Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

Abstract

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM): Caveats with respect to the use of “stem–loop” oligonucleotides as probes for mRNA folding

“…SQRMs were incubated with full-length c-kit mRNA and the results were compared to the previous experiment using the 1.2-kb c-kit RNA fragment as the target (Gifford et al 2005b). All SQRMs were incubated with c-kit RNA, an oligonucleotide (DNA) complementary to the SQRM (positive control) and a scrambled oligodeoxynucleotide (SCR; negative control).…”

“…Of note, all of the SQRMs chosen targeted sequence common to both RNA lengths. The first method, a computer program written in C++, AccessSearch, was used to search 1249 bases of the c-kit coding RNA sequence (bases À21 to 1228; c-kit 1.2 kb) for target sequences with 4 or 5 complementary bases separated by 10-23 bases; when the full complement is synthesized this would form the stem-loop structure of the Type I SQRM (Gifford et al 2005b). A total of 81 sequences were identified, and a set of 10 sites distributed along the 1.2-kb fragment were randomly chosen for mapping experiments (Table 1).…”

“…SQRMs were synthesized as described previously, using fluorescein and 39-C7-4-(49dimethylaminophenylazo)benzoic acid (DABCYL) as the fluorophore-quencher pair (Gifford et al 2005b). Each SQRM has five phosphorothioate nucleotides at each end flanking a central region containing phosphodiester nucleotides, as well as fluorescein and DABCYL moieties conjugated to the 59 and 39 ends, respectively.…”

“…Fluorescence was monitored after reactions were incubated for 30 min at 37°C in a Packard FluoroCount fluorometer using an excitation filter of 490 nm and an emission filter of 525 nm. The signal-tonoise ratios (SQRM incubated with target divided by the SQRM alone) were computed as described previously (Gifford et al 2005b), using the median of ratios method and implemented with the bootstrap algorithm (Brody et al 2002).…”

“…In the course of applying various gene silencing methodologies in living cells, it has become well known that not all sequences within an mRNA are equally accessible for hybridization. In hopes of making silencing more predictable we developed self-quenching reporter molecules (SQRM), oligodeoxynucleotides with fluorophore and quencher moieties at the 59 and 39 ends respectively, to help identify hybridization accessible sequence within a given mRNA (Gifford et al 2005b). As a result, we have hypothesized that these molecules might enable a rational approach to target site selection, and in so doing facilitate the design of more effective DNA, and RNA, antisense oligonucleotide probes and drugs (Gifford et al 2005a,b) The design of Type I SQRMs and their antecedants, the fluorescent reporter molecules known as molecular beacons (MB) (Tyagi and Kramer 1996), is based on the solution structures they are assumed to adopt in vivo.…”