SSD - a free software for designing multimeric mono-, bi- and trivalent shRNAs

RNA interference (RNAi) is a powerful gene silencing technology, widely used in analyses of reverse genetics, development of therapeutic strategies and generation of biotechnological products. Here we present a free software tool for the rational design of RNAi effectors, named siRNA and shRNA designer (SSD). SSD incorporates our previously developed software Strand Analysis to construct template DNAs amenable for the large scale production of mono-, bi- and trivalent multimeric shRNAs, via in vitro rolling circle transcription. We tested SSD by creating a trivalent multimeric shRNA against the vitellogenin gene of Apis mellifera. RT-qPCR analysis revealed that our molecule promoted a decrease in more than 50% of the target mRNA, in a dose-dependent manner, when compared to the control group. Thus, SSD software allows the easy design of multimeric shRNAs, for single or multiple simultaneous knockdowns, which is especially interesting for studies involving large amounts of double-stranded molecules.

Keywords:
Gene silencing; siRNA; multimeric; shRNA; free software

Authorship

Gabriel José de Carli

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.Universidade de São PauloBrazilRibeirão Preto, SP, BrazilUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil.

Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil.Universidade de São PauloBrazilRibeirão Preto, SP, BrazilUniversidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Biologia, Ribeirão Preto, SP, Brazil.

http://orcid.org/0000-0003-3689-4512

Abdon Troche Rotela

Universidad Nacional de Asunción, Facultad de Ciencias Exactas y Naturales, San Lorenzo, Paraguay.Universidad Nacional de AsunciónParaguaySan Lorenzo, ParaguayUniversidad Nacional de Asunción, Facultad de Ciencias Exactas y Naturales, San Lorenzo, Paraguay.

Universidad Nacional de Asunción, Facultad Politécnica, San Lorenzo, Paraguay.Universidad Nacional de AsunciónParaguaySan Lorenzo, ParaguayUniversidad Nacional de Asunción, Facultad Politécnica, San Lorenzo, Paraguay.

http://orcid.org/0000-0001-7390-1780

Greice Lubini

http://orcid.org/0000-0001-6640-3724

Danyel Fernandes Contiliani

http://orcid.org/0000-0002-9225-3384

Nidia Benítez Candia

http://orcid.org/0000-0003-3677-9960

Thiago S. Depintor

http://orcid.org/0000-0003-2610-867X

Fabiano Carlos Pinto de Abreu

http://orcid.org/0000-0003-0112-2520

Zilá Luz Paulino Simões

http://orcid.org/0000-0003-2363-6178

Danilo Fernández Ríos

http://orcid.org/0000-0002-8394-532X

Tiago Campos Pereira

http://orcid.org/0000-0003-2641-7966

Send correspondence to Tiago Campos Pereira. Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil. Laboratório de Genética Molecular da Anidrobiose, Sala 13, Bloco 14 - Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto FFCLRP, Universidade de São Paulo – USP, Av. Bandeirantes, 3900.Bairro Monte Alegre. Ribeirão Preto, SP, Brazil.

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Figures

Figures (3)

Thumbnail

Figure 1
si- and shRNA (SSD) flowchart. SSD software is suitable for designing both siRNAs and multimeric shRNAs. For siRNA design, a target mRNA sequence must be used as ‘input’, while for mshRNA design, either the target mRNA or the previously designed siRNAs sequences can be used as ‘input’. siRNAs may be selected either by ‘position’ within the target mRNA or by `Gibbs free energy’ (i.e., silencing efficiency).

Thumbnail

Figure 2
Predicted secondary structures of DNA templates for multimeric shRNAs, designed by SSD against the Apis mellifera vitellogenin gene. Circularized templates for mono- (a), bi- (b) and trivalent mshRNAs (c). Each base paired region (*) corresponds to a different shRNA.

Thumbnail

Figure 3
Gene silencing mediated by trivalent multimeric shRNA designed by SSD. GFP group was injected with 500 ng of mshRNAs against GFP gene (negative control); Vg group was injected with 500 ng of mshRNAs against Apis mellifera vitellogenin. All experiments were performed in 6-8 biological replicates, each one composed of technical triplicates. Statistical difference was evaluated by ANOVA, with Student T test as post-hoc (* means p < 0.05).

Figure 1 si- and shRNA (SSD) flowchart. SSD software is suitable for designing both siRNAs and multimeric shRNAs. For siRNA design, a target mRNA sequence must be used as ‘input’, while for mshRNA design, either the target mRNA or the previously designed siRNAs sequences can be used as ‘input’. siRNAs may be selected either by ‘position’ within the target mRNA or by `Gibbs free energy’ (i.e., silencing efficiency).

Figure 2 Predicted secondary structures of DNA templates for multimeric shRNAs, designed by SSD against the Apis mellifera vitellogenin gene. Circularized templates for mono- (a), bi- (b) and trivalent mshRNAs (c). Each base paired region (*) corresponds to a different shRNA.

Figure 3 Gene silencing mediated by trivalent multimeric shRNA designed by SSD. GFP group was injected with 500 ng of mshRNAs against GFP gene (negative control); Vg group was injected with 500 ng of mshRNAs against Apis mellifera vitellogenin. All experiments were performed in 6-8 biological replicates, each one composed of technical triplicates. Statistical difference was evaluated by ANOVA, with Student T test as post-hoc (* means p < 0.05).

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SSD - a free software for designing multimeric mono-, bi- and trivalent shRNAs

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[1] Send correspondence to Tiago Campos Pereira. Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Genética, Ribeirão Preto, SP, Brazil. Laboratório de Genética Molecular da Anidrobiose, Sala 13, Bloco 14 - Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto FFCLRP, Universidade de São Paulo – USP, Av. Bandeirantes, 3900.Bairro Monte Alegre. Ribeirão Preto, SP, Brazil.