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1 CircPrime: a web-based platform for design of specific circular RNA primers

3 Fedor Sharko1,2,3,
, Golam Rbbani4,
, Prabhugouda Siriyappagouder4, Joost A.M.

4 Raeymaekers4, Jorge Galindo-Villegas4, Artem Nedoluzhko4,5*, Jorge M.O. Fernandes4,*

1
6 Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prospect
7 33/2, 119071, Moscow, Russia.
2
8 Limited liability company ELGENE, Malaya Kalitnikovskaya 16, 109029, Moscow, Russia.
3
9 National Research Center "Kurchatov Institute", 1st Akademika Kurchatova Square, 123182,
10 Moscow, Russia
4
11 Nord University, Universitetsalléen 11, 8049, Bodø, Norway
5
12 European University at Saint Petersburg, 6/1A Gagarinskaya st., 191187, Saint Petersburg,
13 Russia

14

15
These authors contributed equally to this work

16 * Corresponding authors:

17 Dr. Artem Nedoluzhko, Paleogenomics laboratory, European University at Saint Petersburg,

18 Saint Petersburg, Russia. E-mail: nedoluzhko@gmail.com

19 Prof. Jorge M. O. Fernandes, Faculty of Biosciences and Aquaculture, Nord University, PB

20 1490. 8049 Bodø, Norway. E-mail: jorge.m.fernandes@nord.no

21

22

23

24

25

26

27
bioRxiv preprint doi: https://doi.org/10.1101/2022.12.20.521155; this version posted December 20, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

28 Abstract

29 Background: Circular RNAs (circRNAs) are covalently closed-loop RNAs with critical
30 regulatory roles in cells. The tenth of thousands of circRNAs have been unveiled due to the
31 recent advances in high throughput RNA sequencing technologies and bioinformatic tools
32 development. At the same time, polymerase chain reaction (PCR) cross-validation for circRNAs
33 predicted by bioinformatic tools remains an essential part of any circRNA study before
34 publication.

35 Results: Here, we present the CircPrime web-based platform, providing a user-friendly solution
36 for DNA primer design and thermocycling conditions for circRNA identification with routine
37 PCR methods.

38 Conclusions: User-friendly CircPrime web platform (http://circprime.elgene.net/) works with

39 outputs of the most popular bioinformatic predictors of circRNAs to design specific circular
40 RNA primers. CircPrime works with circRNA coordinates and any reference genome from the
41 National Center for Biotechnology Information database (NCBI).

42 Keywords: Circular RNAs, RNA-sequencing, circRNAs, primer design, RT-PCR, qPCR,

43 validation, PCR, web platform, prediction

44

45 Background

46 In recent years, there is a marked increase in the number of circular RNA (circRNA)-related
47 studies (Figure 1). CircRNAs have become a main focus of non-coding RNA biology research
48 because they affect many genetic regulatory networks. These covalently closed-loop RNA
49 molecules are an integral part of the cell regulome and interact with RNA-binding proteins. They
50 can modulate microRNA expression and indirectly affect gene expression. In addition, some of
51 them contain exon parts and can thus translate into proteins (Kristensen, et al., 2019).

52 Modern sequencing technologies make it now possible to identify hundreds of circRNAs that
53 may be used as biomarkers and therapeutic targets in different applied researches (Nedoluzhko,
54 et al., 2020; Rbbani, et al., 2021; Verduci, et al., 2021). However, in silico prediction of
55 circRNAs leads to numerous false-positives (Hansen, et al., 2016), as well as inconsistencies
56 among different bioinformatic pipelines (Nedoluzhko, et al., 2020). As a result, cross-checking
57 and validation of circRNAs is an essential component of any circRNA study (Das, et al., 2022).

