Engineering Riboswitches in Vivo Using Dual Genetic Selection and

Subscriber access provided by University of South Dakota

Letter

Engineering riboswitches in vivo using dual genetic selection and fluorescence-activated cell sorting Katharine Page, Jeremy Shaffer, Samuel Lin, Mark Zhang, and Jane May Liu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00099 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 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 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Engineering riboswitches in vivo

1

1

Engineering riboswitches in vivo using dual genetic selection and fluorescence-

2

activated cell sorting

3

Katharine Page, Jeremy Shaffer, Samuel Lin, Mark Zhang and Jane M. Liu*

4 5 6 7 8

Department of Chemistry

9

Pomona College

10

645 N. College Avenue

11

Claremont, CA 91711

12 13

*[email protected]

14

(909) 607-8832

15 16

Key words:

17

riboswitches, E. coli, genetic selection, cell sorting

18

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 2 of 25

2

19

Abstract

20

Riboswitches, non-coding RNAs that bind a small molecule effector to control gene

21

expression at the level of transcription or translation, are uniquely suited to meet

22

challenges in synthetic biology. To expand the limited set of existing riboswitches, we

23

developed a riboswitch discovery platform that couples dual genetic selection and

24

fluorescence-activated cell sorting to identify novel riboswitches from a 108 random-

25

sequence library in which the aptamer domain of the ThiM#2 riboswitch was replaced

26

with an N40 sequence. In a proof-of-principle validation, we identified novel riboswitches

27

for the small molecule theophylline. Our best riboswitch (Hit 3-5) displays 2.3-fold

28

activation of downstream gene expression in the presence of theophylline. Random

29

mutagenesis of Hit 3-5, coupled with selections and screens, afforded improved

30

riboswitches displaying nearly 3-fold activation. To the best of our knowledge, this is the

31

first report of in vivo directed evolution of an aptamer domain to generate a functional

32

riboswitch.

33 34

Riboswitches comprise a class of regulatory RNA elements capable of binding small

35

molecule ligands. These naturally occurring riboregulators, found across a range of

36

prokaryotic organisms, act at the transcriptional or translational level to affect gene

37

expression in cis and are mediated not by protein factors, but rather by small molecule

38

binding.1,2 There are two functional components that contribute to riboswitch activity.

39

The first is an aptamer domain (the sensor) that binds the ligand of interest. The second

40

is an expression platform (the actuator), which couples the aptamer’s binding state to

41

the enhancement or repression of downstream gene expression. A riboswitch that


Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



3

42

inhibits expression in the presence of its cognate ligand is termed an OFF switch, while

43

a riboswitch that augments expression is termed an ON switch. Given that these

44

regulators alter gene expression in a ligand-dependent manner and that the sensor and

45

actuator domains are considered modular, riboswitches are particularly well suited for

46

synthetic biology projects involving RNA devices.3–8 A major limitation in developing

47

these RNA tools, however, is the narrow collection of existing riboswitches. Expanding

48

the catalog of riboswitches to accommodate a wide variety of ligands would address a

49

significant bottleneck in the field.

50 51

Powerful in vitro evolution and selection methods (e.g. SELEX) can rapidly identify high-

52

affinity aptamers for small molecules, which can be incorporated as the sensor domain

53

of a synthetic riboswitch.9–13 However, cellular screening of these aptamers after

54

selection is generally required in order to couple the sensors to appropriate actuators to

55

create a functional riboswitch. While this approach to engineering riboswitches from

56

synthetic aptamers has been demonstrated, it does not always work reliably, and it

57

often requires implementing additional rational design strategies.14–18 The difficulty of

58

incorporating an aptamer from SELEX into a genetic switch is not entirely surprising as

59

an aptamer selected in vitro does not necessarily bind the ligand in the same manner in

60

a cellular environment where other interactions also may occur.19 Moreover, natural

61

aptamers that have evolved in a cellular environment typically occupy a set of privileged

62

scaffolds that allow for biological activity, whereas high-affinity aptamers developed via

63

SELEX often are structurally less complex.20 Indeed, an effective riboswitch must do

64

more than just bind its cognate ligand with high affinity; induced changes to secondary




Page 4 of 25

4

65

and tertiary structure must be relayed from the sensor through the actuator, an activity

66

not addressed during in vitro selection.

67 68

The rational reprogramming of naturally occurring riboswitches has been one approach

69

to address the disparity between in vitro selected aptamers and functional cellular

70

riboswitches.14,21,22 Similarly, naturally occurring riboswitches have been tuned through

71

dual genetic selection, for example converting the Escherichia coli TPP riboswitch,

72

which binds thiamine pyrophosphate, from an OFF switch to an ON switch.23–25 In these

73

cases, however, the synthetic riboswitch obtained ultimately recognizes the same ligand

74

as the original riboswitch, or a structurally related one.

75 76

In the present study, we present a procedure for the development of riboswitches that

77

allows for the creation of riboswitches that recognize, theoretically, any ligand of

78

interest. Importantly, the procedure takes place entirely inside a cellular environment,

79

thereby bypassing some of the concerns related to the “SELEX-first” approach.

