Nucleic Acid Photolysis by UV254 and the Impact of Virus

Subscriber access provided by University of Winnipeg Library

Environmental Processes

Nucleic acid photolysis by UV254 and the impact of virus encapsidation Zhong Qiao, Yinyin Ye, Pin Hsuan Chang, Devibaghya Thirunarayanan, and Krista R. Wigginton Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b02308 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

Environmental Science & Technology

1

Nucleic acid photolysis by UV254 and the impact of

2

virus encapsidation

3

4

Zhong Qiao,† Yinyin Ye,† Pin Hsuan Chang,† Devibaghya Thirunarayanan ,† and Krista R.

5

Wigginton†* †

6 7

Department of Civil and Environmental Engineering,

University of Michigan, Ann Arbor, Michigan 48109, USA

8 9 10 11 12 13 14 15 16 17

_____________________________________ *corresponding author: [email protected] Tel. (734) 763-9661 Fax. (734) 764-4292

18

198 words in Abstract + 5174 words in Text + 900 words in Figures + 300 words in Table + 45

19

words in Acknowledgements = Total 6617

Word Counts

20

1 ACS Paragon Plus Environment


21

Abstract:

22

Determining the influence of higher order structure on UVC photolysis will help inform

23

predictions of nucleic acid fate and microorganism inactivation. We measured the direct UV254

24

photolysis kinetics of four model viral genomes composed of single-stranded and double-

25

stranded RNA (ssRNA and dsRNA, respectively), as well as single-stranded and double-stranded

26

DNA (ssDNA and dsDNA, respectively), in ultrapure water, in phosphate buffered saline (PBS),

27

and encapsidated in their native virus particles. The photolysis rate constants of naked nucleic

28

acids measured by qPCR (RT-qPCR for RNA) and normalized by the number of bases measured

29

in a particular sequence exhibited the following trend: ssDNA > ssRNA ≈ dsDNA > dsRNA. In

30

PBS, naked ssRNA bases reacted, on average, 24× faster than the dsRNA bases, whereas naked

31

ssDNA bases reacted 4.3× faster than dsDNA bases. Endogenous indirect photolysis involving

32

1

33

measured rate constants with rate constants reported in the literature shows a general agreement

34

amongst the nucleic acid UV254 direct photolysis kinetics. Our results underscore the high

35

resistance of dsRNA to UVC photolysis and demonstrate the role that nucleic acid structure and

36

solution chemistry play in photoreactivity.

O2 and •OH was ruled out as a major contributing factor in the reactions. A comparison of our

UV254

37

MS2 (ssRNA)

Φ6 (dsRNA)

ΦX174 (ssDNA)

T3 (dsDNA)


Page 2 of 27

Page 3 of 27


38

Introduction

39

Nucleic acids are ubiquitous biopolymers that carry the code for all domains of life, yet several

40

types of nucleic acids are undesirable in the aquatic environment due to their potential impacts

41

on human and ecological health. Disinfection processes often target nucleic acids of problematic

42

microorganisms, but extracellular nucleic acids also pose a potential risk. Antimicrobial

43

resistance genes (ARGs) released from municipal wastewaters and animal farming activities, for

44

example, can transfer resistance to other microorganisms.1 Double-stranded RNA (dsRNA)

45

pesticides in genetically modified crops have the potential to be released into surface waters.2

46

Furthermore, many infectious environmental viruses consist of nucleic acids protected by a

47

simple protein coat.

48 49

The nitrogenous bases in DNA and RNA molecules strongly absorb UV radiation, leading to a

50

number of photochemical reactions. This high photoreactivity of nucleic acids is the main reason

51

why UVC is commonly employed for disinfection in water, air, or on surfaces. The effectiveness

52

of UV at inactivating microorganisms, along with the low levels of disinfection byproducts that

53

form relative to chemical disinfectants, has led to a sustained growth of the UV disinfection

54

industry. By 2019, the UV disinfection market is expected to reach nearly $1 billion, or 1/3 of

55

the total disinfection market.3 Due to the widespread application of UV disinfection, the reaction

56

kinetics that drive the inactivation of microorganisms and nucleic acids should be well-defined.

57 58

The pyrimidine bases in nucleic acids (thymine (T), cytosine (C), and uracil (U)) are more

59

photoreactive with UVC than purine bases (adenine (A), guanine (G)), and the reactions that take

60

place between neighboring thymine bases have been the most extensively studied to-date. The



61

TT dimer products from neighboring pyrimidine bases include the cyclobutane pyrimidine dimer

62

(CPD), pyrimidine pyrimidone photoadducts (i.e., 6−4 products), and their Dewar valence

63

isomers.4 Of these, the CPD products have the highest quantum yields.5,6 In addition to TT

64

dimers, TC, CT, CC, and UU dimer products have also been characterized.5,6 Hydration, protein-

65

nucleic acid linkages, covalent crosslinks between complimentary strands, and backbone breaks

66

also take place in nucleic acids exposed to UVC.7 Pyrimidine photohydrate reactions are the

67

most prevalent of these, and form readily on uracil and cytosine bases, but not on thymine

68

bases.8 Consequently, hydration reactions are more important in RNA than in DNA.

69 70

The physical orientation of bases and the structures that surround them at the time of irradiation

71

impact the base photoreactivity. Dimer formation, for example, requires neighboring pyrimidine

72

bases to have well-aligned double bonds and the correct intrabond separation at the time of UV

73

exposure.9,10 Several studies have characterized the role that the flanking bases of neighboring

74

pyrimidines have on dimer quantum yields,11-13 which directly influence photoreactivity. Purine

75

neighboring bases, in general, and guanine bases at the 5’ end, in particular, reduce dimerization

76

quantum yields.12 Photohydrate formation is not as impacted by the chemistry of the neighboring

77

bases, but the reactions are impacted by access to water molecules.14 The presence of bases in

78

either a single-strand or double-strand can also affect quantum yields; in general, the increased

79

order of double-stranded nucleic acids decreases pyrimidine quantum yields.10,15 An early study

80

reported that viral ssRNA in solution is more susceptible to UVC inactivation than the

81

encapsidated ssRNA genome,16 but to our knowledge, the work has not been extended to other

82

viruses or genome types.


