher Scientific, MA, USA).

191 192

Statistical analysis

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Details on how rate constants were calculated are presented in the SI. To test whether there

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were statistical differences between RNA reaction rate constants measured with RT-qPCR and

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mass spectrometry, multiple linear regression analyses were conducted using StatPlus

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(AnalystSoft Inc., Walnut, CA). The null hypothesis was that the kinetics from each experiment

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were not significantly different. The P values were computed and compared at a confidence level

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of 95%.



199 200

Results and Discussion

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Prior to conducting the reaction kinetics experiments, we developed the stem-loop RT-

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qPCR assay and quantitative MALDI-TOF-MS assay for the two MS2 oligomers. Once

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optimized, both quantitative methods resulted in calibration curves with R2 values greater than

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0.99 and the stem-loop RT-qPCR efficiencies were consistently greater than 0.85 (Figure S3 and

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Figure S4). The calibration curve linear concentration ranges differed between the two

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techniques, so that it was necessary to dilute the RNA samples 20-100× prior to RT-qPCR

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analysis, but no dilution was necessary for the MALDI-TOF-MS analyses.

208 209

Direct Photochemical Reactions with UV254

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We exposed the two RNA oligomers to UV254 doses up to 204 mJ/cm2, and tracked RNA

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reaction kinetics with the quantitative PCR and MS methods. For context, this dose of UV254

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causes approximately 5-log inactivation of MS2 virus.2 The two RNA oligomers degraded

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significantly during the UV254 experiment (Figure 1) and no RNA loss was detected when

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samples were incubated in the dark over the same timeframe. The decay of both oligomers

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measured by MALDI-TOF-MS and RT-qPCR followed first order kinetics over the studied dose

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range (Figure 1), with Oligomer A reacting at a faster rate than Oligomer B (Table 2). In

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particular, the MALDI-TOF-MS results show that 70% of Oligomer A segments reacted after

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204 mJ/cm2 of UV254 irradiation, whereas only 32% of Oligomer B segments reacted.

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Meanwhile, RT-qPCR results also suggested that Oligomer A reacted faster than Oligomer B

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segments following exposure to UV254 (45% and 24%, respectively). Past research on reactions

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in nucleic acids suggests that pyrimidine bases are the most reactive with UVC.30-32 We therefore


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expected Oligomer A to react faster than Oligomer B due to the fact that it contains 17

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pyrimidine bases compared to 7 pyrimidine bases.

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For each of the two oligomers, the first order rate constants measured by MALDI-TOF-MS

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were significantly higher than the rate constants determined by RT-qPCR (Table 2; p < 0.01).

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Specifically, Oligomer A and Oligomer B rate constants measured with MALDI-TOF-MS were

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2.2× and 1.6× higher than rate constants measured with RT-qPCR. This indicates that the

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MALDI-TOF-MS technique is sensitive to different photochemical products than the RT-qPCR

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technique.

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Previous research on nucleic acid photochemistry, primarily with DNA, has identified three

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major direct photochemical pathways.33-35 The first and second pathways involve reactions

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between neighboring pyrimidines that lead to the formation of cyclobutane pyrimidine dimers

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(CPD)36,37 and pyrimidine-pyrimidone 6-4 photoproducts (termed 6-4 products).38,39 A third

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pathway forms pyrimidine photohydrates when the reactions take place in aqueous solutions.40

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Reactions that form pyrimidine photohydrate products result in a mass change of +18.015 Da

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(=H2O), whereas the dimer products do not cause a mass change (Figure S5). In our experiments,

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more damage was detected using MALDI-TOF-MS compared to RT-qPCR, despite the fact that

238

the MALDI-TOF-MS technique was not sensitive to the pyrimidine dimer products. This

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indicates that certain products were not efficiently detected with the RT enzymes.

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Pyrimidine hydrates were the major products detected in the UV-treated samples based on the

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product peaks in the MALDI-TOF-MS and high-resolution ESI-Orbitrap-MS spectra (Figure 2).