58 Reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) are considered the gold
59 standard for identification of circRNA expression in cells (Das, et al., 2022). At the same time,
bioRxiv preprint doi: https://doi.org/10.1101/2022.12.20.521155; this version posted December 20, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

60 primer design for the circRNAs validation differs from the design for the their linear host genes
61 (Vromman, et al., 2022). To date, only a few tools have been published that allow the
62 development of primers for validation of circRNAs. At the same time, they require additional
63 software to be installed in different operating systems – CircPrimer (Zhong, et al., 2018),
64 CircPrimer2.0 (Zhong and Feng, 2022) and circtools (Jakobi, et al., 2019), or work as a web tool
65 with already known circRNAs of model organisms, namely human (Dudekula, et al., 2016) or
66 novel circRNAs for limited number of animal species (Vromman, et al., 2022). Here, to
67 overcome these previous constraints and facilitate the circRNA studies by presenting the user-
68 friendly CircPrime web platform (http://circprime.elgene.net/), which works with outputs of the
69 most popular bioinformatic predictors of circRNAs, such as CIRI2 (Gao, et al., 2018), KNIFE
70 (Szabo, et al., 2015), CIRCexplorer2 (Zhang, et al., 2016), find_circ (Memczak, et al., 2013),
71 circRNA_finder (Westholm, et al., 2014), DCC (Cheng, et al., 2016), mapsplice (Wang, et al.,
72 2010) and common BED files. Importantly, CircPrime is also suitable for non-model organisms
73 that have reference genome assemblies in the National Center for Biotechnology Information
74 database (NCBI).

75

76 Implementation

77 To date, there are several methods for PCR-based identification of different circRNAs types
78 (Figure 2A). One of them is rolling circle amplification (RCA). This method avoids deep RNA
79 sequencing and bioinformatic analysis, but is only capable of identifying a limited number of
80 circRNA types (Boss and Arenz, 2020). The other most commonly used method assumes a
81 longer workflow, which comprises circRNA enrichment, circRNA-library construction, deep
82 sequencing, circRNA prediction, and finally RT-PCR/qPCR validation of bioinformatically
83 predicted circRNAs (Shi, et al., 2022). PCR primers for this validation are designed to target the
84 circRNA fragment overlapping a junction (back-splice) site of a specific circRNA (Figure 2A).

85 We developed CircPrime, as a streamlined pipeline in Python 3 and web platform, which

86 makes use of output files from the most popular circRNAs in silico predictors. The CircPrime
87 script implemented into the web platform currently contains the four main modules shown in
88 Figure 2B and works under the parameters presented in Table 1.

89 After the first step, which includes BED file uploading, CircPrime generates FASTA files
90 using circRNA coordinates and reference genome from the NCBI. Then CircPrime extracts
91 junction regions from the uploaded BED file and develops primer sets with the recommended
92 melting temperature (Tm) for each circRNA in the list (up to 100) using Primer3 (Figure 2B)
bioRxiv preprint doi: https://doi.org/10.1101/2022.12.20.521155; this version posted December 20, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

93 (Koressaar and Remm, 2007; Untergasser, et al., 2012). An example of the CircPrime output is
94 presented as Supplementary Dataset 2.

95 Table 1. Required and default parameters for CircPrime usage.

CircPrime parameter Parameter description

CircRNA BED file Required
Shift range left Default 150 nucleotides
Shift range right Default 150 nucleotides
Optimum length of a primer Default 19 nucleotides
Minimum acceptable length of a primer Default 15 nucleotides
Maximum acceptable length of a primer Default 20 nucleotides
PCR product size Default 150-200 nucleotides
Optimum melting temperature for a primer in Celsius
Minimum acceptable melting temperature for a primer in Celsius
Maximum acceptable melting temperature for a primer in Celsius
Number of primer sets Default 4 sets
96

97 Results

98 The novel CircPrime web-based platform was evaluated to design primer sets for RT-PCR
99 validation of circRNA expression in the muscle transcriptome of a teleost, the Nile tilapia
100 (Oreochromis niloticus). Successfully, we showed that CircPrime significantly simplifies the
101 primer design process for bioinformatically predicted circRNAs without the need to upload a
102 reference genome of the organism studied.

