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/.
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