Page 4 of 27

Page 5 of 27


83

Predicting the kinetics of nucleic acid UVC photolysis is complicated by the fact that many

84

quantum yield values available in the literature were collected with DNA oligomers with

85

repeated base sequences or under varied experimental conditions (e.g., UV wavelength, pH,

86

ionic strength, etc.). Furthermore, RNA photochemistry is comparatively much less represented

87

in the literature than DNA photochemistry, making it difficult to draw conclusions about the

88

overall relative reactivity of single stranded and double stranded RNA and DNA molecules.

89

RNA photochemistry is of particular interest for disinfection purposes because viruses can

90

contain genomes with ssRNA and dsRNA, in addition to ssDNA and dsDNA. In viruses, the

91

dsDNA viruses are typically considered more resistant to UVC radiation than viruses with other

92

genomes types.17 Interestingly, this is not due to relatively lower photoreactivity of dsDNA, but

93

due to the ability of host cells to repair dsDNA.18

94

To address the role that structure plays in nucleic acid photochemistry, we investigated the

95

reactions that take place in single-stranded and double-stranded DNA and RNA viral genomes

96

with UV254. As model systems, we compared photolysis rates of four viral genomes within and

97

outside of virus particles to assess the impact of virus particles incorporation on nucleic acid

98

reactivity. We employed quantitative polymerase chain reaction (qPCR) techniques to target

99

specific regions of the genomes, as these methods are commonly used for detecting nucleic acids

100

in water and tracking microorganism fate through water treatment processes. Finally, we

101

compared our measured reaction rate constants to those predicted with quantum yield values in

102

the literature.

103 104

EXPERIMENTAL METHODS

105

Virus preparation 5 ACS Paragon Plus Environment


106

Four model viruses were selected to represent four types of nucleic acid genomes, namely MS2

107

(ss RNA), φ6 (ds RNA), φX174 (ssDNA), and T3 (dsDNA). The characteristics of these viruses

108

and their genomes are provided in Table 1.

109 110

Escherichia viruses MS2 and T3, and their corresponding E. coli hosts were purchased from

111

American Type Culture Collection (ATCC 15597 and 11303, respectively). MS2 and T3 stock

112

were propagated, enriched, and enumerated based on previously published methods.19 Enriched

113

virus stock solutions were purified using a Fast Protein Liquid Chromatography system (Econo,

114

Bio-Rad) equipped with a HiPrep Sephacryl S-400 HR column (GE). The purified stocks were

115

filter sterilized with 0.22 µm polyethersulfone (PES) membrane filters (Millipore). The final

116

MS2 and T3 virus stocks (~1011 PFU/mL) were stored in phosphate buffered saline (PBS; 5 mM

117

NaH2PO4, 10mM NaCl, pH 7.5) at 4 oC.

118 119

Pseudomonas virus φ6 and its host Pseudomonas syringae pv. phaseolicola were kindly

120

provided by Dr. Linsey Mar at Virginia Tech. φ6 was added to Pseudomonas syringae grown to

121

OD640 of 0.1 in Luria-Bertani (LB) medium (5 g L-1 NaCl) at 26 °C with a multiplicity of

122

infection (MOI) of 2, and the virus and host mixture was incubated for 7 to 9 hours. Due to its

123

lipid envelope, φ6 was concentrated and purified differently than the other three bacteriophages.

124

Cell lysates were filtered through 0.22 µm PES membranes, and were concentrated with a lab-

125

scale tangential flow filtration system (Millipore) with a 30 kDa cellulous filter. The φ6

126

concentrate was purified by centrifuging at 65,700 × g, 4 °C in a 10-40% (w/v) step sucrose

127

gradient for 1.5 hours, then by centrifuging at 65,700 × g, 4 °C in a 40-60% (w/v) linear sucrose

128

gradient for 15 hours overnight. The φ6 virus band was collected with a needle and was

129

transferred to PBS with a 100 kDa centrifugal ultrafilter (Millipore). The final φ6 virus stocks 6 ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27


130

(~1012 PFU/mL) were filter sterilized through 0.22 µm PES membranes, and stored in PBS at -80

131

°C.

132 133

Escherichia virus φX174 and its corresponding bacterial host E. coli ATCC 13706 were kindly

134

provided by Dr. Charles Gerba at the University of Arizona. φX174 virus was propagated by the

135

agar overlay technique. Specifically, soft tryptic soy agar (TSA; 0.7% w/v agar) was mixed with

136

φX174 virus and host bacteria, was overlaid on hard TSA (1.5% w/v agar), and was incubated at

137

37 °C overnight. The soft agar layer was collected and diluted in PBS to release φX174 from the

138

agar. The agar was removed by centrifuging at 3,000 × g for 10 minutes. The supernatant

139

containing φX174 was treated with a chloroform extraction, filtered through a 0.22 µm PES

140

membrane, and stored in PBS at 4 °C.

141 142

UV254 Photolysis

143

UV photolysis of viral genomes were studied when the genomes were within the virus capsids

144

(i.e., encapsidated) and when they were extracted from the virus capsids (i.e., naked nucleic acids

145

in ultrapure nuclease-free water and in nuclease-free PBS). The prepared PBS was heated to 90

146

°C for 30 minutes to eliminate the activity of nucleases. For each UV treatment, the nucleic acids

147

were extracted from the virus stocks immediately before the UV254 treatment with Maxwell 16

148

Viral Total Nucleic Acid Purification Kits (Promega) according to the manufacturer’s

149

instructions. The concentrations of the extracted virus nucleic acids were determined with a

150

Qubit 2.0 (Life Technologies) in ng/mL and converted to genome copies (gc)/µL using the

151

genome’s molecular mass and Avogadro’s constant, and then diluted to a final concentration of

152

~106 gc/µL with ultrapure nuclease free water (ThermoFisher) or nuclease free PBS. The

153

encapsidated nucleic acids were prepared by directly diluting the virus stocks to a similar 7 ACS Paragon Plus Environment


154

genome concentration ~106 gc/µL with PBS. At this concentration, the absorbance of our

155

experimental solutions at 254 nm was below 0.01; it was therefore not necessary to correct the

156

UV254 radiation doses for shielding.

157 158

Samples of the virus in PBS and the extracted nucleic acids in either ultrapure water or PBS (50

159

µL) were added to the wells of 96-well plate with flat bottoms (Costar). The plate was placed

160

approximately 25 cm below four 15 W germicidal UV lamps (model G15T8, Philips) inside a

161

collimated beam unit. The UV irradiance at 254 nm (0.17 mW/cm2) was determined by chemical

162

actinometry measurements.20 The virus and genome solutions were irradiated up to doses of 408

163

mJ/cm2 at room temperature. Aliquots of the experimental solutions were collected from the

164

samples periodically and stored at 4 °C in the dark. Dark control samples were stored at room

165

temperature in the dark for the duration of the longest UV exposure to capture background

166

nucleic acid decay. The experiments were repeated at least four times for every genome type.

167

Following UV treatment, the encapsidated viral nucleic acids were extracted from the virus

168

samples with Maxwell 16 Viral Total Nucleic Acid Purification Kits. The UV-treated samples

169

and dark controls were analyzed by qPCR or RT-qPCR immediately after the completion of the

170

UV experiments.

171 172

qPCR Assay for UV-treated viral genomes

173

Two sets of PCR primers were designed to target two regions of approximately 500 bases or base

174

pairs on each viral genome (Table 1, Table S1). The entire genome of ssRNA of MS2 and

175

dsRNA of φ6 were directly extracted from the purified virus stocks, and were used as standards;

176

gBlock standards containing two target regions (Integrated DNA Technologies) were purchased

177

for φX174 ssDNA and T3 dsDNA. Both experimental samples and standards were quantified in 8 ACS Paragon Plus Environment

Page 8 of 27

Page 9 of 27


178

parallel using qPCR or RT-qPCR assays in a RealPlex2 Mastercycler system (Eppendorf). The

179

reaction rate constants of the target regions were calculated based on a first-order reaction model: ln

𝐶 = 𝑘!"# ∙ 𝐷!"!"# 𝐶!

180

Where 𝐶! is the initial concentration of viral genome segment; 𝐶 is the concentration of the viral

181

genome segment after UV254 treatment; 𝑘!"# is the first-order rate constant; 𝐷!"!"# is the UV254

182

dose (mJ/cm2).

183 184

RT-qPCR assay for MS2 ssRNA

185

The 20 µL one-step RT-qPCR reactions consisted of 10 µL of 2× GoTaq qPCR Master Mix

186

(Promega), 0.4 µL of 50× GoScript RT Mix, 0.6 µL of 10 µM forward primer, 0.6 µL of 10 µM

187

reverse primer, 6.4 µL of nuclease-free water, and 2 µL of RNA sample. The following

188

thermocycling conditions were used: 15 min at 40 °C, 10 min at 95 °C, 45 cycles of 95 °C for 15

189

s, 55 °C for 30 s, and 72 °C for 45 s, followed by a melting curve analysis from 68 to 95 °C for 5

190

minutes.

191 192

RT-qPCR assays for φ6 dsRNA

193

For φ6 genome samples, dsRNA samples were mixed with 10 µM forward primer and 10 µM

194

reverse primer at a volume ratio of 10:1.5:1.5. The sample-primer mixture was heated at 99 °C

195

for 5 minutes, and immediately chilled on ice before the RT-qPCR assays. The 20 µL RT-qPCR

196

reactions consisted of 10 µL of 2× GoTaq qPCR Master Mix, 0.4 µL of 50× GoScript RT Mix,

197

5.2 µL of the pre-treated sample-primer mixture, 4 µL of 5 M Betaine (Sigma-Aldrich), and 0.4

198

µL of nuclease-free water. The following thermocycling conditions were used: 15 min at 40 °C,



199

10 min at 95 °C, 40 cycles of 95 °C for 15 s, 59 °C for 30 s, and 72 °C for 45 s, followed by a

200

melting curve analysis from 60 to 95 °C for 10 minutes.

201 202

qPCR assays for φX174 ssDNA and T3 dsDNA

203

For both φX174 ssDNA and T3 dsDNA, each qPCR reaction was run in 10 µL total volume

204

consisting of 5 µL of EvaGreen qPCR Master Mix (Biotium), 0.25 µL of 25 mg/mL bovine

205

serum albumin (BSA) solution (Sigma-Aldrich), 0.4 µL of 10 µM forward primer, 0.4 µL of 10

206

µM reverse primer, 2.95 µL of nuclease-free water, and 1 µL of DNA samples. The following

207

thermocycling conditions were used for φX174 ssDNA: 5 min at 95 °C, 40 cycles of 95 °C for

208

20 s, 55 °C for 20 s, and 72 °C for 20 s, followed by a melting curve analysis from 68 to 95 °C

209

for 5 minutes. The following thermocycling conditions were used for T3 dsDNA: 2 min at 95 °C,

210

35 cycles at 95 °C for 5 s, 60 °C for 5 s, and 72 °C for 25 s, followed by a melting curve analysis

211

from 55 to 95 °C for 5 minutes.

212 213

Statistical analysis

214

Linear regressions and statistical analyses were conducted using Prism 7 (GraphPad Software,

215

Inc. La Jolla, CA) on pooled data from replicate experiments of each tested region (n ≥ 4).

216

Analysis of Covariance (ANCOVA) tests were conducted to determine whether there were

217

significant differences between the rate constants of two groups (i.e. naked genome vs

218

encapsidated genome, ssDNA vs dsDNA). When p was less than 0.05, we concluded that the rate

219

constants of two groups were significantly different.

220 221

RESULTS AND DISCUSSION


Page 10 of 27

Page 11 of 27


222

Photolysis of naked nucleic acids in ultrapure water. The 500 base (b) regions of the naked

223

ssRNA and ssDNA and the 500 base pair (bp) regions of the naked dsRNA and dsDNA in

224

ultrapure water generally reacted according to first order kinetics, although tailing was observed

225

in region B of MS2 ssRNA and regions A and B of T3 dsDNA (Figure S1). Tailing has been

226

repeatedly observed when nucleic acids are exposed to UV25421-24 and a number of explanations

227

have been suggested, including the presence of aggregated particles (for viruses) and the

228

presence of UV-resistant nucleic acid sequences. We propose that the commonly observed

229

tailing effect is due to pyrimidine dimer reactions reaching photostationary state, as described in

230

early UV photolysis literature.25,26 The reason tailing was observed in region B of MS2 after the

231

same UV254 dose as region A may be due to the greater number of neighboring uracil bases in the

232

sequence (32 in region B, 19 in region A, Table S2). Further research will be necessary to

233

characterize the extent of dimer reversion in different nucleic acids with varied sequences and

234

structures.

235 236

For each region in ultrapure water, we pooled data from replicate experiments (n ≥ 7) and

237

conducted linear regressions on the data that exhibited first order kinetics (Figure 1; Table S3).

