key: cord-0920132-1uqjmeic
authors: Woo, Chang Ha; Jang, Sungho; Shin, Giyoung; Jung, Gyoo Yeol; Lee, Jeong Wook
title: Sensitive one-step isothermal detection of pathogen-derived RNAs
date: 2020-03-09
journal: nan
DOI: 10.1101/2020.03.05.20031971
sha: e05c39ce913edc81f93c9247f9714d6cddd30073
doc_id: 920132
cord_uid: 1uqjmeic

The recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times.

Increasing global trade and travel are considered the cause of frequent emergence and rapid 30 dissemination of infectious diseases around the world. Some life-threatening infectious 31 diseases often have signs and symptoms similar to cold or flu-like syndromes. Early diagnosis 32 is therefore essential to identify the diseases and provide the correct treatment. Immediate and 33 onsite diagnostic decisions also help to prevent the spread of epidemic and pandemic 34 infectious diseases [1] [2] [3] . In order to rapidly diagnose infectious diseases, a nucleic acid-based 35 diagnosis has emerged as an alternative to the conventional culture-based, or immunoassay-36 based, approaches due to their rapidity or specificity 4-6 . 37

To increase sensitivity, current nucleic acid detection methods generally involve a 38 target amplification step prior to the detection step. The conventional amplification method is 39 based on PCR, which requires a thermocycler for delicate temperature modulation. As an 40 alternative to the thermal cycling-based amplification, isothermal amplification methods are 41 available, which rely primarily on a strand-displacing polymerase or T7 RNA polymerase at a 42 constant temperature 7 . However, the complex composition of the isothermal amplification 43 mixtures often renders these approaches incompatible with detection methods and whole 44 diagnosis generally becomes a multi-step process [8] [9] [10] [11] . The diagnostic regimen with multi-step 45 procedures requires additional time, instruments, and reagents, as well as skilled personnel to 46 perform the diagnostic procedure. This aspect limits the broad applicability of nucleic acid 47 diagnostics, especially in situations where rapid and simple detection is required. 48 3 efficiently ligate two DNA probes using a target single-stranded RNA as a splint, enabling 54 the sequence-specific detection of RNA molecule 17, 18 . Because the reaction components of 55 the ligation-dependent methods are relatively simple, we hypothesized that the ligation-56 dependent method could be exploited to establish a one-step RNA detection platform when 57 combined with compatible amplification and signal generation methods in a single reaction 58 mixture. 59

In this study, we developed a one-step, ligation-dependent isothermal reaction 60 cascade that enables rapid detection of RNAs with high sensitivity, termed SENsitive Splint-61 We designed a reaction cascade that allows the one-step diagnostic test, in which all reaction 76 steps for nucleic acid detection occur simultaneously in a single tube (Fig. 1) . The cascade 77

consists of four core components, which includes only two enzymes: a set of oligonucleotide 78 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 4 probes, SplintR ligase, T7 RNA polymerase, and a fluorogenic dye. The components were 79 mixed in a buffer solution with ribonucleotides. Upon addition of the pathogen-derived RNA 80 sample, the reaction steps of ligation, transcription, and dye-aptamer binding enabled 81 detection, amplification, and signal production, respectively. 82

Two single-stranded DNA probes were designed to include several functional parts 83 involved in amplification, detection, and signal generation, thereby eliminating the need for 84 human intervention during the entire diagnostic process (Fig. 1) using the full-length, ligated probe as a DNA template, which can be bound with the 101 fluorogenic dye to emit fluorescence as an output (Fig. 1) . Notably, the reaction scheme of 102 SENSR inherently supports two mechanisms that could amplify the signal: 1) multiple 103 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 5 transcription events from the full-length, ligated probe by T7 RNA polymerase and 2) the 104 presence of target RNA sequence on the full-length transcript which could be utilized as an 105 additional splint for unligated probes in the reaction mixture. Accordingly, SENSR could 106 enable sensitive RNA detection without any pre-amplification steps. 107 108

In this study, we used MRSA as a model case to validate each reaction step that constitutes 110 SENSR. MRSA is of particular interest because it requires significant effort to minimize 111 healthcare-related infections and prevent future infectious diseases of drug-resistant 112 pathogens 22 . 113

First, we designed a pair of probes that target the mecA transcript of MRSA following 114 the probe design process described in the previous section (Supplementary Note 1 and  115   Supplementary Tables 1 and 3) , and the RNA-splinted ligation between the two probes was 116 tested. The probes were ligated using SplintR ligase with or without the target RNA, and the 117 reaction resultants were further amplified with a pair of PCR primers and analyzed (see 118

Methods section). The correct size of the PCR product was obtained only when the two 119 probes and target RNA were added together to the ligation mixture (Fig. 2a) . This result 120 indicates that our probes were successfully ligated only in the presence of the target RNA. 121

