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

• Published in: Journal of the American Chemical Society Vol. 127; no. 38; pp. 13293 - 13299
• Main Authors: Hrdlicka, Patrick J, Babu, B. Ravindra, Sørensen, Mads D, Harrit, Niels, Wengel, Jesper
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 28.09.2005; Amer Chemical Soc
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chemistry; Chemistry, Multidisciplinary; DNA - analysis; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Microscopy, Fluorescence - methods; Molecular Structure; Nucleic acids; Nucleic Acids - analysis; Oligonucleotide Probes - chemistry; Oligonucleotides; Oligonucleotides, Antisense - chemical synthesis; Oligonucleotides, Antisense - chemistry; Physical Sciences; Pyrenes - chemistry; RNA - analysis; Science & Technology; Temperature; POLYMERASE-CHAIN-REACTION; ELECTRON-TRANSFER; THIAZOLE ORANGE; DNA PROBES; FLUOROGENIC PROBES; LABELED OLIGONUCLEOTIDES; MOLECULAR BEACONS; QUENCHED PROBES; LIGHT-UP PROBES; MONOMER FLUORESCENCE; Biochemical analysis; Functionalization; Fluorescent labelling; Pyrene derivatives; Thermal denaturation; Qualitative analysis; Oligonucleotide; Oligodeoxyribonucleotide; Quantum yield; Nucleic acid; Fluorescence spectrometry
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja052887a

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 λ 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.