238

Control samples stored in the dark did not react over the timeframe of the experiments,

239

suggesting that the background decay was negligible (Figures S1). The first order rate constants

240

of the two measured regions in naked dsRNA were not statistically different (p = 0.15), nor were

241

the two regions of naked dsDNA (p = 0.32; Table S4). The two regions analyzed in ssRNA had

242

statistically different kinetics (p = 0.001), as did the two regions in ssDNA (p < 0.0001; Table

243

S4). This was likely due to the different number of neighboring pyrimidine bases in the two

244

regions of ssRNA and the two regions of ssDNA (Table S2).



245 246

φ6 and T3 are dsRNA and dsDNA viruses, respectively, and MS2 and φX174 are ssRNA and

247

ssDNA viruses, respectively; thus, the analyzed 500 bp regions in φ6 and T3 have twice as many

248

bases as the 500 b regions of MS2 and φX174. It was therefore not possible to directly compare

249

the measured rate constants of the single-stranded regions with the double-stranded regions. By

250

comparing the ssRNA regions with the ssDNA regions, and the dsRNA regions with the dsDNA

251

regions, it was evident that the DNA regions reacted faster than the RNA regions. Specifically,

252

the two ssDNA regions reacted ~2× faster than the two ssRNA regions, and the two dsDNA

253

regions reacted ~8× faster than the two dsRNA regions. A study on the UV254 photolysis of short

254

DNA and RNA hairpin structures with HPLC also concluded that DNA is more photoreactive

255

than RNA.27

256 257

Viral nucleic acids can act as sensitizers with UV254, leading to other reactions in virus

258

particles.28 We therefore assessed the role that 1O2 and ·OH played in the UV254 photolysis of

259

naked T3 dsDNA in ultrapure water (see SI). The reaction kinetics of the naked dsDNA in D2O

260

were no different than it was in ultrapure water (Figure S2, p = 0.83), and no difference in

261

reaction kinetics was observed between dsDNA with methanol and dsDNA in ultrapure water

262

(Figure S2, p = 0.95). These results demonstrate that neither 1O2 nor ·OH was contributing

263

significantly to the decay of the naked dsDNA. We therefore concluded that the observed nucleic

264

acid reactions in ultrapure water were due to direct photolysis.

265 266

Photolysis of naked nucleic acids in PBS. For the naked ssRNA, dsRNA, and dsDNA regions,

267

the photolysis rate constants in PBS were substantially lower than in ultrapure water (Figure 1,


Page 12 of 27

Page 13 of 27


268

Table S3). Specifically, the ssRNA rate constants were, on average, 1.7× higher in ultrapure

269

water than PBS, the dsRNA rate constants were, on average, 5.3× higher in ultrapure water, and

270

the dsDNA rate constants were, on average, 2.2× higher in ultrapure water. For ssDNA, only

271

region A had faster kinetics in ultrapure water based on ANCOVA analysis, and the difference in

272

this case was minor (1.1×, p = 0.04). The reason that the photoreactivities of MS2 ssRNA, φ6

273

dsRNA, and T3 dsDNA were much more impacted by solution chemistry than φX174 ssDNA is

274

not clear, but may involve the circularity of the φX174 ssDNA genome. The MS2 and T3

275

genomes are linear, and the φ6 genome is linear and segmented.

276 277

Our PBS buffer consisted of 5 mM NaH2PO4 and 10 mM NaCl (pH 7.5), whereas our

278

suspensions of nucleic acids in ultrapure water solutions were approximately pH 5.8. Pyrimidine

279

dimer quantum yields are generally not impacted by pH6,14 and early work with cytosine and

280

uracil hydrates suggest their quantum yields are relatively consistent above pH 5.29 It is therefore

281

unlikely that our observed differences in ultrapure water and PBS were due to pH. In terms of

282

ionic strength, Douki observed a 20% decrease of TT dimer quantum yields and a 300% increase

283

in TC dimer quantum yields when 20 mM NaCl was added to calf thymus DNA in pure water.6

284

In that study, the DNA dimer quantum yields were consistent when the NaCl concentrations

285

increased above 20 mM. Pyrimidine photohydrate quantum yields are also impacted by ionic

286

strength, as well as buffer type.29 These observed effects of ionic strength and buffer type on

287

quantum yield are likely related to nucleic acid structure. Hydrate formation, for example,

288

requires water molecules to access bases; this occurs more readily when double helix RNA

289

structures are denatured.29 Dimer formation requires that neighboring pyrimidines are favorably

290

aligned at the time of excitation, and this can be inhibited when structures are highly ordered.10



291

Here, the nucleic acids in PBS likely had more ordered structures than the nucleic acids in

292

ultrapure water, thus inhibiting dimer and hydrate formation.

293 294

Predicted photolysis rate constants. We next predicted the theoretical photolysis rate constants,

295

𝑘!"#$ , for the eight nucleic acid regions based on a base composition method. Specifically, we

296

used the sequences in our ssRNA, dsRNA, ssDNA, and dsDNA regions and the RNA and DNA

297

extinction coefficients and quantum yields in the literature (Table S2), and summed the rate

298

constants of the major potential reactive bases in the regions:

299 𝑘!"#$,! =

2.3 𝑏 ∙ 𝜀!"# ∙ Φ 𝑈 !

!

𝑘!"#$ =

𝑘!"#$,! = !

!

2.3 𝑏 ∙ 𝜀!"# ∙ Φ 𝑈

300 301

Here, 𝑘!"#$ (cm2 mJ-1) is the predicted rate constant of the nucleic acid regions, 𝑘!"#$,! (cm2 mJ-

302

1

303

UU, and CC for RNA, and C, TT, CT, TC, and CC for DNA, 𝜀!"# (M-1 cm-1) is the extinction

304

coefficient of the nucleic acid monomer or dimer at 254 nm in M-1 cm-1, Φ (mol eins-1) is the

305

quantum yield of a particular reaction, U is a constant of the number of joules per einstein at 254

306

nm (4.7 × 105 J eins-1), and b is the number of reactive nucleic acid monomers or dimers in the

307

nucleic acid region.30 For the dsRNA and dsDNA regions, the sequences of both strands were

308

included in the 𝑘!"#$ calculation.

