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Biomedical Optics & Medical Imaging

Oligonucleotide molecular beacons for intracellular diagnosis and therapy

An oligonucleotide sequence conjugated to a fluorophore–quencher pair, and then loaded in polymethyl methacrylate nanoparticles, can be used as a theranostic agent.
24 February 2017, SPIE Newsroom. DOI: 10.1117/2.1201611.006665

The protein survivin—a member of the inhibitor of apoptosis protein family—plays a key role in the regulation of the cell cycle, apoptosis (programmed cell death), and cell migration.1 This protein is also expressed at very high levels in most tumor types.1 The detection of survivin is therefore of vital importance in cancer diagnosis, and there is the potential to achieve this with the use of ‘theranostic’ agents. Such agents have both therapeutic and diagnostic properties and are expected to play a significant role in future personalized medicine.2

Oligonucleotide optical switches are suitable for theranostic applications because they can bind to specific nucleic acid sequences and can generate/modify optical emissions when they interact with these sequences.3 For example, oligonucleotide molecular beacons (MBs)—oligonucleotide sequences labeled with a fluorophore at one end and a quencher (characterized by an absorption band that overlaps with the emission band of the fluorophore) at the other end—are used widely as optical switches4 (particularly in intracellular sensing). This is because they produce a fluorescence signal instantaneously upon hybridization with a complementary target.5

The MB structure (see Figure 1) is designed to adopt a hairpin configuration (stem–loop) when the complementary sequence is not present. In this situation, the fluorescence of the fluorophore is inhibited because it is located close to the quencher. If the fluorophore is excited in this hairpin configuration, it does not emit fluorescence. Rather, energy is transferred to the quencher along a non-radiative pathway because the requirement (i.e., a distance of a few angstroms between the fluorophore and quencher) for this transfer is satisfied. When hybridization occurs with a complementary sequence, the stem–loop structure opens up and the fluorophore and quencher become sufficiently distant that non-radiative energy transfer does not occur and the fluorophore emits when excited.


Figure 1. Illustration of the structure and operation of an oligonucleotide molecular beacon (MB). The MB switches from a hairpin (‘off’) configuration to an open (‘on’) configuration when it hybridizes with a target sequence. This causes the generation of an optical signal because the fluorophore (right, in yellow) is sufficiently distant from the quencher (left, in gray). RNA: Ribonucleic acid.

In our work, we have thus investigated a new type of oligonucleotide MB for use as an optical switch. In particular, to bind to, detect, and inhibit the messenger ribonucleic acid (mRNA) sequence that encodes survivin, an appropriate combination of oligonucleotide, fluorophore, and quencher is required. Indeed, we selected the oligonucleotide 5'-CGACGGAGAAAGGGCTGCCACGTCG-3' from among several previously reported sequences,6 because the underlined segment constitutes the specific sequence needed to bind to survivin mRNA. Classical fluorophores (such as fluorescein and cyanine-based dyes), however, are unstable under prolonged exposure to light, which can impair their performance over time. We therefore used the nitrogen-containing heterocyclic fluorescent dye Atto 647N (which absorbs at 644nm and emits at 669nm) as the fluorophore for labeling the 5' end of our MB, because it has a more rigid structure and is more stable than classical fluorophores. Atto 647N thus enables the generation of a more reliable signal. Crucially for intracellular applications, the excitation and emission wavelengths of Atto 647N and the time taken for its emission signal to decay are relatively insensitive to pH, temperature, and atmospheric humidity. We also selected BlackBerry Quencher 650 (BBQ650)—which is non-fluorescent and absorbs between 550 and 750nm, with a maximum around 650nm—as the corresponding quencher for labeling the 3' end of the oligonucleotide. Furthermore, we used a target DNA sequence—complementary to that in the MB (5'-CCCCTGCCTGGCAGCCCTTTCTCAAGGACC-3')—and a random DNA sequence (5'-ATCGGTGCGCTTGTCG-3') in buffer solutions for our tests.

