Molecular beacons of xeno-nucleic acid for detecting nucleic acid

Abstract

Molecular beacons (MBs) of DNA and RNA have aroused increasing interest because they allow a continuous readout, excellent spatial and temporal resolution to observe in real time. This kind of dual-labeled oligonucleotide probes can differentiate between bound and unbound DNA/RNA in homogenous hybridization with a high signal-to-background ratio in living cells. This review briefly summarizes the different unnatural sugar backbones of oligonucleotides combined with fluorophores that have been employed to sense DNA/RNA. With different probes, we epitomize the fundamental understanding of driving forces and these recognition processes. Moreover, we will introduce a few novel and attractive emerging applications and discuss their advantages and disadvantages. We also highlight several perspective probes in the application of cancer therapeutics.

Keywords: Diagnostics.; Molecular beacon; Nucleic acid; Xeno-nucleic acid.

Figure 1

a) Representation of an ideal-DNA conformation with the main structural dimensions. b) DNA base-pairs indicating the pattern of hydrogen bonds. The minor groove is the side of the base pairs facing towards the sugar-phosphate backbone, and the major groove is the side facing away. (Reprinted with permission from ref. 27. Copyright 2011, Wiley-VCH Verlag GmbH & Co. KGaA.)

Figure 2

The mechanism of MBs in sensing a DNA/RNA sequence (probes refer to immobilized sequences; DNA/RNA targets refer to sequences in the sample being captured). a) The intercalated dye of probe can fold back and intercalate between the formed Watson-Crick base pairs or serves as a base surrogate that is forced to intercalate adjacent to the expected mutation site. b) Upon hybridization with complementary sequence, the stem-loop hairpin structure of MB opens, which separates the reporter dye and the quencher and results in increased fluorescence intensity; c) In the single-stranded state the donor and the acceptor are separated from each other. When the probe encounters a target RNA, the MB undergoes a spontaneous conformational reorganization that forces the stem together, leading to a fluorescence resonance energy transfer (FRET) signal change.

Figure 3

Schematic chemical model of PNA (blue) hybridized with DNA (red), with the hydrogen bonding between complementary nucleobases depicted by dotted lines (A= adenine, G= guanine, C= cytosine and T= thymine). (Reprinted with permission from ref. 38. Copyright 2006, Wiley-VCH Verlag GmbH & Co. KGaA.)

Figure 4

Schematic representation of four different complexes formed by PNA binding to duplex DNA. a) Conventional triplex. b) Triplex invasion complexes are formed at homopurine DNA targets with complementary homopyrimidine PNAs. c) Duplex invasion complexes are formed with some homopurine PNAs. d) Double duplex invasion complexes. (Reprinted with permission from ref. 47. Copyright 2001, Elsevier B. V.)

Figure 5

The base sequence and chemical structure of the PNA-DNA adduct. The DNA part of the adduct has three functional moieties: (1) a 3'-terminal biotin which serves to immobilize the entire structure by binding to streptavidin-coated mitrotitre wells; (2) a quencher moiety (DABCYL) at the 3'-terminus of the DNA stem structure; (3) a sequence which pairs with 10 bases in the PNA part of the adduct, forming the 10-base pair stem structure. The PNA part of the adduct, which is joined to the DNA part via a disulfide bond, also comprises three functional moieties: (1) a 15-base probe sequence in the unstructured loop domain; (2) 10 bases capable of pairing with the DNA part, where one base in the PNA probe performs double duty, also being part of the 10-base stem structure; (3) a fluorescent moiety (AMCA), coupled to the free terminus of the PNA. (Reprinted with permission from ref. 68. Copyright 1998, Elsevier B. V.)

Figure 6

FIT probes with TO bind to Aeg and D-Orn backbones that are employed in PNA. In both molecules, the chromophores are aligned in a comparable six-atom distance from adjacent base pairs. B= adenine, thymine, guanine, cytosine; R, Ac, Ac-Lys-Lys; R', Gly-NH₂. (Reprinted with permission from ref. 70. Copyright 2008, Elsevier B. V.)

Figure 7

a) Structure of the PNA probe (B denotes nucleobases). The asymmetric cyanine dye thiazole orange (TO) is conjugated to a PNA with the sequence lys⁺-CCTTTTTCTT. b) Chemical structure of the thiazole orange derivative (TO-N'-10-COOH). c) Chemical structure of Aeg(TO) employed in PNA. (Reprinted with permission from ref. 71, 72. Copyright 2000, 2000. Elsevier B. V., Elsevier B. V.)

Figure 8

Near-field fluorescence images for the single target DNA fluorescence in situ hybridization samples by fluorescence resolution of 50 nm. a) The fluorescence images of DNA molecule (stained with molecular probe); b) The fluorescence images of Alexa 532 pigment, and c) The overlapped images of a and b. In the image of c, the YOYO-1 and A532 fluorescence signals are artificially colored in blue and red, respectively. (Reprinted with permission from ref. 76. Copyright 2004, American Chemical Society.)

Figure 9

Chemical structures of PNA intercalator dye (BO, TO) and CLSM images of influenza A infected MDCK cells simultaneously stained with the matrix protein 1 (M1) specific BO-probe (a) and the neuraminidase (NA) specific TO probe (b) at indicated time points post infection. White bars correspond to 10 μm. (Reprinted with permission from ref. 77. Copyright 2012, American Chemical Society.)

Figure 10

Schematic chemical model of LNA (blue) hybridized with DNA (red) in antiparallel orientation, with the hydrogen bonding between complementary nucleobases depicted by dotted lines.

Figure 11

a) Predicted structure for 2'OMe-RNA 2209 that specifically targets oskar mRNA. b) Oskar mRNA localization (red) in a fixed Drosophila melanogaster egg chamber at mid-oogenesis, as detected with the 2'OMe-RNA 2209 molecular beacon. Oskar mRNA is transported from the nurse cells to the oocyte through ring canals via a microtubule-dependent process. Action was stained with Phalloidin-FITC (green). (Reprinted with permission from ref. 95. Copyright 2012, American Chemical Society.)