) is the predicted rate constant of the reactive nucleic acid monomer or dimer 𝑖, including C, U,

309


Page 14 of 27

Page 15 of 27


310

The resulting predicted rate constants of the eight regions exhibited trends similar to the

311

experimental rate constants (𝑘!"# ) of the naked nucleic acid regions (Figure 1). For both the

312

experimental and predicted rate constants, the two ssDNA regions had the fastest kinetics, on

313

average, followed by the dsDNA and ssRNA regions, and the dsRNA regions were the least

314

reactive. The trends observed between the measured rate constants of the two regions in ssDNA

315

(region B > region A) and the two regions in ssRNA (regions B > region A) were also predicted

316

based on the different numbers of reactive monomers and dimers in their sequences (Table S2).

317 318

For each region, the predicted rate constant was larger than the measured values in ultrapure

319

water and in PBS. The predictions were closest for ssDNA, with the predicted rate constants

320

within a factor of 2 of the measured rate constants in both ultrapure water and PBS. The

321

predictions for dsRNA were the furthest from the measured values, with up to a 24-fold

322

difference between the predicted rate constant and the measured rate constant. The differences

323

were more pronounced when ssRNA, dsRNA, and dsDNA were in PBS than when in ultrapure

324

water (Figure 1). The discrepancies between predicted rate constants and measured rate constants

325

have several possible explanations. First, many of the quantum yield values available in the

326

literature were measured on short oligomers with repeated bases (e.g. UUUUUUU), and these

327

likely react differently than longer sequences with mixed bases. Quantum yield values available

328

in the literature were measured under a range of temperatures, solution pH, UVC wavelengths, or

329

ionic strengths that differed from our experimental conditions; these parameters can affect

330

photolysis quantum yields.31,32

331



332

Quantum yields can also be impacted by the technique by which they are measured. In many of

333

the early reports, quantum yields were measured using the nucleic acid base chromosphere

334

decay, and this can suffer from interference by the formed products.14 The molecular methods

335

used here may not detect all of the reactions that take place in the RNA and DNA. Updated

336

quantum yield measurements under a variety of experimental conditions and a better

337

characterization of PCR method tolerance to nucleic acid photolysis products would help address

338

some of the observed discrepancies.

339 340

We present each 𝑘!"#$ value with the contributing reactions (𝑘!"#$,! ) identified by color (Figure

341

1; Table S2); this highlights the relative importance of hydration and dimerization reactions in

342

RNA versus DNA, as predicted with quantum yields in the literature. In both the ssRNA and

343

dsRNA regions, most of 𝑘!"#$ (~70%) is due to hydration reactions, with the rest from

344

dimerization reactions. In DNA regions, the opposite is true, with most of 𝑘!"#$ (~85%)

345

resulting from dimerization reactions. Another interesting observation from the photolysis

346

predictions is the relative contributions of TT dimers in the ssDNA and dsDNA reactions. TT

347

dimers are commonly assumed to be the most prevalent UV photoproducts in DNA, but here, the

348

sum of the predicted C hydrate contribution and TC dimer contribution is greater than the TT

349

dimer contribution. We should note that C hydrates can revert back to cytosines under some

350

conditions, and that was not included in our predictions.29 The CT, TC, and CC quantum yields

351

used in 𝑘!"#$ were based on a study in which DNA photoproducts were quantified following the

352

exposure of calf thymus DNA to UV254.5 We used the relative yields of TC, CT, or CC dimer

353

products to TT dimer products in that study, along with the well-established TT cyclobutane

354

dimer quantum yield for UV254 to estimate TC, CT, and CC quantum yields (Table S2). We were 16 ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27


355

unable to locate quantum yield values for UC and CU reactions in RNA; the predicted RNA rate

356

constants would have been even higher if those reactions had been included.

357 358

Single stranded versus double stranded photoreactivity. As mentioned above, the measured

359

rate constants of the single stranded and double stranded nucleic acid regions were not directly

360

comparable due to the regions containing different numbers of bases. We therefore normalized

361

the rate constants by the number of bases in the analyzed regions (Figure 2) to observe the

362

impact of double-strand structures on RNA and DNA reactivity. In ultrapure water, RNA bases

363

reacted, on average, 7.7× faster in the single-stranded regions than in the double-stranded

364

regions; in buffer, the difference in kinetics increased to 24×. For DNA in ultrapure water, the

365

single-stranded DNA regions reacted, on average, 2.1× faster than the double-stranded genome,

366

whereas in buffer, that difference increased to 4.3×. In the case of DNA, some of the observed

367

differences were likely due to the higher proportion of adjacent TT bases in the two ssDNA

368

regions (36 and 48 TT pairs in 500 bases; Table S2) compared to the two dsDNA regions (46 and

369

47 TT pairs in 1000 bases; Table S2). The ss and ds RNA sequences, however, had similar

370

proportions of reactive bases, thus the lower photoreactivity of dsRNA compared to ssRNA was

371

likely due to the impact of the double helix.

372 373

Together, these results demonstrate that the double helix has a greater impact on RNA

374

photoreactivity than on DNA photoreactivity, and that the impact of the double helix was

375

enhanced in buffer solution compared to ultrapure water. An early study of RNA exposed to

376

UV280 found that hydrates formed 10× faster in polyU oligomers in ssRNA than in polyU:polyA

377

in dsRNA, while dimers formed 5× faster in ssRNA compared to dsRNA.15 By comparison, in



378

DNA, reported TT dimer quantum yields differ by less than 2× in ssDNA and dsDNA.10,33 Our

379

results underscore the high resistance of dsRNA to UV254 photolysis compared to other nucleic

380

acids, particularly in buffered solutions. The resistance of dsRNA rotavirus to UVC inactivation

381

has been noted previously.34

382 383

Photolysis of encapsidated nucleic acids. To explore how the incorporation of nucleic acids in

384

viral particles impacts the rate of direct photolysis by UV254, we conducted experiments with the

385

same nucleic acids analyzed above, but encapsidated in virus particles and suspended in PBS

386

(Figure 1). We hypothesized that that genomes compressed inside a protein capsid would be less

387

photoreactive, due to possible decreased availability of H2O molecules for hydrate formation, the

388

restriction of motions necessary for dimer formations, and the potential for energy transfer to

389

virus proteins. In fact, for three of the four viruses tested, namely MS2 (ssRNA), φ6 (dsRNA),

390

and φX174 (ssDNA), encapsidation did not impact the reaction kinetics of the nucleic acids

391

(Table S4; p > 0.05). We observed slightly faster reaction kinetics (~1.2×) for both T3 dsDNA

392

regions when encapsidated, although the ANCOVA analysis p values were barely below our

393

0.05 cutoff for statistical significance (Table S4). These results of little or no effect from