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Products of Oligomer A included a single pyrimidine photohydrate (mass difference of +18.02

243

Da) and a double pyrimidine photohydrate (mass difference of +36.03 Da; Figure S6). The

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concentration of the single pyrimidine photohydrate product, monitored as the peak height of the



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product relative to the internal standard, reached a maximum at a UV254 dose of 81.6 mJ/cm2 and

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then decreased until the final dose of 204 mJ/cm2 (Figure 2). A single pyrimidine photohydrate

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product of Oligomer B was also detected, but its intensity relative to the internal standard was

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lower than the corresponding Oligo A photohydrate product (Figure 2). A double pyrimidine

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photohydrate product of Oligomer B was not detected.

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Our results agree with an early report on RNA photochemistry that suggested hydrated

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residues are the major photoproducts after large doses of UVC irradiation.41 More recent studies

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tend to assume that pyrimidine dimers in RNA are the major photoproduct,42,43 likely because

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that is true for DNA.44 The discrepancy between RNA and DNA products may be due to the fact

254

that the rate-limiting step of dimerization is the conformational change that creates favorable

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base alignment at the time of excitation, and this may be more prevalent with DNA.45

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The RT enzyme was inhibited or halted by certain RNA products, which may include

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pyrimidine dimers, pyrimidine hydrates, or some other products that have not been identified.

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Pyrimidine dimers are bulkier modifications than the hydrates (Figure S5) and thus may be more

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likely to impact the reverse transcriptase. Although the impact of pyrimidine dimers on RT has

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not been reported, DNA polymerase enzymes can be stopped by certain DNA modifications,

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depending on the type of DNA modification46 and on the specific polymerase. Taq polymerases,

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for example, do not read over pyrimidine dimer lesions, whereas A- and B- family polymerases

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do replicate sequences with pyrimidine dimers.47

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The decrease of RT-qPCR response through water treatment processes and environmental

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processes is often assumed to correlate with the loss of virus infectivity.21,48 There are issues with

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making this assumption, including that the inactivation pathway might not target the genome,2,49

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the RT-qPCR measures only a fraction of the viral genome,7,50 and that the RT enzyme might not


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detect the same type of damage that inactivates the viral RNA genome. Whereas publications

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have addressed the first two points, the specific RNA chemistry that inactivates the virus and

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how that compares to RT-qPCR remains largely unexplored.

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Within the host cell, the genome of (+)ssRNA viruses (e.g., MS2, poliovirus, norovirus) must

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be sufficiently intact to serve as messenger RNA for the host cell ribosomes to make new virus

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proteins and to serve as a template for RNA dependent RNA polymerases to make new RNA

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genomes. With RT-qPCR, on the other hand, the RNA must be sufficiently intact for reverse

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transcriptase to make a complimentary DNA strand that is then amplified by PCR. Previously, a

276

one-hit genome inactivation model was suggested for MS2 treatment with UV254 when RNA

277

damage was monitored by RT-qPCR.7 In other words, the RNA modifications detected by RT-

278

qPCR were sufficient to explain the extent of MS2 inactivation. In our experiments, much more

279

RNA damage was detected by mass spectrometry than by RT-qPCR (Figure 1). Assuming the

280

one-hit model with RT-qPCR detection is accurate for MS2 and other (+)ssRNA viruses, our

281

data suggests a large fraction of UV254-induced RNA reaction products do not inactivate viruses.

282 283

Direct Photochemical Reactions with Simulated Sunlight

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Neither Oligomer A or Oligomer B decreased significantly in concentration after 5 hours of

285

simulated sunlight exposure, regardless of the method used to quantify the oligomer

286

concentration (Figure 3). This dose of UVB solar irradiation (5100 J/m2 UVB and 1.2 x 105 J/m2

287

UVA) is equivalent to approximately 1.5 hours of noontime irradiation in Ann Arbor, Michigan

288

during the summer (Figure S1). Previous research suggests that direct photolysis plays a role in

289

virus inactivation in sunlit waters, with UVB wavelengths causing most of the photoinactivation;

290

20,51,52

we therefore anticipated reactions in the RNA oligomers.



