RESUMO
The design of new RNA sequences that retain the function of a model RNA structure is a challenge in bioinformatics because of the structural complexity of these molecules. RNA can fold into its secondary and tertiary structures by forming stem-loops and pseudoknots. A pseudoknot is a set of base pairs between a region within a stem-loop and nucleotides outside of this stem-loop; this motif is very important for numerous functional structures. It is important for any computational design algorithm to take into account these interactions to give a reliable result for any structures that include pseudoknots. In our study, we experimentally validated synthetic ribozymes designed by Enzymer, which implements algorithms allowing for the design of pseudoknots. Enzymer is a program that uses an inverse folding approach to design pseudoknotted RNAs; we used it in this study to design two types of ribozymes. The ribozymes tested were the hammerhead and the glmS, which have a self-cleaving activity that allows them to liberate the new RNA genome copy during rolling-circle replication or to control the expression of the downstream genes, respectively. We demonstrated the efficiency of Enzymer by showing that the pseudoknotted hammerhead and glmS ribozymes sequences it designed were extensively modified compared to wild-type sequences and were still active.
Assuntos
RNA Catalítico , RNA Catalítico/química , RNA/genética , RNA/química , Pareamento de Bases , Algoritmos , Nucleotídeos , Conformação de Ácido NucleicoRESUMO
Pseudoknots are important motifs for stabilizing the structure of functional RNAs. As an example, pseudoknotted hammerhead ribozymes are highly active compared to minimal ribozymes. The design of new RNA sequences that retain the function of a model RNA structure includes taking in account pseudoknots presence in the structure, which is usually a challenge for bioinformatics tools. Our method includes using "Enzymer," a software for designing RNA sequences with desired secondary structures that may include pseudoknots. Enzymer implements an efficient stochastic search and optimization algorithm to sample RNA sequences from low ensemble defect mutational landscape of an initial design template to generate an RNA sequence that is predicted to fold into the desired target structure.
Assuntos
Biologia Computacional/métodos , Desenho Assistido por Computador , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/genética , Biologia Sintética/métodos , Algoritmos , Sequência de Bases , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Técnicas In Vitro , Cinética , Motivos de Nucleotídeos/genética , Reação em Cadeia da Polimerase/métodos , RNA/genética , Dobramento de RNA/genética , RNA Catalítico/metabolismo , Software , Transcrição GênicaRESUMO
We present a new publicly accessible web-service, RiboSoft, which implements a comprehensive hammerhead ribozyme design procedure. It accepts as input a target sequence (and some design parameters) then generates a set of ranked hammerhead ribozymes, which target the input sequence. This paper describes the implemented procedure, which takes into consideration multiple objectives leading to a multi-objective ranking of the computer-generated ribozymes. Many ribozymes were assayed and validated, including four ribozymes targeting the transcript of a disease-causing gene (a mutant version of PABPN1). These four ribozymes were successfully tested in vitro and in vivo, for their ability to cleave the targeted transcript. The wet-lab positive results of the test are presented here demonstrating the real-world potential of both hammerhead ribozymes and RiboSoft. RiboSoft is freely available at the website http://ribosoft.fungalgenomics.ca/ribosoft/.