394

encapsidation contradict an early report on the UV photolysis of tobacco mosaic virus ssRNA;

395

that research found that the ssRNA genome was 6x more sensitive to UV when outside of the

396

virus capsid than when inside the capsid.16

397 398

Literature review of nucleic acid photolysis kinetics. A number of previous studies have

399

measured the UV254 photodegradation of viral nucleic acids and extracellular DNA with qPCR or

400

RT-qPCR.24,34-43 It is normally difficult to compare these results because the regions they target


Page 18 of 27

Page 19 of 27


401

have a range of sizes. We therefore normalized the literature rate constants by the number of

402

bases in the analyzed regions and compared the normalized rate constants from different studies

403

and from different genome types (Table S5). The nucleic acid UV254 photolysis rate constants in

404

the literature generally agree with one another and with our data (Figure 3). The compiled data

405

confirms that the ssDNA encapsidated genomes are the most reactive with UV254 and that the

406

encapsidated dsRNA genomes are the least reactive with UV254. The average rate constants per

407

base (mean ± 95% C.I.) for encapsidated ssRNA, dsRNA, ssDNA, and dsDNA were 3.1 × 10-5

408

± 1.0 × 10-5, 4.3 × 10-6 ± 5.8 × 10-6, 1.8 × 10-4 ± 1.9 × 10-5, and 1.7 × 10-5 ± 8.3 × 10-6 cm2 mJ-1

409

base-1, respectively. The average rate constants per base for naked dsDNA was 4.8 × 10-5 ± 2.2 ×

410

10-5 cm2 mJ-1 base-1. The scatter observed for each genome type is likely due to variations in the

411

analyzed region sequences (e.g., number of TT sequences) and differences in experimental

412

conditions between laboratories (e.g., buffer, temperature, etc.).

413 414

Environmental Implications. Our research demonstrates the relatively high resistance of

415

dsRNA compared to the other genome types. The dsRNA rate constants measured here were

416

~80× lower than the ssDNA rate constants, which exhibited the highest rate constants on a per

417

base basis. The dsRNA rate constants from this work will help with future efforts to disinfect

418

dsRNA viruses and help predict the environmental fate of dsRNA plant incorporated protectants,

419

which are increasingly applied to combat agricultural pests. Our results suggest that dsRNA

420

viruses and dsRNA plant-incorporated protectants likely persist after other forms of nucleic acids

421

have degraded from photochemical reactions.

422



423

Another important conclusion of this work is the large impact that higher order structure and

424

solution chemistry plays in nucleic acid photochemistry, particularly for RNA. This complicates

425

efforts to predict the UV254 inactivation rate constants of virus genomes using published nucleic

426

acid quantum yields in combination with the genome size and sequence. The ssDNA of φX174

427

was the only genome unaffected by the solution chemistry. Future research will be needed to

428

determine if this is the case for all ssDNA viruses or for all circular genomes.

429 430

Finally, it is worth discussing the implications of employing qPCR and RT-qPCR methods to

431

quantify intact RNA and DNA, respectively. These two approaches employ different enzymes to

432

detect damage. Namely, qPCR uses polymerase to copy intact DNA regions and RT-qPCR uses

433

reverse transcriptase to convert intact RNA regions to complimentary DNA. The relative

434

tolerances of polymerase and reverse transcriptase to photolysis products is not currently known,

435

but our earlier work demonstrated that the photolysis kinetics of short RNA oligomers were

436

faster when measured with a mass spectrometry method than when measured with RT-qPCR.44

437

We selected qPCR and RT-qPCR methods for this research, as opposed to HPLC or LC-MS

438

methods, in order to obtain region-specific rate constants in the genomes; this information is lost

439

when genomes are digested and individual bases are quantified by HPLC or LC-MS.

440

Furthermore, the hydrate products that form with UV254 can undergo facile dehydration, a

441

reaction that is acid-catalyzed.8 Consequently, acid digestion steps and acidic mobile phases in

442

LC separations will affect the measured UV254 reaction rates and this is avoided with qPCR and

443

RT-qPCR. Future research should characterize the specific reactions measured by the

444

polymerases used in qPCR and the reverse transcriptases used in RT-qPCR, as well as the

445

photoproducts that impact the biological activity of RNA and DNA.


Page 20 of 27

Page 21 of 27


446 447

SUPPORTING INFORMATION

448

The Supporting Information is available free of charge via the internet at http://pubs.acs.org.

449

Additional materials and methods on experiments probing the role of reactive oxygen

450

species; figures of UV254 photolysis kinetics of encapsidated and naked viral nucleic

451

acids; UV254 photolysis kinetics of naked dsDNA genomes in ultrapure water, D2O and

452

methanol; table that lists the viruses, primer sets, and the target genome regions; table

453

that describes the prediction of the UV photolysis rate constants; table with reaction rate

454

constants from linear regressions; p values obtained from ANCOVA tests; UV254

455

photolysis rate constants of encapsidated viral nucleic acids and plasmids reported in

456

previous literature.

457 458

AUTHOR INFORMATION

459

Corresponding author

460

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

461 462

ACKNOWLEDGMENT

463

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

464

NSF PIRE Award 1545756. We thank Professor Rose Cory from the University of Michigan for

465

her technical assistance and Kathryn Langenfeld from the University of Michigan for her

466

assistance with virus preparations.

467 468

REFERENCES:



469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514

(1)

(2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12) (13) (14) (15) (16) (17) (18)