80

Specifically, we constructed a riboswitch plasmid library in which the aptamer domain of

81

the ThiM#2 riboswitch23 (an ON switch that responds to TPP) was replaced by a

82

randomized region of 40 nucleotides. The plasmids were introduced into E. coli and the

83

resulting library was subjected to multiple rounds of dual genetic selection and

84

fluorescence-activated cell sorting (FACS) screening to generate riboswitches

85

responsive to theophylline. We were able to generate theophylline-responsive

86

riboswitches after only three rounds of selections and screens. An additional round of

87

mutagenesis, selections and screens of one of the switches further increased its


Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



5

88

activity. Our de novo development of novel theophylline riboswitches provides a general

89

platform from which many more synthetic riboswitches may be discovered, addressing a

90

major obstacle to broadening the scope of riboswitch-based technology.

91 92

Results and Discussion

93

To make a library from which to select and screen for novel ON riboswitches, we started

94

with a ThiM#2 riboswitch construct,23 which contains an aptamer region that recognizes

95

TPP, an expression platform that allows the riboswitch to function as an ON switch, and

96

a tetA-gfp+ fusion that acts as a selectable marker and reporter.24 As our aim was to

97

identify novel riboswitches for theophylline, we replaced the aptamer region upstream of

98

the ThiM#2 expression platform by cloning in a random 40-nucleotide sequence,

99

generating a theoretical diversity of 1024 sequences (Figure 1A; Table S1). The

100

expression platform of ThiM#2 between the aptamer and ribosome binding site (RBS)

101

was retained. We introduced our plasmid library into E. coli, generating a 108 library of

102

transformants.

103 104

Our riboswitch discovery platform consists of genetic selections using the dual

105

selectable marker, tetA, and high-throughput fluorescence screens using GFP+.13,23–27

106

Bacteria expressing tetA are resistant to tetracycline and susceptible to heavy metal

107

toxicity. Thus, in order to identify members of the library that activate gene expression in

108

a theophylline-dependent manner, we performed “positive” selections with tetracycline

109

and theophylline and “negative” selections with Ni2+ and without theophylline. In addition

110

to selections, we performed screens using FACS in which we isolated the most and




Page 6 of 25

6

111

least fluorescent library members following positive and negative selections,

112

respectively (Figure S1). FACS has been shown to identify improved riboswitch variants

113

and, in addition, provides an opportunity to identify the library members exhibiting the

114

most extreme levels of ON and OFF gene expression in a manner not afforded to

115

selections.28,29 Thus, we proposed that subjecting our riboswitch library to high-

116

throughput screens in addition to genetic selections would increase the robustness of

117

our engineering platform.

118 119

We sought to identify theophylline riboswitches from our library by performing three

120

complete rounds of selection, incorporating FACS screening (ON-sort) after positive

121

selection in rounds two and three (Figure S2). Cultures were supplemented with 1 mM

122

theophylline when appropriate. As we were selecting for function from a random-

123

sequence library, we incrementally increased the stringency of positive selection by

124

starting with a low concentration of tetracycline in round one and increasing the

125

tetracycline concentration in rounds two and three, using 30, 40, and 50 µg/mL,

126

respectively. Ni2+ concentrations were kept constant at 0.3 mM in each negative

127

selection. These chosen concentrations were based on prior selections performed on a

128

TPP switch.24 Following the third round of selections and screens, dilutions of the

129

surviving library members were plated and eight isolates were randomly selected for

130

sequencing and fluorescence assays (Table S2). Of those interrogated, Hit 3-5

131

displayed the greatest fold activation (Figure 1, Figure S3). We then repeated the

132

selections and FACS screens on our N40 library with one modification. After negative

133

selections of rounds two and three, we incorporated an “OFF-sort” through FACS


Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



7

134

screening as a way to identify members with low levels of fluorescence in its “OFF”

135

state (Figure S2). After three rounds, eight randomly-selected post-selection library

136

members were sequenced and assayed for fluorescence. Hit 3-8 was enriched to half of

137

the sequences analyzed and was the only isolate that displayed riboswitch activity

138

(Figure 1). Hit 3-5 and Hit 3-8 display a 2.3 and 2.1-fold activation of gene expression,

139

respectively (Figures 1B, 1C and S4). Interestingly, Hit 3-8 displays much lower levels

140

of gene expression in both its “ON” and “OFF” states compared to Hit 3-5 (Figure 1B).