291

The rate of MS2 inactivation was reportedly 0.22 h-1 in sensitizer-free water with UVA/UVB

292

intensities similar to those used here.20 The explanation for our lack of detectable reaction is

293

most likely due to the short RNA segments, which are only 0.7% of the length of the MS2

294

genome. Although the data on RNA reactions due to solar radiation is scarce, the rate constants

295

for pyrimidine dimer formation in dsDNA from UVA or UVB radiation was reportedly 1.4×10-7

296

and 1.0×10-4 per kbp per J/m2 respectively.53 Rate constants for the formation of other DNA

297

photoproducts with UVA and UVB were not readily available in the literature. We applied these

298

reaction rate constants for DNA pyrimidine dimer formation to the full MS2 RNA genome and

299

our 24-mer oligomers. For the MS2 genome, the predicted pyrimidine formation rates were

300

approximately 2 – 5× faster than the MS2 direct photoinactivation rates reported by Silverman et

301

al.20 There are several potential explanations for this discrepancy, including that reactions may

302

be slower in RNA than in DNA, that incorporation in a virus particle may influence the RNA

303

reaction kinetics, that there were differences in the UVA/UVB spectra emitted by the lamps, and

304

that other products are responsible for inactivating the MS2 genome. Regardless of the reason for

305

the discrepancy, the DNA pyrimidine formation rate constants did an adequate job of predicting

306

MS2 inactivation with sunlight. When these same rate constants were applied to our 24-mer

307

oligomers, calculations predicted a ~3% decrease in oligomer concentration due to pyrimidine

308

dimer formation after 5 hours of solar simulator irradiation. This is in agreement with our lack of

309

observed oligomer decay with RT-qPCR and mass spectrometry over the experiment timeframe.

310

Future experiments should expose RNA oligomers in our size range to much higher solar UV

311

doses (e.g., >50,000 J/m2 of solar UVB) in order to readily observe reactions in the oligomers.

312 313

Indirect Photochemical Reactions with Simulated Sunlight


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When irradiated in the presence of the Rose Bengal sensitizer, Oligomers A and B decreased

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in concentration according to first order kinetics and Oligomer B decreased more rapidly than

316

Oligomer A (Figure 4). This trend in reactivity with 1O2 is opposite than what was observed in

317

the direct photochemical reactions and is most likely due to the relative number of guanine bases

318

in the oligomers (10 in Oligomer B and 1 in Oligomer A). Whereas uracil and cytosine are the

319

most sensitive bases to direct photo-oxidation, guanine bases are the most reactive with 1O2 and

320

other oxidants.54

321

There was no statistical difference in reaction kinetics measured by quantitative MALDI-TOF-

322

MS and RT-qPCR (Figure 4, Table 2), suggesting that the same 1O2-induced RNA damage is

323

detected by both techniques. It should be noted that reactions between the oligomers and 1O2

324

resulted in products that interfered with the MALDI oligomer peaks. Consequently, we limited

325

the oligomer measurements by MALDI-TOF-MS to the initial 50% of the Oligomer A and

326

Oliogmer B reactions (Figure 4). This product interference was not observed with RT-qPCR

327

measurements, so the oligomer reactions were monitored by RT-qPCR over the entire

328

experimental timeframe (i.e., 100 minutes). Previous research demonstrated that oxidative

329

damage in RNA inhibited the synthesis of cDNA by RT, although the products were not

330

identified.55 Here, following thirty minutes of 1O2 exposure, ESI-Orbitrap-MS detected a major

331

product for Oligomer A and two major products for Oligomer B (Figure 5). The Oligomer A

332

product had a mass of 7469.579 Da; this product, which is 16.05 Da heavier than the reactant,

333

likely involves the formation of an 8-hydroxyguanosine (8-OHG) adduct, which is a common

334

marker for RNA oxidation. Products of Oligomer B included a species with mass change of

335

+6.00 Da (Product B1) and a species with a mass change of +13.00 Da (Product B2; Figure 5).

