Multilabeled Pyrene-Functionalized 2‘-Amino-LNA Probes for Nucleic Acid Detection in Homogeneous Fluorescence Assays

Abstract

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.

Locked Nucleic Acids: Properties, Applications, and Perspectives

“…Moreover, amino-LNA may serve as an excellent branching point for decorating the LNA sequences with various entities, and several relevant studies have already been conducted [112,113]. Elsewhere in the field of nanotechnology, LNA sequences have been used to block a polymerase extension reaction in the development of a universal surface DNA computer [114].…”

Nucleic Acid Fluorescent Probes for Biological Sensing

“…110 Due to the change of arrangement state of the labeled pyrene molecule upon hybridization, the probe generates bright fluorescence. 111 This probe is demonstrated to be applicable for in vitro transcription assays and direct microscopic observation of probes bound to mRNA in its native form in living cells.…”

Nucleic Acid Mimetics

“…Stacking of the fluorophores via duplex formation between complementary strands results in the enhancement of excimer emission at a wavelength of 500 nm. Increase in emission is an indicator of successful duplex formation, and incorporation of H ligands into the GNA strands can be used as a Cu(II) "turnon" sensor as shown in Figure 20 84 On hybridization and intercalation of pyrene into the minor groove of the double helix, a large increase in the thermal stability of the duplexes was observed, together with an increase in the emission intensity up to 69-fold for two monomers separated on a strand as shown in Figure 22. Emission of the pyrene was virtually sequence independent; however, the position of the fluorophore impacted the emission intensity.…”