141

We postulate that incorporation of an OFF-sort following negative selection may allow

142

the isolation of rare switches that have a low basal level of gene expression. Moreover,

143

our platform uncovered theophylline riboswitches with unique aptamer sequences, and

144

both of these aptamers are different from the aptamers identified from in vitro selections

145

using SELEX (Figure 1A and S5).30

146 147

One of these in vitro selected aptamers, mTCT8-4, has been used to create a

148

theophylline-responsive riboswitch.12,31 We replaced the 5’ untranslated region of Hit 3-

149

5, up to the ribosome binding site, with a sequence coding for the mTCT8-4-based

150

riboswitch and compared the two constructs (Figure 1A). The mTCT8-4-based

151

riboswitch displayed an impressive 15.8-fold activation upon ligand addition, compared

152

to the 2.3-fold activation achieved by Hit 3-5 (Figure 1B). At the same time, the mTCT8-

153

4 aptamer was selected after eight rounds of SELEX, compared to the three rounds of

154

dual genetic selection used here. Additional high-throughput screens were also used to

155

identify an ideal actuator sequence linking the aptamer to the RBS, a process that is not

156

always successful in generating a riboswitch given the difference between the cellular




Page 8 of 25

8

157

milieu in which the aptamer is expected to function and the in vitro conditions from

158

which the aptamers was selected.14–18,31 Overall, our results suggest two things: one,

159

aptamers engineered in vivo and in vitro may converge to different solutions; and, two,

160

certain ligand-binding RNAs capable of eliciting modest biological activity can be

161

identified within a cellular environment without the use of in vitro engineering methods.

162 163

We also attempted to obtain riboswitches responsive to theophylline from our starting

164

108 library using a “selection-only” or a “sort-only” approach. None of the randomly

165

selected eight clones that survived three rounds of selection in the selection-only

166

method demonstrated any fold-activation in response to theophylline (data not shown).

167

Intriguingly, the sort-only protocol resulted in enrichment of ThiM#2 clones to five of the

168

seven sequenced isolates. As our N40 library was constructed using a ThiM#2 construct

169

as a template, it is not surprising that there may have been residual copies of the parent

170

plasmid that were not detected when we sequenced only 8 of the initial library members

171

(Table S1). It is unclear, however, why ThiM#2 was able to become enriched through

172

FACS screening as theophylline does not appear to induce the TPP riboswitch to

173

activate gene expression (Figure 1C). Regardless, our results indicate that the

174

combination of genetic selection with FACS screening is an efficacious method for

175

identifying new riboswitches from random-sequence libraries.

176 177

Following the identification of Hits 3-5 and 3-8, we tested whether riboswitch activity

178

was retained in different genetic constructs. First, we cloned the lacZ gene in place of

179

the tetA-gfp+ fusion. Hits 3-5 and 3-8 demonstrate a 1.9 and 1.7-fold activation,


Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



9

180

respectively, in this pLac-rbsw-lacZ context (Figure 2A). We then tested riboswitch

181

activity in a construct with an arabinose-inducible promoter, replacing the pLac promoter

182

that was used in our original library construction and in pLac-rbsw-lacZ. In beta-

183

galactosidase assays, Hits 3-5 and 3-8 both display a 1.7-fold activation in this pBAD-

184

rbsw-lacZ construct (Figure 2B). These results demonstrate that our engineered

185

riboswitches function in diverse contexts and that their switching activity is independent

186

of the genes that they regulate.

187 188

To characterize the specificity of Hits 3-5 and 3-8, we tested riboswitch activity to

189

caffeine and TPP. Caffeine is structurally similar to theophylline and only differs by a

190

methyl group on N-7. TPP, on the other hand, is structurally distinct from theophylline.

191

Using the pLac-rbsw-tetA-gfp+ constructs, neither Hits 3-5 nor 3-8 display activity to

192

TPP (Figure 1C). However, both hits show slightly increased activity to caffeine

193

compared to theophylline (Figure 1C). Because we did not incorporate into our platform

194

a feature that identifies theophylline binders and simultaneously excludes caffeine

195

binders, this finding is unsurprising. As a comparison, experiments of previously

196

reported theophylline aptamers isolated by SELEX incorporated three rounds of a

197

“counterselection” in which RNA library members capable of binding both theophylline

198

and caffeine were discarded, resulting in sequences with a 1000-fold selectivity for

199

theophylline over caffeine.30 When those SELEX-selected aptamers were incorporated

200

into riboswitches, they maintained selectivity.12 While our hits do show promiscuity to

201

the general methylxanthine structure, they are able to discriminate against a distinctly

202

different target compound.




Page 10 of 25

10

203 204

The dose-responses of Hits 3-5 and 3-8 to theophylline and caffeine were examined in

205

fluorescence assays using the pLac-rbsw-tetA-gfp+ constructs. The EC50 values of Hit

206

3-8 to theophylline (0.9 mM) and caffeine (0.8 mM) are slightly lower than those of Hit 3-

207

5 (6.6 and 1.0 mM, respectively) (Figure 3). For comparison, a riboswitch developed

208

using the mTCT8-4 aptamer was reported to have an EC50 of 0.25 mM.12 Using a SYBR

209

Green I assay,32,33 the Kd of the full-length 5’ UTR of Hit 3-5 for theophylline and

210

caffeine was determined to be 238 and 299 µM, respectively (Figure S6). Using the

211

same assay, we determined the Kd of the mTCT8-4 aptamer to be 373 nM, in line with

212

previously measured values.34

213 214

The switching activity of the original ThiM#2 in response to TPP is thought to be a result

215

of translational regulation;23 however, northern analysis indicates that for Hit 3-5

216

addition of theophylline to the growth medium increased tetA-gfp+ mRNA levels ~3-fold,

217

indicative of regulation of gene expression via effects on either transcriptional read

218

through or mRNA stability (Figure S7). The relatively low binding affinity of Hits 3-5 and

219

3-8 may be a result of a mechanism that relies more on fast binding to allow for

220

increased transcription processivity, rather than a highly stable binding-event that would

221

be needed to affect translation.18

222 223

We set out to determine if our platform could be used to improve the activity of Hit 3-5.