336

The masses of Product B1 and Product B2 are not indicative of common RNA adducts reported



337

in the literature, such as 8-hydroxyadenosine, 5-hydroxycytidine, and 5-hydroxyuridine. The

338

products were not resolved with chromatography, thus the fragmentation data was inconclusive.

339

Reported reaction rate constants for 1O2 with RNA monomer bases offer an opportunity to

340

relate our results with established chemical kinetics.54,56 By summing up the reported rate

341

constants of the individual bases in our oligomers, we predicted second order rate constants of

342

7.8 x 106 M-1s-1 for Oligomer A and 1.3 x 107 M-1s-1 for Oligomer B (Table S2). The predicted

343

rate constants are 7× and 2× higher than the measured rate constants, respectively. This may be

344

due to inaccuracies in the reported 1O2 rate constants (see discussion in SI) or due to the impact

345

of primary structure on base reactivity. The predicted rates do agree with our finding that 1O2 is

346

more reactive with Oligomer B than with Oligomer A.

347

The impact that RNA oxidation products have on viral RNA-dependent RNA polymerases

348

has not been studied, but the major DNA oxidation product 8-oxoG leads to mutations during

349

transcription by DNA-dependent RNA polymerases.57,58 If RNA-dependent RNA polymerases

350

undergo the same error, oxidation products in the genome may cause mutations that lead to non-

351

infective viruses. Likewise, oxidized mRNAs can cause ribosome stalling and thus result in

352

defective proteins synthesis;58-61 in viral RNA, this may lead to incomplete or flawed viral

353

capsids that are unable to recognize and interact with host cells.

354 355

Environmental Implications

356

We studied the photochemical reactivity of purified MS2 RNA oligomers to understand the

357

influence of genome sequence on RNA reactivity during water disinfection processes. Our

358

results demonstrate that different regions of viral RNA genomes have distinct photoreactivities

359

and regions that are most susceptible to direct photolysis may be least susceptible to indirect


Page 16 of 25

Page 17 of 25


360

photolysis. Furthermore, not all of the photochemical reactions that take place in RNA were

361

readily detectable by RT-qPCR or MS, which has direct implications for analytical techniques

362

used to define reaction kinetics. Because the detection of RNA modifications that cause virus

363

inactivation are of most interest, future research efforts should seek out the RNA products that

364

inhibit RNA-dependent RNA polymerases and ribosomes.

365

RNA bases incorporated in a virus particle likely react differently than RNA bases in an

366

oligomer. We expect that the direct photolysis reaction rate constants observed in our 24-mers

367

with UV254 would vary slightly when they are incorporated in the full virus, with additional

368

products forming due to interactions between the RNA and capsid proteins. With indirect

369

photolysis involving 1O2, we expect that reaction rate constants would be significantly reduced

370

when the oligomers are incorporated into the virus particle due to the fact that the protein capsid

371

and RNA genome reduce the accessibility of 1O2 to the RNA oligomers. RNA-protein

372

interactions in virus particles likely cause additional RNA oxidation products. The exact impact

373

that RNA higher-level order has on RNA base photochemistry reactivity remains to be

374

investigated. Filling these remaining fundamental knowledge gaps on RNA photochemistry and

375

the biological significance of photochemical products will not only be important for

376

understanding the inactivation of waterborne viruses, but across all domains of life in natural and

377

engineered waters exposed to photochemical stresses.

378

379

Supporting Information Available. The accompanying SI material includes descriptions of our

380

rate constant calculations, spectra of the solar simulator output and solar spectrum in Ann Arbor,

381

MI, a figure of 1O2 concentration measurements, calibration curves for quantitative MALDI-

382

TOF-MS and RT-qPCR methods, chemical structures of RNA photochemical and oxidation

383

products, a high-resolution M.S. spectra for a UV double photohydrate product, a table of our LC



Page 18 of 25

384

gradient profile, and a table of our 1O2 rate constant predictions. This information is available

385

free of charge via the Internet at http://pubs.acs.org.