We then used the ligated probe as a DNA template to test whether transcription could 122 occur. The ligation mixture was added at a 1/10 ratio to the in vitro transcription reaction 123 mixture with T7 RNA polymerase. Only when the target RNA was present in the ligation 124 reaction was the full-length transcript (92 nt) observed from transcription, thereby confirming 125 both target-dependent ligation and the subsequent transcription (Fig. 2b) . 126

Finally, we confirmed that the transcript from the full-length ligated probe could 127 produce fluorescence upon binding to the fluorogenic dye. The reaction mixture of sequential 128 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 6 ligation and transcription reactions were purified, and an equal amount of RNAs from each 129 combination was incubated with the fluorogenic dye (Fig. 2c) . The RNA product from the 130 reaction mixture with the two probes and target RNA produced higher fluorescence than that 131 of the other combinations. Therefore, we confirmed that the target RNA could be detected 132 the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is Since the one-step and one-pot isothermal reaction condition was established, we then 157 assessed the sensitivity and turnaround time of SENSR. We evaluated the sensitivity by 158 measuring fluorescence from one-step reactions containing the mecA probe pair and synthetic 159 mecA RNA in the range of 0.1 aM to 220 nM (Fig. 3a) . Notably, the detection limit was as 160 With the fast and sensitive RNA detection using SENSR, we next attempted to reconfigure 174 this platform for the detection of RNA markers from various pathogens. Target RNA 175 sequences for SENSR are specified by only two hybridization regions (UHS and DHS) of 176 probes, which makes the probe design process fast and straightforward without many 177 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is able to detect the Influenza A RNA target with similar sensitivity and linearity (Fig. 4e) . 201

Finally, we designed a probe pair for a recently emerging pathogen, SARS-CoV-2. The target 202 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 9 sequence was selected based on the standard real-time RT-PCR assay for the SARS-CoV-2 26 , 203 which aimed at the RNA-dependent RNA polymerase (RdRp) gene (Supplementary Table 3) . 204

Again, SENSR successfully detected its target RNA as low as 0.1 aM, which corroborates the 205 high adaptability of this method to various RNA markers (Fig. 2f) . 206

Taken together, we demonstrated that SENSR could be easily reconfigured to detect 207 various RNA markers of pathogens by redesigning the probes. The probe design process is 208 simple and requires a small amount of computation using open web-based software. All probe 209 pairs tested showed high sensitivity and linearity for detecting RNA markers, reinforcing the 210 robustness of the probe design process and the wide expandability of SENSR. 211 212 Direct detection of a pathogen using SENSR 213

Next, we employed SENSR for the detection of RNA samples derived from the live cells of a 214 pathogen. We targeted MRSA, whose marker RNA was detected by SENSR. Methicillin-215

Sensitive Staphylococcus aureus (MSSA) that contains no target mRNA was used as a 216 negative control. MRSA and MSSA cells were heated to 95 °C to lyse the cells and to release 217

RNAs. The samples were then diluted and added to SENSR reaction to investigate the 218 specificity and sensitivity (Fig. 5a ). We observed a significant difference in fluorescence 219 intensity between MRSA and MSSA (Fig. 5b) . The RNA sample from only 2 CFU per 100 220 μL reaction of MRSA, not MSSA, was clearly detected by SENSR, thereby indicating its 221 high sensitivity and specificity even with samples of the living pathogen. Finally, the 222 performance of SENSR was further validated using samples prepared in human serum (Fig.  223 5c). The sensitivity and specificity of SENSR were unaffected by the presence of human 224 serum (Fig. 5d) , indicating the suitability of SENSR in practical applications. 225 226 Dual target detection using orthogonal SENSR probes 227 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint fluorescence for Influenza A virus. Indeed, the presence of either target RNA (1 nM) was 253 easily detected by the fluorescence pattern (Fig. 6b) . Across various concentrations of each 254 target RNA, the SENSR probes specifically produced fluorescence that responded only to 255 respective targets, thereby enabling orthogonal dual detection of two pathogens (Fig. 6c) . 256

Lastly, we applied the orthogonal dual detection to the SARS-CoV-2, which has many 257 related viruses with high sequence homology. Simultaneous detection of multiple target sites 258 along its genome would enable specific discrimination of this emerging pathogen from 259 others. In addition to the previously demonstrated SARS-CoV-2 probe pair (Fig. 4f) CoV-2 RNA, thereby exhibiting higher fluorescence intensity compared to that of the related 267 viral RNA sequences (Fig. 7b) . We then tested the orthogonal dual detection of two target 268 regions using the SARS-CoV-2-MG1 and SARS-CoV-2-BR2 probe pairs. Dual SENSR 269 assay effectively detected the target RNA and maintained the specificity of each probe pair 270 (Fig. 7c) . Therefore, the dual SENSR assay could be used to assist diagnostic decision 271 making by providing two detection results that can complement each other. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint based methods, including real-time-PCR, require a complex procedure, expensive 278 instruments, and skilled expertise. Various isothermal amplification methods for RNA have 279 been introduced to replace traditional methods 7,8 , but they generally require numerous 280 reaction components, often making them expensive and incompatible with the signal 281 production step. 282

