MicroRNA Sponge Production Using PCR-Based Concatemerization of Short DNA Oligonucleotides

Abstract

Micro RNAs (mi Rs) are small, non coding RNAs encoded within our genome that regulate gene expression by silencing messenger RNA (mRNA) transcripts. Since being discovered in humans in 2001, much has been learned concerning the many cellular activities that mi Rs can affect. Mi Rs have been implicated in almost every cellular activity, including cell fate determination, stress response, metabolism, apoptosis, and carcinogenesis. Importantly, alterations in mi R activity due to mutation or mis expression have been repeatedly shown to result in tumorigenesis and disease. However, because of their relatively recent discovery, therapeutic tools to suppress mi Rs continue to be developed. One extremely promising way to inhibit mi Rs is to use mi R sponges which consist of ~15 mi R “target” sites that can specifically bind and inactivate particular mi Rs. Unfortunately, mi R sponge production has proven to be problematic thus far as current production methods involve either costly commercial synthesis or low throughput ligation-based cloning. However, we have recently developed a novel, PCR-based method that allows sponge production in a rapid, high throughput manner. In all, PCR amplification, cloning, sequence confirmation and large scale production are readily achievable in less than two weeks and at a significantly lower cost than current methods.