386

Author Information

387

Corresponding author

388

*Phone: +1- (734)-763-9661; e-mail: [email protected]

389 390

Acknowledgment

391

This research was supported by National Science Foundation (NSF) BRIGE Award #1329576.

392

We thank James Windak from University of Michigan for his help with MALDI-TOF-MS

393

analysis, Thomas Yavaraski and Sergey Chernyak from University of Michigan for their

394

assistance with ESI-Orbitrap-MS measurements, and Collin Ward and Rose Cory for their help

395

with simulated sunlight experiments.

396

Oligomer B

0.0

0.0

-0.2

-0.2

log10 (C/C0)

log10 (C/C0)

Oligomer A

-0.4

-0.6

0

50

100

UV dose

150

-0.4

-0.6

200

RT-qPCR MALDI-TOF 0

50

(mJ/cm2)

100

UV dose

150

200

(mJ/cm2)

397 398

Figure 1. Reactions of two MS2 viral RNA oligomers with UV254 irradiation measured by RT-

399

qPCR and quantitative MALDI-TOF-MS. Experiments were run in triplicate. Experimental

400

conditions: [RNA segment]0 = 4 µM in nuclease free water, pH 6.2.



A

746.39570 z=10

4×10 6

745.69643 z=10

6×10

5

4×10 5 2×10 5

783.91028 z=10 783.81022 z=10

784.20851 z=10 784.11044 z=10

784.60712 z=10 784.70719 z=10 784.80703 z=10

783.71022 z=10

8 78

4.

6

4

4. 78

4.

2

78

4.

0

78

8 3.

4. 78

8

0 7. 74

6

6.

6.

74

4 6.

m/z

401

74

2

0 6.

6.

74

74

8

0

5. 74

5.

6

0

74

746.49544 z=10 746.59542 z=10 746.79476 z=10

745.79620 z=10

2×10 6

784.40746 z=10 784.50724 z=10

784.30785 z=10

8×10 5

746.29608 z=10

745.89620 z=10

B

78

6×10 6

74

Intensity

8×10 6

1×10 6

746.09633 z=10 746.19592 745.99647 z=10 z=10

Intensity

1×10 7

Intensity ratio of product peak to IS peak

Page 19 of 25

0.8 0.6

Oligo A

C

Oligo B

0.4 0.2 0.0

0

50

100

150

200

UV dose (mJ/cm2)

m/z

402

Figure 2. Pyrimidine photohydrates resulting from reactions of oligomers with UV254. A) High-

403

resolution mass spectrum of Oligomer A pyrimidine photohydrate product with -10 charge. B)

404

High-resolution mass spectrum of Oligomer B pyrimidine photohydrate product with -10 charge.

405

C) Ratio of pyrimidine photohydrates peak intensities to internal standard peak intensities (26-

406

mer internal standards with constant concentration of 2 µM), measured with MALDI-TOF-MS.

407

Experimental error bars represent standard error (n = 3 experiments); some error bars are smaller

408

than the symbols. Oligomer B

0.1

0.1

-0.1

-0.1

log10 (C/C0)

log10 (C/C0)

Oligomer A

-0.3

-0.5

-0.5 0

409

-0.3

1

2

3

Time (h)

4

0

5

RT-qPCR

DarkControl (qPCR)

MALDI-TOF

DarkControl (MALDI)

1

3

2

4

5

Time (h)

410

Figure 3. Reaction of two MS2 viral RNA oligomers with simulated solar irradiation measured

411

by RT-qPCR and quantitative MALDI-TOF-MS. Control experiments were conducted in dark

412

environment at the same time. Experiments were run in duplicate. Experimental conditions:

413

[RNA segment]0 = 1.3 µM in nuclease free water, pH 6.2.