In contrast, SENSR satisfies many desirable requirements for onsite diagnostic tests 283 for pathogens, such as short turnaround time (30 min), low limit of detection (0.1 aM), 284 inexpensive instrumentation and reagents, and a simple diagnostic procedure. SENSR 285 integrates all component reaction steps using the specially designed probes that contain all 286 required functional parts: promoter, hybridization sequence to target, and an aptamer 287 template. Even with the multifaceted features of the SENSR probes, the design process is 288 systematic and straightforward. Therefore, new SENSR assay can be promptly developed for 289 emerging pathogens as exemplified by the successful design of SENSR assay for SARS-290

The probe design is unique in that two DNA probes are designed to expose single-292 stranded target recognition parts, enabling hybridization of the target RNA and the probes at 293 37℃. The hybridization sequences were systematically selected using the nucleic acid design 294 software Primer-BLAST and NUPACK to minimize any structure formation while 295 maximizing hybridization to the target RNA. The efficient hybridization between the probes 296 and target RNA is one of the reasons for enabling high sensitivity during the isothermal 297

The promoter probe is programmed to form a stem-loop structure and the stem part 299 forms a double-stranded T7 promoter sequence that initiates transcription by recruiting T7 300 RNA polymerase. Since the two strands of T7 promoter part are physically connected by the 301 loop, the probability of formation of a functional double-stranded promoter is higher in the 302 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 13 stem-loop structured design than when each strand of the promoter is not connected by the 303 loop. Thus, the hairpin structured, self-assembling promoter sequence in the promoter probe 304 can facilitate hybridization and subsequent transcription more efficiently. 305

The initiated transcription elongates through the single-stranded DNA as a template to 306 amplify target RNAs containing aptamer RNAs. The use of a fluorogenic RNA aptamer 307 facilitated SENSR development by enabling fast and straightforward signal generation. 308

Compared to conventional fluorescent protein outputs, the use of RNA aptamers as reporters 309 can reduce the time it takes to observe the signal 34 . 310

The simple enzyme composition is another reason to enable one-step and one-pot 311 detection. The fewer the enzymes, the easier it is to optimize in terms of temperature and 312 buffer composition. In designing the detection scheme, we deliberately tried to reduce the 313 number of enzymes, thus creating one of the simplest isothermal detection schemes based on 314 two enzymes: SplintR ligase for target detection and T7 RNA polymerase for amplification. 315

In addition to the results shown in this study, we expect that SENSR has a broad range 316 of possibilities for pathogen detection. First, SENSR can be easily implemented in the initial 317 screening of infectious diseases at places where a large number of people gather and 318 transfer 35,36 . With a short turnaround time and a simple reaction composition, SENSR is an 319 ideal diagnostic test for rapid and economical screening. Second, SENSR will be a valuable 320 platform for the immediate development of diagnostic tests for emerging pathogens 1,37 321 because of the simple probe design process and broad adaptability of SENSR. In this work, 322 we demonstrated the successful application of SENSR to six pathogens, using minimal 323 redesign based on the highly modular structure of the probes. In theory, SENSR detection 324 probes can be designed for any RNA as long as the target nucleic acid sequence is available. 325

This feature provides SENSR a significant advantage over antibody-based diagnostics to 326 rapidly respond to the outbreak of infectious disease. The nucleic acid probe synthesis is 327 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 14 more scalable than animal antibody production. Therefore, SENSR is more suitable for rapid 328 mass production of diagnostic kits than antibody-based diagnostics. Future efforts on 329 automated probe design will be needed to accelerate the development of SENSR assays for 330 newly emerging pathogens. 331

In conclusion, SENSR is a powerful diagnostic platform for RNA detection, which 332 offers a short turnaround time, high sensitivity and specificity, and a simple assay procedure, 333 and eliminates the need for expensive instrumentations and diagnostic specialists. Given the 334 simple probe design process, and its rapid development, SENSR will be a suitable diagnostic 335 method for emerging infectious diseases. 336 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is Inhibitor (20 U), 2 μL T7 RNA polymerase (50 U), and 8.7 μL RNase-free water was 355 incubated at 37 °C for 16 h. The resulting reaction products were treated with 1 μL of DNase 356 I (RNase-free) for 1 h at 37 °C. The transcript was purified using the Riboclear TM (plus!) 357 RNA kit (GeneAll, Seoul, Republic of Korea) and quantified using the absorbance at 260 nm. 358