103 In this study, we successfully applied circRNAs list predicted by CIRI2 (Gao, et al., 2018)
104 and CircPrime web platform to design circRNA primer pairs and validate their RT-PCR
105 efficiency using total RNA extract from the Nile tilapia skeletal muscle tissue (see details in
106 Supplementary Material). We expect that this bioinformatic tool will play a relevant role on
107 varied studies describing circRNAs expression and their possible functionality. CircPrime is
108 applicable both on model and non-model organisms, including even those with a poor genome
109 assembly and annotation.

110

111

112
bioRxiv preprint doi: https://doi.org/10.1101/2022.12.20.521155; this version posted December 20, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

113 Conclusions

114 Herein, we present a Circprime web platform (http://circprime.elgene.net/) for PCR primer
115 design and PCR conditions development for validation of circRNAs predicted based on RNA-
116 sequencing data using different types of bioinformatics tools. We expect that this web tool will
117 be convenient for users who intend to analyze the expression of circRNAs in animal and plant
118 transcriptomes.

119

120 Figure legends

121 Figure 1. Exponential increase in the number of publications mentioning “circRNA” in their title.
122 X-axis shows years from 2014 until 2021; Y-axis represents the number of publications. Source:
123 Web of Science, accessed 6 October 2022.

124

125 Figure 2. An overview of the CircPrime pipeline. (A) Types of possible variants for primer
126 design for circular RNA validation. (B) The main steps of CircPrime pipeline and tools
127 combined in it.

128

129 Availability and requirements

130 Project name: CircPrime

131 Project home page: http://circprime.elgene.net/

132 Operating system(s): Platform independent

133 Programming language: Python 3.10

134 Other requirements: None

135 License: GNU GPL Version 3

136

137 List of abbreviations

138 PCR: Polymerase chain reaction

139 CircRNA: Circular RNA

140 RT-PCR: Reverse transcription PCR

bioRxiv preprint doi: https://doi.org/10.1101/2022.12.20.521155; this version posted December 20, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

141 qPCR: Quantitative PCR

142 NCBI: National Center for Biotechnology Information database

143 RCA: Rolling circle amplification.

144

145 Declarations

146 Any restrictions to use by non-academics: no license needed

147 Ethics approval and consent to participate: This research was approved by the Nord University
148 (Bodø, Norway) ethical committee. The experimental procedures involving animals were
149 performed in accordance with the regulation and instructions of the Norwegian Animal Research
150 Authority (FOTS ID 1042). All procedures involving animals were conducted according to the
151 EU Directive 2010/63 on the use of animals for scientific purposes.

152 Consent for publication: Not applicable

153 Availability of data and materials: The user-friendly CircPrime tool for circular RNA primer
154 development is written in Python 3 and implemented on a web-based platform. It is freely
155 available online at http://circprime.elgene.net/. The RNA-seq dataset generated and analysed
156 during the current study is available in the GEO (NCBI) repository, under the accession number
157 PRJNA826285

158 Competing interests: The authors declare there are no competing interests.

159 Funding: This study has received funding from the European Research Council (ERC) under the
160 European Union’s Horizon 2020 research and innovation programme (grant agreement no
161 683210) and from the Research Council of Norway under the Toppforsk programme (grant
162 agreement no 250548/F20). Fedor Sharko was partly supported by the state task of the Federal
163 Research Center of Biotechnology RAS.

164 Authors' contributions: F.S. – wrote tool script and implemented it on a web-based platform,
165 wrote and approved the final draft; G.R. – performed the experiments, prepared figures and/or
166 tables, and approved the final draft; P.S. - performed the experiments, prepared figures and/or
167 tables, and approved the final draft; J.R. – conceived and designed the experiments and approved
168 the final draft; J.G. – conceived and designed the experiments and approved the final draft; A.N.
169 – conceived and designed the experiments, performed the experiments, analyzed the data,
170 prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final
bioRxiv preprint doi: https://doi.org/10.1101/2022.12.20.521155; this version posted December 20, 2022. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY-NC-ND 4.0 International license.

171 draft. J.M.O.F. – conceived and designed the experiments, authored or reviewed drafts of the
172 paper, approved the final draft, administrated project and acquisited funding.

173 Acknowledgements: Not applicable

174

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