Native mRNA antisense-accessible sites library for the selection of antisense oligonucleotides, PNAs, and siRNAs

Abstract

A procedure for rapidly generating a library of antisense-accessible sites on native mRNAs (mRNA antisense-accessible sites library [MASL]) is described that involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific polymerase chain reaction (PCR). Antisense phosphorothioate oligodeoxynucleotides (ODNs), peptide nucleic acids (PNAs), and small interfering RNAs (siRNAs) can then be identified by screening against the antisense-accessible sites. The utility of this methodology is demonstrated for the identification of more effective inhibitors of inducible nitric oxide synthase (iNOS) induction than have previously been reported. This method may also be useful for constraining folding calculations of native mRNAs and for designing mRNA imaging probes.

Dual Peptide Nucleic Acid- and Peptide-Functionalized Shell Cross-Linked Nanoparticles Designed to Target mRNA toward the Diagnosis and Treatment of Acute Lung Injury

“…These SCKs were subjected to reaction, coincidentally, with PNAs and TAT that were functionalized with maleimide, at pH 6.5 for 6 h at 0 °C. Two anti-iNOS PNAs, PNA240 and PNA480, and mismatch controls were selected for conjugation to the SCKs (Table 2), from a set of PNAs previously shown to have similar affinity for iNOS mRNA, but differing abilities to inhibit iNOS expression 67 . In each case, the unreacted thiols were capped using 4-maleimido butyric acid.…”

“…The ratio of bound to free ODN vs concentration was then fit to a standard equation (Equation 1), from which the dissociation constant (K d ) could be determined. As can be seen in Table 3, ODNs designed on the basis of binding sites identified by the RT-ROL mapping assay (iNOS240, iNOS480) 67 showed very high affinity with dissociation constants (K d ’s) of 7.3 ± 1.2 and 5.2 ± 2.5 nM (Table 3). …”

Imaging Genetic Information

“…This latter approach has been extended to determine the accessible sites on mRNAs produced by in vitro transcription [69] or isolated directly from cells ( Fig. 13.4) [71]. This mapping method uses a library of random 10-mer sequences fused to a PcR tag to prime reverse transcriptase from sites accessible to the primer.…”

“…The affinity of chemical analogs of the highest-affinity antisense ODNs can then be determined directly or through a competition assay. These techniques have been successfully applied for the identification of highaffinity sites for the unr mRNA that is overexpressed in mcF-7 cells [69] and iNOS mRNA that is overexpressed in the mouse macrophage cell line in an inflammatory response to gIFN and LPS [71]. The problem with using in vitro transcribed RNA is that it lacks the 5′-capping sequence and may not fold in the same way as in vivo and would certainly lack any bound proteins that may alter the conformation of the mRNA or block otherwise accessible sites.…”

“…The strand displacement probe was designed to target iNOS mRNA, which is upregulated over 100-fold in response to LPS/γ-IFN. The antisense binding site on the iNOS mRNA had been determined through the use of an RT-ROL assay on endogenous mRNA recovered from whole cell extracts of mouse macrophage cell line [71]. The strand displacement probe was constructed from a neutral, high-affinity, degradation-resistant PNA strand bearing a fluorescein fluorophore to a five-nucleotide shorter negatively charged partially complementary DNA strand bearing a DABcYL fluorescence quencher.…”