Vikesland, P. J.; Pruden, A.; Alvarez, P. J. J.; Aga, D.; Bürgmann, H.; Li, X.-D.; Manaia, C. M.; Nambi, I.; Wigginton, K.; Zhang, T.; Zhu, Y.-G. Toward a comprehensive strategy to mitigate dissemination of environmental sources of antibiotic resistance. Environ. Sci. Technol. 2017, 51 (22), 13061–13069. Parker, K. M.; Sander, M. Environmental fate of insecticidal plant-incorporated protectants from genetically modified crops: knowledge gaps and research opportunities. Environ. Sci. Technol. 2017, 51 (21), 12049–12057. Disinfection market set for billion dollar growth up to 2019. waterworld.com. 2018. Douki, T. The variety of UV-induced pyrimidine dimeric photoproducts in DNA as shown by chromatographic quantification methods. Photochem. Photobiol. Sci. 2013, 12 (8), 1286–1302. Douki, T.; Cadet, J. Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions. Biochemistry 2001, 40 (8), 2495–2501. Douki, T. Low ionic strength reduces cytosine photoreactivity in UVC-irradiated isolated DNA. Photochem. Photobiol. Sci. 2006, 5 (11), 1045–1051. Görner, H. New trends in photobiology: Photochemistry of DNA and related biomolecules: Quantum yields and consequences of photoionization. J. Photoch. Photobio. B. 1994, 26 (2), 117–139. Lemaire, D. E.; Ruzsicska, B. P. Quantum yields and secondary photoreactions of the photoproducts of dTpdT, dTpdC and dTpdU. Photochem. Photobiol. 1993, 57 (5), 755– 757. McCullagh, M.; Hariharan, M.; Lewis, F. D.; Markovitsi, D.; Douki, T.; Schatz, G. C. Conformational control of TT dimerization in DNA conjugates. A molecular dynamics study. J. Phys. Chem. B. 2010, 114 (15), 5215–5221. Schreier, W. J.; Schrader, T. E.; Koller, F. O.; Gilch, P.; Crespo-Hernández, C. E.; Swaminathan, V. N.; Carell, T.; Zinth, W.; Kohler, B. Thymine dimerization in DNA is an ultrafast photoreaction. Science 2007, 315 (5812), 625–629. Pan, Z.; Hariharan, M.; Arkin, J. D.; Jalilov, A. S.; McCullagh, M.; Schatz, G. C.; Lewis, F. D. Electron donor–acceptor interactions with flanking purines influence the efficiency of thymine photodimerization. J. Am. Chem. Soc. 2011, 133 (51), 20793– 20798. Pan, Z.; McCullagh, M.; Schatz, G. C.; Lewis, F. D. Conformational control of thymine photodimerization in purine-containing trinucleotides. J. Phys. Chem. Lett. 2011, 2 (12), 1432–1438. Law, Y. K.; Forties, R. A.; Liu, X.; Poirier, M. G.; Kohler, B. Sequence-dependent thymine dimer formation and photoreversal rates in double-stranded DNA. Photochem. Photobiol. Sci. 2013, 12 (8), 1431–1439. Fisher, G. J.; Johns, H. E. Pyrimidine photodimers; 1976; pp 225–294. Pearson, M.; Johns, H. E. Suppression of hydrate and dimer formation in ultravioletirradiated poly (A + U) relative to poly U. J. Mol. Biol. 1966, 20 (2), 215–229. McLaren, A. D.; Takahashi, W. N. Inactivation of infectious nucleic acid from tobacco mosaic virus by ultraviolet light (2537 A). Radiat. Res. 1957, 6 (5), 532. Gerba, C. P.; Gramos, D. M.; Nwachuku, N. Comparative inactivation of enteroviruses and adenovirus 2 by UV light. Appl. Environ. Microbiol. 2002, 68 (10), 5167–5169. Weitzman, M. D.; Carson, C. T.; Schwartz, R. A.; Lilley, C. E. Interactions of viruses 22 ACS Paragon Plus Environment

Page 22 of 27

Page 23 of 27

515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560


(19)

(20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35)

with the cellular DNA repair machinery. DNA Repair 2004, 3 (8-9), 1165–1173. Pecson, B. M.; Martin, L. V.; Kohn, T. Quantitative PCR for determining the infectivity of bacteriophage MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: advantages and limitations of an enzymatic treatment to reduce false-positive results. Appl. Environ. Microbiol. 2009, 75 (17), 5544–5554. Rahn, R. O. Potassium iodide as a chemical actinometer for 254 nm radiation: use of iodate as an electron scavenger. Photochem. Photobiol. 1997, 66 (6), 885–885. Beck, S. E.; Rodríguez, R. A.; Hawkins, M. A.; Hargy, T. M.; Larason, T. C.; Linden, K. G. Comparison of UV-induced inactivation and RNA damage in MS2 phage across the germicidal UV spectrum. Appl. Environ. Microbiol. 2016, 82 (5), 1468–1474. Mattle, M. J.; Kohn, T. Inactivation and tailing during UV254 disinfection of viruses: contributions of viral aggregation, light shielding within viral aggregates, and recombination. Environ. Sci. Technol. 2012, 120830135926005. McKinney, C. W.; Pruden, A. Ultraviolet disinfection of antibiotic resistant bacteria and their antibiotic resistance genes in water and wastewater. J. Am. Chem. Soc. 2012, 46 (24), 13393–13400. Chang, P. H.; Juhrend, B.; Olson, T. M.; Marrs, C. F.; Wigginton, K. R. Degradation of extracellular antibiotic resistance genes with UV254 treatment. Environ. Sci. Technol. 2017, 51 (11), 6185–6192. Helleiner, C. W.; Pearson, M. L.; Johns, H. E. The ultraviolet photochemistry of deoxyuridylyl (3′→5′) deoxyuridine. Proc. Natl. Acad. Sci. U.S.A. 1963, 50 (4), 761– 767. Johns, H. E.; Pearson, M. L.; LeBlanc, J. C.; Helleiner, C. W. The ultraviolet photochemistry of thymidylyl-(3′→5′)-thymidine. J. Mol. Biol. 1964, 9 (2), 503–IN1. Kundu, L. M.; Linne, U.; Marahiel, M.; Carell, T. RNA is more UV resistant than DNA: The formation of UV‐induced DNA lesions is strongly sequence and conformation dependent. Chem. Eur. J. 2004, 10 (22), 5697–5705. Wigginton, K. R.; Menin, L.; Sigstam, T.; Gannon, G.; Cascella, M.; Hamidane, H. B.; Tsybin, Y. O.; Waridel, P.; Kohn, T. UV radiation induces genome‐mediated, site‐ specific cleavage in viral proteins. ChemBioChem 2012, 13 (6), 837–845. Fisher, G. J.; Johns, H. E. Pyrimidine Photohydrates. In Photochemistry and Photobiology of Nucleic Acids; Elsevier, 1976; pp 169–224. Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental organic Chemistry 2 nd Edition. Brown, I. H.; Johns, H. E. Photochemistry of uracil. Intersystem crossing and dimerization in aqueous solution. Photochem. Photobiol. 1968, 8 (4), 273–286. Evans, N. A.; McLaren, A. D. Quantum yields for inactivation of tobacco mosaic virus nucleic acid by ultraviolet radiation (254 nm.). J. Gen. Virol. 1969, 4 (1), 55–63. Tarmer, Z.; Wierzchowski, K. L.; Shugar, D. Influence of polynucleotide secondary structure on thymine photodimerization. Acta Biochim Pol 1696, 16 (1), 83–107. Li, D.; Gu, A. Z.; He, M.; Shi, H.-C.; Yang, W. UV inactivation and resistance of rotavirus evaluated by integrated cell culture and real-time RT-PCR assay. Water Res. 2009, 43 (13), 3261–3269. Ho, J.; Seidel, M.; Niessner, R.; Eggers, J.; Tiehm, A. Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation. Water Res. 2016, 103, 141–148. 23 ACS Paragon Plus Environment