The purified RNA was used immediately for the downstream reaction or stored at -80 °C. The 359 RNA transcripts were assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa 360 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 16 Clara, CA, USA) using an RNA 6000 nano kit (Agilent Technologies) following the 361 manufacturer's direction. All primers are listed in Supplementary Table 4 RNase-free water. The mixture was heated to 95 °C for 3 min, then slowly cooled to room 377 temperature. This was followed by the addition of 1 μL 10 SplintR buffer and 0.5 μL of 378 SplintR ligase (25 U), and incubation of the mixture at 37 °C for 30 min. The reaction was 379 terminated by heating at 95 °C for 10 min. The ligated product was amplified through PCR 380 reaction with LigChk_F and LigChk_R primers (Supplementary Table 4 ). The PCR products 381

were assessed by an Agilent 2100 Bioanalyzer using a DNA 1000 kit (Agilent Technologies) 382 according to the manufacturer's protocol. 383 384 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint The ligation resultants were amplified with a pair of PCR primers (LigChk_F,R in 555 Supplementary Table 4 ) and analyzed using Bioanalyzer. The ligation reaction occurred 556 when only the promoter probe, reporter probe, SplintR ligase, and target RNA were all 557 present. A full-length probe combining the promoter and reporter probes was amplified with 558 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint the same set of PCR primers and used as a size control, as indicated by the red arrow. b, 559

Transcription reaction. The ligated mixtures were used as a DNA template to validate 560 transcription. The transcript was obtained only in the presence of target RNA and all other 561 components, demonstrating both target-dependent ligation and the subsequent transcription. 562

The red arrow points to the correct size of the transcript. c, Fluorescence reaction. After 563 sequential ligation and transcription reactions, the reaction mixture with the correct size of 564 the transcript produced higher fluorescence compared to other conditions that lack one of the 565 necessary components. Fluorescence tests are four experimental replicas (two-tailed student's 566 test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; bars represent mean ± s.d). 567

All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is four experimental replicas (two-tailed student's test; * P < 0.05, ** P < 0.01, *** P < 0.001, 575 **** P < 0.0001; bars represent mean ± s.d). 576

All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is SARS-CoV-2, respectively. All probe pairs tested showed high sensitivity and linearity to 583 detect RNA markers. All tests are four experimental replicas (two-tailed student's test; * P < 584 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; bars represent mean ± s.d). 585 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is samples. The detection limit of SENSR is as low as 2 CFU per 100 µL reaction. c, Detection 591 of bacterial cell diluted in human serum as a proxy clinical sample. Bacteria-contained human 592 serum was thermally lysed and subjected to the SENSR reaction. d, An obvious distinction in 593 the fluorescence intensity between MRSA-and MSSA-contained human serum was 594 observed. The detection limit of SENSR is as low as 2 CFU per 100 µL reaction. All tests are 595 four experimental replicas (two-tailed student's test; * P < 0.05, ** P < 0.01, *** P < 0.001, 596 **** P < 0.0001; bars represent mean ± s.d). 597 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint 

Better tests, better care: improved diagnostics for infectious 455 diseases

A review of methods for the detection of pathogenic 457 microorganisms

Laboratory-guided detection of disease outbreaks: three 459 generations of surveillance systems

Recent advances in the development of nucleic acid 461 diagnostics

Standardization of nucleic acid tests for clinical measurements of bacteria 463 and viruses

Emerging pathogens: challenges 465 and successes of molecular diagnostics

Isothermal amplified detection of DNA and RNA

Low-Cost Detection of Zika Virus Using Programmable 471

Nucleic acid detection with CRISPR-Cas13a/C2c2

Multiplexed and portable nucleic acid detection platform with 475

Recent developments in ligase-mediated amplification and detection

Discriminatory bases that enable specific detection of SARS-CoV-2 612 against viruses with highly similar sequences are marked by bold letters. Grey shades indicate 613 mismatches between the sequences of SARS-CoV-2 and other viruses. b, Singleplex 614 detection of 1 aM SARS-CoV-2 RNA by SENSR. 229E, Human coronavirus 229E

Human coronavirus NL63; OC43, Human coronavirus OC43; HKU1, Human coronavirus 616 HKU1; Bat-SARS-1, Mg772933 Bat SARS-related coronavirus

All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is One-pot dual detection of SARS-CoV-2 by orthogonal probe pairs, SARS-CoV-2-MG1 and -620 BR1. All tests are two experimental replicas. Fold changes were calculated by dividing the 621 normalized fluorescence values by that with no target RNA. 622 All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.05.20031971 doi: medRxiv preprint