We postulate that inability to predict mRNA folding seriously impairs the design of antisense nucleic acids (ASNA) capable of efficienty squelching gene expression. A rational method for identifying mRNA sequence to which ASNA could hybridize might help solve this problem. We therefore developed an mRNA mapping strategy employing stem loop structures known as molecular beacons (MB). MB consist of fluorophore/quencher pairs at the 5' and 3' ends of self-complementary bases flanking an intervening sequence (IVS) antisense to a specific mRNA. When hybridized to a target, the MB can emit a fluorescent signal thereby reporting the presence of accessible sequence. To target MB, we developed a computer algorithm designed to locate palindromic sequences 5 bases in length separated by intervening sequence (IVS) of -18 bases. The Myb mRNA sequence was scanned with this program and several sites to which palindromic MB could be targeted were identified. When five of these MBs were hybridized to full length, in vitro transcribed Myb RNA, one gave -80% fluorescence, where 100% was defined as the signal obtained from a DNasel digested MB. Other sequences to which MB were directed gave -15-30% maximal fluorescence, while control MB gave 10% fluorescence. AS ON were then synthesized corresponding to accessible, and non-accessible regions of the Myb mRNA as predicted by the MB guided mapping. When transfected into a hamster fibroblast cell line (TK-tsl 13) engineered to express c-myb, AS ON targeted to maximally accessible sequence diminished Myb protein expression, as measured by Western blotting, by -70%, while ODN targeted to inaccessible regions diminished expression by -20%. As an alternative strategy, we also developed an algorithm to calculate the free energy of binding for the arms of DNAzymes complementary to the c-myb transcript. An MB was developed from the sequence of one of these DNAzymes that exhibited 70% of maximum fluorescence when incubated with Myb mRNA in vitro. This DNAzyme cleaved synthetic targets corresponding to those found in the c-myb gene with high efficiency. These results suggest that mRNA folding is a critical parameter to consider when designing ASNA. We conclude that MB guided mapping, in combination with relevant thermodynamic considerations, can give highly useful information for development of more efficient antisense drugs.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology