Recyclable single-stranded DNA template for synthesis of siRNAs.
RNA interference is a post-transcriptional silencing mechanism triggered by the bioavailability and/or exogenous introduction of double-stranded RNA (dsRNA) into cells. Here we describe a novel method for the synthesis of siRNA in a single vessel. The method employs in vitro transcription and a single-stranded DNA (ssDNA) template and design, which incorporates upon self-annealing, two promoters, two templates, and three loop regions. Using this method of synthesis we generated efficacious siRNAs designed to silence both exogenous and endogenous genes in mammalian cells. Due to its unique design the single-stranded template is easily amenable to adaptation for attachment to surface platforms for synthesis of siRNAs. A siRNA synthesis platform was generated using a 3' end-biotinylated ssDNA template tethered to a streptavidin coated surface that generates stable siRNAs under multiple cycles of production. Together these data demonstrate a unique and robust method for scalable siRNA synthesis with potential application in RNAi-based array systems.
Departments of Urology, Tulane University Health Sciences Center, New Orleans, Louisiana, USA.
Mussa M
MM
Ali
Demian
D
Obregon
Krishna C
KC
Agrawal
Mahmoud
M
Mansour
Asim B
AB
Abdel-Mageed
D001483:Base Sequence; D045744:Cell Line, Tumor; D004277:DNA, Single-Stranded / Q000737:chemistry*; D020868:Gene Silencing; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
D004277:DNA, Single-Stranded; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034741:RNA, Small Interfering