SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

<div><p>Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine <i>Hras</i> mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes <i>in vivo</i> molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.</p></div

Point mutation detection by Hras allele specific FRET probes in NAR, 308 and MEF cells.

<p>A - anchor<b>/</b>A,T <b>-</b> allele specific probe<b>/</b>A,U synthetic target oligo or no (O) target. After oligos transfection, cells were suspected to Acceptor Photobleach (AP) analysis. NAR cells containing Anchor and wt (A) probe showed relatively high FRET with full matched A synthetic target (blue left bar). A single mismatch in target sequence showed no FRET (U, red bar). When no target was present in the cells (NAR, green bar), no FRET was observed, indicating no FRET background. WT specific (A) probe induced a positive FRET response in both 308 and MEF cells, indicating presence of the wt allele (308– purple bar, MEF - orange bar). Mutant T probe exhibited positive FRET response only in 308 mutated cells (turquoise) as opposed to wt MEF cells (light blue).</p

Single cell Acceptor photobleach analysis.

<p>The Anchor (FRET donor, AF488) and mutant Probe (FRET acceptor, TR) were co-transfected to the same cell and fluorescence was measured before (upper panel) and after (lower panel) red photobleaching. As a result of photobleaching, the acceptors red signal decreases. When energy transfer occurred, the donor green emission increased after the acceptors photobleach. <b>A.</b> In 308 cells, which endogenously carry the Hras point mutation, an increase in green donors signal was observed after acceptor photobleach whereas in <b>B.</b> Hras wt MEF cells, no increase in donors signal was observed.</p

Hras oligo probes sequences and modifications.

<p>Key: <b>A,C,U/T,G –</b>2′OMe RNA<b>, a,c,u,g –</b> RNA<b>, <u>A,C,T,G</u></b> - LNA residues<b>, Bold font -</b> SNP site<b>, TR –</b> Texas Red<b>, AF –</b> Alexa Fluor<b>, “*” -</b> phosphorothioate <b>(PS)</b> internucleotide linkage<b>, PSEnd-</b> incidates phosphorothioate bonds only on terminal linakges of the molecule<b>, targ-</b> target sequence.</p

Influence of chemical modifications on target specificity and FRET intensity.

<p>Three differentially modified FRET couples were incubated with: no target (left bar), mismatch target (middle bar) or full match target (right bar) and FRET was measured. Targ – target, Anc - Anchor Alexa Fluor 488 (AF488), PS- ODNs heavily modified with PS bonds, PSEnd- PS modifiIcation only at the edge of the ODN, pT- mutant specific probe T marked with either Texas Red (TR) or Alexa Fluor 594 (<b>AF594</b>) as acceptor fluorophore.</p

Point mutation in Hras gene in DMBA/TPA treated cells.

<p>(<b>A</b>) The A>T substitution causes the appearance of XbaI consensus site (<b>B</b>) XbaI digestion of Hras in MEF (left lane) and 308 cells (right lane). Two restriction fragments of 300 and 400 bp emerge in mutated Hras resulting from cleavage of full length 700 bp replicon in the newly formed XbaI restriction site. (<b>C</b>) Sequencing of Hras codon 61 reveals A>T transversion in of 308 cell line which is derived from early DMBA/TPA induced papillomas <b>(ii).</b> Mouse embryonic fibroblasts (MEFs) hold the typical A allele <b>(i)</b>.</p

FRET probes are SNP specific <i>in vitro</i> at 37°C.

<p>Multicomponent plots indicating fluorescent signals from the anchor (blue line) and the allele specific probe (red line) as a course of temperature increase (37–75°C). <b>A</b> – Anchor/<b>A, T</b> allele specific probe or O – no probe/<b>A, U</b> – target RNA oligo complement to A and T probes respectively. Perfect match between the probe and target results in an energy transfer between the donor anchor and acceptor probe. When both the donor anchor and acceptor probe are bound to target at 37°C, their proximity causes energy transfer resulting in red fluorescence. As a course of temperature increase, the oligos denaturate from target (51°C), energy transfer ceases and only the donor fluorescence can be detected, causing a typical “8” shaped FRET plot to be generated (<b>A, B</b>). A single mismatch in target sequence results in low affinity of the oligos therefore no FRET response is detected (<b>C, D</b>). No target control (<b>E</b>), no donor control (<b>F</b>), no acceptor control (<b>G</b>).</p

Secondary structure prediction of Hras target sequences.

<p>Predicted folding state of Hras <i>synthetic targets</i> are shown in structures (<b>A–C</b>)<b>.</b> Green underline - anchor binding site. Red underline - Probe binding site. Yellow frame - codon 61. (<b>A</b>) and (<b>B</b>) represent the wt A allele whereas C represents the U mutant allele. The 40 bp synthetic target sequence is predicted to create a hairpin structure under physiological conditions. Structure (<b>D</b>) represents the energetically favored structure of <i>endogenous</i> Hras mRNA. According to this structure, the mutated codon is in a hairpin structure.</p

Chemical modifications on the nucleic acid backbone increase their nuclease stability.

<p>(<b>A</b>) PS bonds include replacement of non-bridging oxygen with sulfur. (<b>B</b>) 2′OMe are modification of the 2′-position of the ribose with a methyl group. (<b>C</b>) LNA bases contain a methylene bridge between the 2′-<i>O</i> with the 4′-C of the ribose and “locks” the sugar conformation.</p

A FRET pair contains two fluorophores with overlapping spectrum.

<p>When the donor is excited, FRET enables the fluorescence emission of the acceptor. It should be noted that the background noise arising from the leakage between the donor and acceptor channels need to be deducted.</p