224

We re-synthesized the 40 nt aptamer domain and the 16 nt expression platform of Hit 3-

225

5 with a 24% mutagenesis rate, which would theoretically result in an average of 13


Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



11

226

point mutations per sequence. This new library was incorporated into the pLac-tetA-gfp+

227

backbone and introduced into E. coli, resulting in a 107 library. Prior to selections and

228

screens on this library, we sequenced eight clones and measured activity to

229

theophylline (Table S3, Figure S8). As expected, random mutagenesis generally ablates

230

riboswitch activity. This library was then subjected to three rounds of selection, including

231

both ON- and OFF-sorting from the second round, forward. The concentrations of

232

theophylline (1 mM), tetracycline (30, 40, and 50 μg/mL), and Ni2+ (0.3 mM) used were

233

the same as those used in the original selections that yielded Hits 3-5 and 3-8. After just

234

two rounds, the activity of several isolates had improved, compared to the parent. One

235

isolate demonstrated nearly 3-fold activation, a 35% increase from Hit 3-5 (Figure 4A).

236

While one additional round of dual selection and sorting did result in enrichment of

237

specific sequences, the fold-activation did not significantly improve (data not shown).

238

The seven isolated sequences each had on average two mutations in the expression

239

platform region of the original ThiM#2 template (Table S4). It is expected that efficient

240

coupling of the sensor and the actuator would be required for an effective riboswitch.

241 242

Additional analysis of the isolates that demonstrated improved activation, along with Hit

243

3-5, point to specific nucleotides that are strictly conserved in the 56 nt region that

244

includes the aptamer domain and expression platform [e.g. nucleotides 9-13 (GUCAA)

245

and 39-42 (UUGC)] (Figure 4B). Overall, in the analyzed active isolates, 27 out of 56 nt

246

are strictly conserved, whereas the randomly selected and sequenced members of the

247

mut389 starting library only have 7 nt that are “conserved” by chance (Table S3). The




Page 12 of 25

12

248

strictly conserved residues in the active isolates, as well as other residues that are

249

highly conserved, likely contribute to riboswitch function.

250 251

We have presented a platform to discover new riboswitches in which the selection of the

252

sensor and actuator domains takes place inside the cell. Thus, from the start, the

253

complexities of the cellular environment and how it may impact RNA folding and RNA-

254

ligand binding are factored in to the selection process. The starting 108 library size was

255

likely critical to the success of our approach.15 Previous efforts to identify riboswitches

256

through dual genetic selection or FACS-screens involved starting libraries on the order

257

of 104-105.23–25,28,29 In each of those cases, however, the starting library included an

258

aptamer domain either provided by nature or selected via SELEX. The complexity of the

259

problem we addressed – identifying an aptamer domain in vivo – warranted a larger

260

starting library, which are typically limited by the transformation efficiency of bacteria to

261

~109. In comparison, in vitro selections of aptamers often start with libraries of at least

262

1013 unique molecules.35–37 At the same time, solution frequencies as low as 1 in 108

263

have been previously observed for in vitro selection of aptamers against small

264

molecules.36,37 In the example presented here, the solution-frequency we encountered

265

deemed a 108 library adequate for identifying aptamer domains for the small molecule

266

theophylline via an in vivo platform. An additional round of mutagenesis of one of our

267

original hits, which increased the total numbers of sequences sampled, was able to

268

further improve the activity of our selected riboswitch.

269


Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



13

270

The choice of theophylline as the ligand of interest to validate our approach is grounded

271

upon the following rationale: First, since theophylline is a molecule with a known

272

riboswitch, we reasoned that a riboswitch ‘solution’ was indeed possible. Second, the

273

existing theophylline riboswitch has been extensively studied and reengineered by

274

many different research groups, giving us additional opportunities for comparison of

275

potential hits using our platform.12,28–31,38 Indeed, the riboswitches that we identified

276

contain sequences entirely distinct from previously studied theophylline riboswitches

277

and affect transcription of downstream genes, rather than translation.12,26,31 We

278

anticipate that the general platform provided here, involving the combination of a large

279

(108-109) starting library to identify modest initial hits, followed by additional directed

280

evolution, will serve as a template for identifying other novel riboswitches.

281 282

Methods

283

Reagents. All oligonucleotides were purchased from Integrated DNA Technologies;

284

primers used in this study are listed in Table S5. All cloning enzymes were purchased

285

from New England Biolabs. All PCR reactions were performed using Q5 DNA

286

Polymerase. Gibson Assembly Master Mix or Hi-Fi DNA Assembly Master Mix was

287

used for DNA fragment assembly to generate plasmid constructs. Theophylline,

288

caffeine, and thiamine were purchased from Sigma Aldrich.