Page 20 of 25

Oligomer A 0.1

log10 (C/C0)

0.0 -0.1 -0.2 -0.3 0

20

40

60

80

100

Time (mins)

RT-qPCR MALDI-TOF DarkControl (qPCR)

Oligomer B

DarkControl (MALDI)

log10 (C/C0)

0.0 -0.5 -1.0 -1.5

0

20

40

60

80

100

Time (mins) 414 415

Figure 4. Reaction of two MS2 viral RNA oligomers with 1O2 measured by RT-qPCR and

416

quantitative MALDI-TOF-MS. Control experiments were conducted in dark environment at the

417

same time. Experiments were run in triplicate. Experimental conditions: [RNA segment]0 = 1.0

418

µM in 1.5 mg/L Rose Bengal solution, pH 6.6.


Page 21 of 25


A

B

0 mins

2.0×10

5

930.6835 [OligoA-8H]8-

2.0×10

1.0×10 5 5.0×10 4 0.0

1.0×10 5 5.0×10 4

930

931

m/z

932

0.0 1564

933

1566

4

Intensity

Intensity

2×10 4 1×10 4 931

m/z

932

6×10 4

0 1564

933

1566

4×10

4

930.6832 [OligoA-8H]8-

3×10 4

3×10 4 2×10 4 1×10 4 0

419

932.6895 [Product A-8H]8-

930

931

m/z

1568

m/z

30 mins

Intensity

Intensity

5×10

1572

4×10 4

30 mins 4

1570

1566.7908 [Product B1-5H]5-

2×10 4

932.6878 [Product A-8H]8-

930

1572

1565.5847 [OligoB-5H]5-

8×10 4

3×10 4

0

1570

15 mins

930.6828 [OligoA-8H]8-

4×10 4

1568

m/z

15 mins 5×10

0 mins

1.5×10 5

Intensity

Intensity

1.5×10 5

1565.5812 [OligoB-5H]5-

5

932

1566.7603 [Product B1-5H]51565.5733 [OligoB-5H]5-

2×10 4

1×10 4

0 1564

933

1568.1608 [Product B2-5H]5-

1566

1568

m/z

1570

1572

420

Figure 5. High-resolution mass spectra of two MS2 viral RNA oligomers treated with 1O2 for 0,

421

15 and 30 minutes, obtained by ESI-Orbitrap-MS under full-scan negative-ion mode. A)

422

Oligomer A and reaction products. A) Oligomer B and reaction products.

423



Page 22 of 25

424

Table 1. The sequences and masses of the RNA oligonucleotides from MS2 genome and

425

corresponding internal standards. RNA segment

Sequence

Oligomer A

5’- 982AUCCAUAUCACACCCUUUUCCACG1005 -3’

Oligomer A Internal Standard

5’- AUCCAUAUCACACCCUUUUCCACGAU -3’

Oligomer B Oligomer B Internal Standard

5’- 168UGGAAGCAGGGAUCGCAGGCGCAA191-3’ 5’- UGGAAGCAGGGAUCGCAGGCGCAAAU-3’

Average Mass Monoisotopic Mass (Da) 7453.521 7449.990 8088.898 8085.067 7832.846 7829.137 8468.224 8464.214

426 427 428

Table 2. First order rate constants of oligomer reactions with UV254 and second order rate

429

constants of oligomer reactions with 1O2 measured with RT-qPCR and quantitative MALDI-MS.

430

Errors reflect the 95% confident internal values of rate constants, based on a single linear

431

regression of triplicate experimental data. Arrows indicate there are significant differences

432

between rate constants (p < 0.05; multiple linear regression test). UV254 (mJ-1cm2) MALDI-MS RT-qPCR

RNA Segment

O2 (M-1s-1) MALDI-MS RT-qPCR

Oligomer A

5.7 x 10-3 ± 2.5 x 10-4

2.6 x 10-3 ± 2.0 x 10-4

1.1 x 106 ± 6.1 x 104

1.1 x 106 ± 1.4 x 105

Oligomer B

1.9 x 10-3 ± 1.4 x 10-4

1.2 x 10-3 ± 3.4 x 10-4

5.9 x 106 ± 6.1 x 105

6.5 x 106 ± 3.3 x 105

433

434 435 436

437 438 439 440 441

1

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Direct and Indirect Photochemical Reactions in Viral RNA Measured

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