Synthesis and Characterization of a Magnetically Active 19F Molecular Beacon

Abstract

Gene expression is used extensively to describe cellular characteristics and behaviors; however, most methods of assessing gene expression are unsuitable for living samples, requiring destructive processes such as fixation or lysis. Recently, molecular beacons have become a viable tool for live-cell imaging of mRNA molecules in situ. Historically, beacon-mediated imaging has been limited to fluorescence-based approaches. We propose the design and synthesis of a novel molecular beacon for magnetic resonance detection of any desired target nucleotide sequence. The biologically compatible synthesis incorporates commonly used bioconjugation reactions in aqueous conditions and is accessible for laboratories without extensive synthesis capabilities. The resulting beacon uses fluorine (¹⁹F) as a reporter, which is broadened, or turned "off", via paramagnetic relaxation enhancement from a stabilized nitroxide radical spin label when the beacon is not bound to its nucleic acid target. Therefore, the ¹⁹F NMR signal of the beacon is quenched in its hairpin conformation when the spin label and the ¹⁹F substituent are held in proximity, but the signal is recovered upon beacon hybridization to its specific complementary nucleotide sequence by physical separation of the radical from the ¹⁹F reporter. This study establishes a path for magnetic resonance-based assessment of specific mRNA expression, providing new possibilities for applying molecular beacon technology in living systems.

Figure 1

ESI-MS analysis of oligonucleotides at each step of the reaction (products 1–3). Predicted masses were verified for the starting oligonucleotide (A, MW: 9824 Da), the oligonucleotide with reduced thiol group (1) (B, MW: 9734 Da), the oligonucleotide modified with TFB (2) (C, MW: 9988 Da), and the fully modified oligonucleotide (3) (D, MW: 10,154 Da).

Figure 2

¹⁹F NMR spectra of TFB-only oligonucleotide (2) (green, top) in the presence of unconjugated nitroxide radical (red, middle) compared to conjugated nitroxide radical which forms the full beacon (blue, bottom). The ¹⁹F resonance is unaffected by radicals in solution but is broadened in a distance-dependent manner once the radical is bound to the other end of the beacon stem. Exponential line broadening of 3 Hz applied.

Figure 3

¹⁹F NMR spectra of 13 μM fully modified beacon (3) alone (top, green), in the presence of 150 μM mismatched target (middle, red), and the addition of 150 μM matched target (bottom, blue). Spectra remained unchanged upon the addition of mismatched target, but matched target DNA hybridized to the beacon disrupting paramagnetic broadening of fluorine resonance by physical separation of the ¹⁹F from the spin label. Exponential line broadening of 3 Hz applied. Data is representative of behavior observed in 3 additional replicate experiments.

Figure 4

NMR spectra of ¹⁹F molecular beacon (3) in the presence of excess matched target at full beacon concentrations of 4, 5, 7, 10, and 13 μM (A). Signal-to-noise ratio was linearly correlated with hybridized beacon concentration, allowing for prediction of the lower limit of detection for the beacon when signal is twice as high as noise under these same processing conditions if all of the available beacon is bound (B). Signal-tonoise ratio was also assessed for a different batch of ¹⁹F molecular beacon (3) at 12 μM with increasing concentrations of matched target added (C). Data points above the red line correspond to the observance of a discernible signal while those concentrations below a signal-to-noise ratio of 2 were undetectable. Exponential line broadening of 3 Hz applied to each spectrum. Signal-to-noise ratios were determined using Bruker TopSpin NMR

Scheme 1

Conjugation of Trifluorobromoacetyl and Nitroxide Radical to Oligonucleotide