289 290

Bacterial Strains and Growth Conditions. All strains used in this study are described

291

in Table S6. Strains were grown at 37 °C with orbital shaking at 250 rpm in LB broth or

292

M9 minimal medium containing 0.8% w/v glycerol. Unless otherwise stated, the minimal




Page 14 of 25

14

293

medium source was supplemented with 0.1% casamino acids, 0.1 mM CaCl2, 2 mM

294

MgSO4, and 0.1 mM thiamine. Carbenicillin was used as an antibiotic at 50-100 μg/mL.

295 296

Library Construction and Plasmid Cloning. In order to amplify pthiM#2-tetA-gfp+, a

297

variant of pthiM#2-tetA-gfpuv,24 without its 34-base pair aptamer domain, PCR was

298

performed with primers pthiM#2tetAgfp forward and pthiM#2tetAgfp reverse (0.5 μM,

299

each), using the pthiM#2-tetA-gfp+ template (0.5 – 1 ng/μL). PCR was performed with

300

an initial denaturation step of 30 s at 98 °C followed by 25 cycles of 10 s at 98 °C, 30 s

301

at 67 °C, and 2.5 min at 72° C. The PCR product was treated twice, consecutively, with

302

DpnI (40 units at 37 °C for 60 min, each). The DNA was purified using the QIAquick

303

PCR Purification kit (QIAGEN) and quantified by gel electrophoresis. This backbone

304

was assembled (3:1 insert to vector ratio) with an oligonucleotide containing a

305

randomized (machine-mixed) 40 base pair region (5’-

306

CGTGAAGGCTGAGAAATACCCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

307

NNNNNNNNGCTATTACAAGAAGATCAGGAGCAAAC-3’). The assembled product

308

(Figure S9) was purified using the QIAquick PCR Purification kit and eluted in 40 μL

309

water. Transformation of the plasmid library into TOP10 E. coli cells (Invitrogen) was

310

performed by electroporation at 2000 V. Transformed cells were recovered in 2x YT for

311

30 minutes at 37 °C with aeration at 250 rpm. Dilutions (10-3, 10-4, and 10-5) of the

312

transformed cells were plated on LB agar plates containing 50 μg/mL carbenicillin and

313

library size was estimated from the number of colonies formed. The remaining library

314

was grown overnight in 2x YT with 50 μg/mL carbenicillin and then stored at -80 °C in

315

20% glycerol the following day. The 10-5 plate contained 1880 colonies, providing an


Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



15

316

estimate of library size of 1 x 108. Sequencing of 8 randomly-selected isolates afforded

317

eight unique sequences (Table S1), none of which were the starting pthiM#2-tetA-gfp+

318

plasmid.

319 320

To construct the mutagenized Hit 3-5 (m389) library, the same procedure as above was

321

used, but using the following machine-mixed oligonucleotide during DNA assembly: 5’ -

322

GCTGAGAAATACCCGCACTGTTCGTCAAGAAAGCATCATTGTGACTGTGTAGATTG

323

CTATTACAAGAAGATCAGGAGCAAACTATG, in which the underlined sequence,

324

corresponding to the aptamer and expression platform regions, was mutagenized at a

325

24% rate. The resulting library was transformed into E. coli, plated and recovered, as

326

above. The 10-4 and 10-5 plates contained 1100 and 142 colonies, respectively,

327

providing an estimate of library size of 1 x 107. Sequencing of 8 randomly-selected

328

isolates afforded eight unique sequence, each of which was different from the parental

329

sequence (Table S3).

330 331

Dual Genetic Selection and Fluorescence-Activated Cell Sorting. The library was

332

diluted to an optical density (OD600) of 0.05 (~108 cells) in 20 mL M9 medium with 100

333

μg/mL carbenicillin. Prior to positive and negative selections, the cultures were

334

incubated at 37 °C with aeration at 250 rpm for 4-6 hours with or without a ligand of

335

interest (1 mM). The incubated cultures were diluted to an OD600 of 0.05 (~108 cells) in

336

20 mL fresh M9 medium containing either the ligand of interest (1 mM) and tetracycline

337

(30–50 μg/mL) for positive selections or nickel chloride (0.3 mM) for negative selections

338

and incubated at 37 °C with orbital shaking at 250 rpm for 12-20 hours.




Page 16 of 25

16

339 340

Fluorescence-activated cell sorting (FACS) was performed following selections in the

341

second and third rounds. Selection cultures were washed and subsequently diluted

342

1:100 (~107 cells/mL) in 1 x M9 salts. FACS was carried out using a Biorad S3™ Cell

343

Sorter with excitation using a 488 nm laser and fluorescence detected using a 525/30

344

bandpass filter. Approximately 106 cells were interrogated and of those, the 5-10% most

345

(ON) or least (OFF) fluorescent were collected in LB broth with 50 μg/mL carbenicillin

346

and recovered at 37 °C with aeration at 250 rpm for 12-20 hours.