We find that the fluorescence spectrum of the MB (at a concentration of 1μM)—Figure 2(a)—increased in intensity upon interaction with the target sequence at increasing concentrations (between 0.1 and 1nM). However, we observed no enhancement in the signal after interaction with the random sequence at a concentration of 1nM. We also show—Figure 2(b)—the calibration curve that we obtained after the hybridization reaction between the MB (at 1μM) and its target sequence at increasing concentrations (0.1–1000nM). These results demonstrate that our MB can be used to detect endogenous nucleic acids and it could therefore be employed as a diagnostic agent. We have also conducted an in vitro study from which we showed that our oligonucleotide—when introduced (with the use of lipofectamine) into A375 human cutaneous melanoma cells—was selective for its mRNA target and acted as a pro-apoptosis agent.7 Our MB thus has the potential to act as a theranostic agent, i.e., because it can serve as a sensing probe and be used to silence its target mRNA.


Figure 2. (a) Fluorescence spectra of the MB (at a concentration of 1μM) after interaction with various concentrations of its target (complementary DNA sequence) and a random DNA sequence. The spectra recorded in the presence of the random DNA sequence and in the absence of the target (buffer) effectively coincide. a.u.: Arbitrary units. (b) Calibration curve obtained after the reaction between the MB (1μM) and increasing concentrations (conc) of its target (0.1–1000nM).

Single-stranded DNA such as our MB, however, cannot be taken up effectively by cells without the use of a carrier or a specific transfection agent.8 We therefore selected polymethyl methacrylate nanoparticles (PMMA NPs) to enable the cellular uptake of our MB because of their high biocompatibility (they have previously been shown as highly biocompatible, even when used in vivo, because they are nontoxic and are excreted in feces at levels of up to 80 or 100%9) and to avoid possible enzymatic degradation of the MB.10, 11 We used quaternary ammonium salts to functionalize the outer shells of the PMMA NPs, which provided a positively charged surface and thereby promoted the uptake of the negatively charged MB. We also labeled the core of the NPs with fluorescein (excitation wavelength 488nm, emission wavelength 525nm) to allow us to track their cellular uptake. Confocal microscopy images of A549 human lung adenocarcinoma epithelial cells that we obtained (Figure 3) show the successful cellular uptake of the MB (loaded onto PMMA NPs) after incubation for 90 minutes (in agreement with literature results8). Moreover, when we introduced our MB into healthy cells (adult human dermal fibroblasts) that had low levels of survivin mRNA, an insignificant fluorescence signal was generated.12 This indicates that the in vitro interaction between our MB and mRNA is highly specific.


Figure 3. Confocal microscopy images of A549 human lung adenocarcinoma epithelial cells obtained at a magnification of ×40. These images illustrate the cellular uptake of the MB—loaded onto polymethyl methacrylate nanoparticles (PMMA NPs)—after incubation for 90 minutes. (a) Fluorescence emitted by the PMMA NPs at an excitation wavelength of 488nm. (b) Fluorescence emitted by the MB after interacting with the survivin messenger RNA target sequence, at an excitation wavelength of 635nm. (c) Merged image of (a) and (b).

In summary, we have demonstrated that our oligonucleotide MB can be used simultaneously as an optical switch, a sensing probe, and a drug (by silencing its target mRNA sequence). Our MB can therefore potentially be used as a theranostic agent. Moreover, we have demonstrated that the use of PMMA NPs (as carriers to deliver the MB into cells) may also lead to a decrease in the enzymatic degradation of the MB in vivo. In future work, we intend to study if our MB can be used for the quantitative determination of targets in vivo. In addition, we will investigate functionalization of PMMA NPs with agents that enable targeting of specific cells and more-selective delivery of the MB. This would allow the therapeutic concentration of the MB to be reduced, which—together with the use of NPs—would consequently also reduce its systemic toxicity.

This research study was conducted in collaboration with Pisa University's Department of Translational Research and of New Surgical and Medical Technologies, and Department of Pharmacy, as well as the Institute for Organic Synthesis and Photoreactivity, CNR (all Italy).


Ambra Giannetti, Barbara Adinolfi, Sara Tombelli, Cosimo Trono
Institute of Applied Physics, CNR
Sesto Fiorentino, Italy

Ambra Giannetti's research focuses on the treatment of nanoparticles for the delivery of molecular switches into cells as theranostic agents. She also works on the study and development of optical biosensors and biosurfaces, including the treatment of protein and DNA for the preparation of active surfaces.


References:
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