561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591

(36)

(37)

(38) (39) (40) (41) (42) (43) (44)

Beck, S. E.; Rodríguez, R. A.; Linden, K. G.; Hargy, T. M.; Larason, T. C.; Wright, H. B. Wavelength dependent UV inactivation and DNA damage of adenovirus as measured by cell culture infectivity and long range quantitative PCR. Environ. Sci. Technol. 2013, 48 (1), 591–598. Calgua, B.; Carratalà, A.; Guerrero-Latorre, L.; de Abreu Corrêa, A.; Kohn, T.; Sommer, R.; Girones, R. UVC inactivation of dsDNA and ssRNA viruses in water: UV fluences and a qPCR-based approach to evaluate decay on viral infectivity. Food Environ. Virol. 2014, 6 (4), 260–268. Simonet, J.; Gantzer, C. Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers. Appl. Environ. Microbiol. 2006, 72 (12), 7671–7677. Dancho, B. A.; Chen, H.; Kingsley, D. H. Discrimination between infectious and noninfectious human norovirus using porcine gastric mucin. Int. J. Food Microbiol. 2012, 155 (3), 222–226. Yoon, Y.; Chung, H. J.; Wen Di, D. Y.; Dodd, M. C.; Hur, H.-G.; Lee, Y. Inactivation efficiency of plasmid-encoded antibiotic resistance genes during water treatment with chlorine, UV, and UV/H2O2. Water Res. 2017, 123, 783–793. Wigginton, K. R.; Pecson, B. M.; Sigstam, T.; Bosshard, F.; Kohn, T. Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity. Environ. Sci. Technol. 2012, 46 (21), 12069–12078. Ye, Y.; Chang, P. H.; Hartert, J.; Wigginton, K. R. Reactivity of enveloped virus genome, proteins, and lipids with free chlorine and UV254. Environ. Sci. Technol. 2018, 52 (14), 7698–7708. Vazquez-Bravo, B.; Gonçalves, K.; Shisler, J. L.; Mariñas, B. J. Adenovirus replication cycle disruption from exposure to polychromatic ultraviolet irradiation. Environ. Sci. Technol. 2018, 52 (6), 3652–3659. Qiao, Z.; Wigginton, K. R. Direct and indirect photochemical reactions in viral RNA measured with RT-qPCR and mass spectrometry. Environ. Sci. Technol. 2016, 50 (24), 13371–13379.


Page 24 of 27

Page 25 of 27


Naked, water

Naked, PBS

Encapsidated, PBS

C hydrates U hydrates CC dimers UU dimers TT dimers TC dimers CT dimers

ns

*

0.08

ns

0.16 ****

****

0.06 0.04 0.02 0.00

592

0.20

ns

0.12 ****

*

*

ns

**** ns

A ssRNA (MS2)

B

**** ns

**** ns

A

B

dsRNA (φ6)

0.08 0.04

A

B

ssDNA (φX174)

A

B

kpred (cm2 mJ-1)

kexp (cm2 mJ-1)

0.10

0.00

dsDNA (T3)

593

Figure 1. Measured UV254 rate constants of virus genome regions A and B in naked forms

594

(white and black bars) and in encapsidated forms (grey bars) and predicted rate constants of the

595

same genome regions (bars with patterns). Error bars represent standard error for replicate

596

experiments (n ≥ 4). Asterisks indicate rate constants are significantly different (one asterisk for

597

0.01 < p < 0.05, four asterisks for p < 0.0001) and ns indicates that rate constants are not

598

significantly different (p > 0.05). The ANCOVA analysis p values are presented in Table S4.

599


kexp (cm2 mJ-1 base-1)


Naked, water

1.8×10-4

Naked, PBS

Page 26 of 27

Encapsidated, PBS

1.2×10-4

6.0×10-5

0.0

A

B

ssRNA (MS2)

600

A

B

A

dsRNA (φ6)

B

ssDNA (φX174)

A

B

dsDNA (T3)

601

Figure 2. Experimental first order rate constants of target viral genome regions normalized by

602

the number of bases in corresponding region. Error bars represent standard error.

k (cm2 mJ-1 base-1)

10-3

10-4

10-5

603

Encapsidated

ds D N A

ds D N A

A N D ss

ds R N A

ss

R

N

A

10-6

Naked


Page 27 of 27


604

Figure 3. Comparison of the first order rate constants measured in this study on two regions of

605

each genome with first order rate constants reported in the literature normalized by the number

606

of bases in the PCR amplicon. Black points represent data from this study conducted in PBS,

607

gray points represent data from this study conducted in ultrapure water, and white points

608

represent data from the literature.

609 610

Table 1. Four model viruses used in this study, including details of the genome regions analyzed. Region Location

Region size (bases or base pairs)

Region A Region B

944-1439 2693-3189

496 b 497 b

Enveloped

Region A Region B

S1141-S1639 L1510-L1993

499 bp 484 bp

Nonenveloped

Region A Region B

571-1074 1717-2209

504 b 493 b

Family/Genus*

Genome type

MS2

Leviviridae/ Levivirus

ssRNA (linear)

3.6 kb

~25

Nonenveloped

φ6

Cystoviridae/ Cystovirus

dsRNA (segmented)

2.9 kbp (S) 4.1 kbp (M) 6.4 kbp (L)

~85

5.4 kb

~25

Virus

φX174

Microviridae/ ssDNA Phix174microvirus (circular)

Genome Particle size Enveloped size (nm) /nonenveloped

Podoviridae/ dsDNA ~50 × 20 Region A 1678-2186 509 bp 38.2 kbp Nonenveloped T7virus (linear) (tailed) Region B 11826-12324 499 bp * Family and genus were based on the International Committee on Taxonomy of Viruses (ICTV), https://talk.ictvonline.org/taxonomy/ T3

611

Regions Analyzed


Nucleic Acid Photolysis by UV254 and the Impact of Virus

Recommend Documents