347 348

Fluorescence Assays. Fluorescence assays were performed following rounds of

349

selections and screens. Strains were incubated overnight in M9 medium. Cultures were

350

diluted to an OD600 of 0.05 and incubated until they reached an OD600 of ~0.2-0.3. The

351

cultures were split into two fractions and incubated with or without theophylline (1 mM),

352

caffeine (1 mM), or thiamine (0.1 mM) for 1-2 hours. Cultures were either diluted 1:5-

353

1:10 in 1 X PBS or 0.5-1 mL were pelleted (8,000 x g, 5 min) and resuspended in 1 x

354

PBS. Samples (200 μL) were transferred to a 96-well optical-bottom plate and relative

355

fluorescence (excitation at 480 nm, emission at 520 nm) and OD600 were measured

356

using a Biotek Synergy 4 plate-reader. The relative fluorescence units (RFU) of each

357

sample were normalized to the OD600 reading of each sample to provide the reported

358

RFU/OD600 value. Assays were performed with three or five biological replicates in

359

technical duplicate or triplicate. For fluorescence assays testing the dose response of

360

potential riboswitches, concentrations of theophylline ranged from 0.025 - 2 mM.

361

Nonlinear regression and statistical analyses were performed using Prism GraphPad.


Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



17

362 363

Beta-Galactosidase Assays. Strains were grown, as above for the fluorescence

364

assays. After 1-2 h of treatment of with ligand of interest, OD600 measurements of

365

cultures (250 μL) were recorded in clear 96-well plates (Costar) using a Biotek Synergy

366

4 plate-reader. Cells (100 μL) were lysed for at least 25 minutes with lysis buffer (10 μL)

367

containing PopCulture Reagent (Novagen) and lysozyme (ThermoFisher). Cell lysate

368

(30 μL) was incubated (28 °C) with substrate solution (150 μL; 60 mM Na2HPO4, 40 mM

369

NaH2PO4, 1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG), 2.7 μL/mL β-

370

mercaptoethanol) and absorbance at 420 nm was recorded every 30 seconds for 1 hour

371

using a Biotek Synergy 4 plate-reader. Reported values are in units of mean

372

OD420/min/OD600. Results are representative of at least three biological replicates.

373

Statistical analyses were performed using Prism GraphPad.

374 375

Author Contributions

376

K.P, J.S. and J.M.L. performed all of the experiments with assistance from S.L and M.Z.

377

K.P. and J.M.L wrote the paper, with editorial assistance from all authors. J.M.L.

378

conceived and supervised the study.

379 380

Supporting Information 

381

The Supporting Information is available free of charge on the ACS Publications website

382

at DOI: XXXXX.

383

Further experimental details, including plasmid cloning, sequencing and RNA analysis;

384

additional figures, tables, and their references (PDF)




Page 18 of 25

18

385 386

Acknowledgements

387

We would like to thank Dr. Yohei Yokobayashi for providing the original plasmid

388

expressing a thiM#2-tetA-gfpuv fusion. The research was funded by Pomona College

389

and an NSF grant to J.M.L. (CBET-1258307). J.M.L. is a Henry-Dreyfus Teacher-

390

Scholar. We thank Erick Velasquez and Marek Zorawski for their technical assistance at

391

the start of this project and Greg Copeland for critical readings of the manuscript.

392 393

References

394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421

(1) Serganov, A., and Nudler, E. (2013) A decade of riboswitches. Cell 152, 17–24. (2) Mehdizadeh Aghdam, E., Hejazi, M. S., and Barzegar, A. (2016) Riboswitches: From living biosensors to novel targets of antibiotics. Gene 592, 244–259. (3) Zhou, L.-B., and Zeng, A.-P. (2015) Exploring lysine riboswitch for metabolic flux control and improvement of L-lysine synthesis in Corynebacterium glutamicum. ACS Synth. Biol. 4, 729–734. (4) Blount, K. F., Wang, J. X., Lim, J., Sudarsan, N., and Breaker, R. R. (2007) Antibacterial lysine analogs that target lysine riboswitches. Nat. Chem. Biol. 3, 44. (5) Weigand, J. E., and Suess, B. (2009) Aptamers and riboswitches: perspectives in biotechnology. Appl. Microbiol. Biotechnol. 85, 229. (6) Fowler, C. C., Brown, E. D., and Li, Y. (2010) Using a riboswitch sensor to examine coenzyme B12 metabolism and transport in E. coli. Chem. Biol. 17, 756–765. (7) Bell, C. L., Yu, D., Smolke, C. D., Geall, A. J., Beard, C. W., and Mason, P. W. (2015) Control of alphavirus-based gene expression using engineered riboswitches. Virology 483, 302–311. (8) Michener, J. K., and Smolke, C. D. (2014) Synthetic RNA switches for yeast metabolic engineering. Methods Mol. Biol. Clifton NJ 1152, 125–136. (9) Werstuck, G., and Green, M. R. (1998) Controlling gene expression in living cells through small molecule-RNA interactions. Science 282, 296–298. (10) Weigand, J. E., Sanchez, M., Gunnesch, E.-B., Zeiher, S., Schroeder, R., and Suess, B. (2008) Screening for engineered neomycin riboswitches that control translation initiation. RNA 14, 89–97. (11) Hanson, S., Berthelot, K., Fink, B., McCarthy, J. E. G., and Suess, B. (2003) Tetracycline-aptamer-mediated translational regulation in yeast. Mol. Microbiol. 49, 1627–1637. (12) Desai, S. K., and Gallivan, J. P. (2004) Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. J. Am. Chem. Soc. 126, 13247–13254.


Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Engineering riboswitches in vivo 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467

19

(13) Jang, S., Jang, S., Xiu, Y., Kang, T. J., Lee, S.-H., Koffas, M. A., and Jung, G. Y. (2017) Development of artificial riboswitches for monitoring of naringenin in vivo. ACS Synth. Biol. 6, 2077–2085. (14) Wu, M.-C., Lowe, P. T., Robinson, C. J., Vincent, H. A., Dixon, N., Leigh, J., and Micklefield, J. (2015) Rational re-engineering of a transcriptional silencing PreQ1 riboswitch. J. Am. Chem. Soc. 137, 9015–9021. (15) Jijakli, K., Khraiwesh, B., Fu, W., Luo, L., Alzahmi, A., Koussa, J., Chaiboonchoe, A., Kirmizialtin, S., Yen, L., and Salehi-Ashtiani, K. (2016) The in vitro selection world. Methods 106, 3–13. (16) Berens, C., and Suess, B. (2015) Riboswitch engineering—making the allimportant second and third steps. Curr. Opin. Biotechnol. 31, 10–15. (17) Groher, Florian, Bofill-Bosch, C., Schneider, C., Braun, J., Jager, S., Geißler, K. H., and Seuss, B. (2018) Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator. Nucleic Acids Res. 46, 2121-2132. (18) Etzel, M., and Mörl, M. (2017) Synthetic riboswitches: from plug and pray toward plug and play. Biochemistry 56, 1181–1198. (19) Kim, Y.-B., Wacker, A., Laer, K. von, Rogov, V. V., Suess, B., and Schwalbe, H. (2017) Ligand binding to 2΄-deoxyguanosine sensing riboswitch in metabolic context. Nucleic Acids Res. 45, 5375–5386. (20) Porter, E. B., Polaski, J. T., Morck, M. M., and Batey, R. T. (2017) Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors. Nat. Chem. Biol. 13, 295–301. (21) Dixon, N., Duncan, J. N., Geerlings, T., Dunstan, M. S., McCarthy, J. E., Leys, D., and Micklefield, J. (2010) Reengineering orthogonally selective riboswitches. Proc. Natl. Acad. Sci. 107, 2830–2835. (22) Robinson, C. J., Vincent, H. A., Wu, M.-C., Lowe, P. T., Dunstan, M. S., Leys, D., and Micklefield, J. (2014) Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species. J. Am. Chem. Soc. 136, 10615–10624. (23) Nomura, Y., and Yokobayashi, Y. (2007) Reengineering a natural riboswitch by dual genetic selection. J. Am. Chem. Soc. 129, 13814–13815. (24) Muranaka, N., Sharma, V., Nomura, Y., and Yokobayashi, Y. (2009) An efficient platform for genetic selection and screening of gene switches in Escherichia coli. Nucleic Acids Res. 37, e39–e39. (25) Zhou, L.-B., and Zeng, A.-P. (2015) Engineering a lysine-ON riboswitch for metabolic control of lysine production in Corynebacterium glutamicum. ACS Synth. Biol. 4, 1335–1340. (26) Sharma, V., Nomura, Y., and Yokobayashi, Y. (2008) Engineering complex riboswitch regulation by dual genetic selection. J. Am. Chem. Soc. 130, 16310–16315. (27) Nomura, Y., and Yokobayashi, Y. (2007) Dual selection of a genetic switch by a single selection marker. Biosystems 90, 115–120. (28) Lynch, S. A., and Gallivan, J. P. (2009) A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res. 37, 184–192. (29) Fowler, C. C., Brown, E. D., and Li, Y. (2008) A FACS-based approach to engineering artificial riboswitches. ChemBioChem 9, 1906–1911. (30) Jenison, R. D., Gill, S. C., Pardi, A., and Polisky, B. (1994) High-resolution molecular discrimination by RNA. Science 263, 1425–1429.



Engineering riboswitches in vivo 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490

Page 20 of 25

20

(31) Lynch, S. A., Desai, S. K., Sajja, H. K., and Gallivan, J. P. (2007) A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. Chem. Biol. 14, 173–184. (32) McKeague, M., Velu, R., Hill, K., Bardóczy, V., Mészáros, T., and DeRosa, M. C. (2014) Selection and characterization of a novel DNA aptamer for label-free fluorescence biosensing of ochratoxin A. Toxins 6, 2435–2452. (33) McKeague, M., De Girolamo, A., Valenzano, S., Pascale, M., Ruscito, A., Velu, R., Frost, N. R., Hill, K., Smith, M., and McConnell, E. M. (2015) Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation. Anal. Chem. 87, 8608–8612. (34) Chang, A. L., McKeague, M., Liang, J. C., and Smolke, C. D. (2014) Kinetic and equilibrium binding characterization of aptamers to small molecules using a label-free, sensitive, and scalable platform. Anal. Chem. 86, 3273–3278. (35) Ruscito, A., and DeRosa, M. C. (2016) Small-molecule binding aptamers: Selection strategies, characterization, and applications. Front. Chem. 4. (36) Ellington, A. D., and Szostak, J. W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822. (37) Ellington, A. D., and Szostak, J. W. (1992) Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. Nature 355, 850-852. (38) Zimmermann, G. R., Jenison, R. D., Wick, C. L., Simorre, J.-P., and Pardi, A. (1997) Interlocking structural motifs mediate molecular discrimination by a theophyllinebinding RNA. Nat. Struct. Mol. Biol. 4, 644–649.


Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



21

491 492 493 494 495 496 497 498 499 500 501 502 503

Figure 1. Selections and screens on a riboswitch library uncovered two unique theophylline riboswitches. (A) The N40 plasmid library consists of a degenerate 40nucleotide sequence, the ThiM#2 expression platform, RBS, and tetA-gfp+ fusion. (B) Hit 3-5, Hit 3-8, and mTCT8-4 regulate expression of the tetA-gfp+ fusion in response to theophylline (1 mM) and display a 2.3, 2.1, and 15.8-fold activation, respectively. (C) Within the context of pLac-rbsw-tetA-gfp+, Hits 3-5 and 3-8 exhibit a 3.3 and 2.9-fold activation to caffeine (1 mM), respectively, and do not exhibit activity to thiamine (0.1 mM). Data are representative of three biological replicates. Bars indicate mean values and error bars indicate standard deviation. *** p < 0.001, **** p < 0.0001 by two-tailed ttest. “rbsw” stands for riboswitch and is indicative of the ThiM#2, 3-5, 3-8, or mTCT8-4 riboswitches in the construct.




Page 22 of 25

22

504

505 506 507 508 509 510 511 512

Figure 2. Hits 3-5 and 3-8 regulate lacZ expression in response to theophylline indendent of promoter. (A) Hits 3-5 and 3-8 display a 1.9 and 1.7-fold activation, respectively, in the context of pLac-rbsw-lacZ. (B) Hits 3-5 and 3-8 display a 1.7 -fold activation in the context of pBAD-rbsw-lacZ. Data are representative of three biological replicates. Bars indicate mean values and error bars indicate standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by two-tailed t-test comparing ON and OFF states.


Page 23 of 25


23

A

B 5

513 514 515 516 517 518 519 520

4

pLac-3-5-tetA-gfp+

4

caffeine

3

theophylline

2 1

Fold Activation

Fold Activation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pLac-3-8-tetA-gfp+ caffeine

3

theophylline

2 1

0 10-5 10-4.5 10-4 10-3.5 10-3 10-2.5

0 10-5 10-4.5 10-4 10-3.5 10-3 10-2.5

log[Ligand], M

log[Ligand], M

Figure 3. Hits 3-5 and 3-8 display a dose-response to theophylline and caffeine. EC50 values of riboswitches were obtained from fitting of the fold activation to log[Ligand]. EC50 of Hits 3-5 and 3-8 are, 6.6 mM and 0.9 mM, respectively, to theophylline and 1.0 mM and 0.8 mM, respectively, to caffeine. Data are representative of three biological replicates. Bars indicate mean values and error bars indicate standard deviation.




Fold Activation

A

B

4 3

24

*

**

**

2 1 0

m ut 38 m 9-2 ut 38 -1 m 9-2 ut 38 -2 m 9-2 ut 38 -3 m 9-2 ut 38 -4 m 9-2 ut 38 -5 m 9-2 ut 38 -7 92H 8 it 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

521 522 523 524 525 526 527 528 529 530

Isolate

Figure 4. Two rounds of selections and sorts on the mutagenized Hit 3-5 library resulted in riboswitch variants, several of which had improved fold-activation compared to the parental sequence (average fold-activation of 2.2 in these assays, as indicated by the dotted line). (A) Bars indicate mean values and error bars represent standard deviation of 2-3 biological replicates. * p < 0.05, ** p < 0.01 by two-tailed t-test compared to fold-activation of Hit 3-5. (B) Multiple sequence alignment of mut389-2-1, 2, 3, 4, 5, 7 and 8, along with Hit 3-5. Highlighted nucleotides indicate differences from Hit 3-5.


Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Engineering riboswitches in vivo using dual genetic selection and fluorescenceactivated cell sorting Katharine Page, Jeremy Shaffer, Samuel Lin, Mark Zhang and Jane M. Liu Table of contents graphic:

+ ON 1. Nickel 2. Cell Sorting (OFF)

Selections + FACS

1. Tetracycline 2. Cell Sorting (ON)

OFF


Engineering Riboswitches in Vivo Using Dual Genetic Selection and

Recommend Documents