Pyrene-functionalized oligonucleotides and locked nucleic acids (LNAs): tools for fundamental research, diagnostics, and nanotechnology

Abstract

Pyrene-functionalized oligonucleotides (PFOs) are increasingly explored as tools in fundamental research, diagnostics and nanotechnology. Their popularity is linked to the ability of pyrenes to function as polarity-sensitive and quenchable fluorophores, excimer-generating units, aromatic stacking moieties and nucleic acid duplex intercalators. These characteristics have enabled development of PFOs for detection of complementary DNA/RNA targets, discrimination of single nucleotide polymorphisms (SNPs), and generation of π-arrays on nucleic acid scaffolds. This critical review will highlight the physical properties and applications of PFOs that are likely to provide high degree of positional control of the chromophore in nucleic acid complexes. Particular emphasis will be placed on pyrene-functionalized Locked Nucleic Acids (LNAs) since these materials display interesting properties such as fluorescence quantum yields approaching unity and recognition of mixed-sequence double stranded DNA (144 references).

Fig. 1

Position-dependent pyrene characteristics in nucleic acid duplexes.

Fig. 2

Pyrene-functionalized monomers and anticipated location of the pyrene moiety in nucleic acid duplexes.

Fig. 3

Structure of LNA-T and α-L-LNA-T monomers. Numbering of sugar ring carbons is shown for LNA.

Fig. 4

Pyrene-functionalized LNAs and anticipated location of the pyrene moiety in nucleic acid duplexes.

Fig. 5

Placement of pyrene moieties in the minor groove using N2'-functionalized 2'-amino-LNA. Molecular modelling structure of duplex between 5'-d(GCA 37A37 CAC) and 3'-d(CGT ATA GTG). Monomer 37: 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA-T. (Reproduced with permission from ref. 90. Copyright 2005 American Chemical Society).

Fig. 6

Placement of pyrene moieties in the duplex core using N2'-functionalized 2'-amino-α-L-LNA. Different representations of molecular modelling structure of duplex between 5'-d(GTG A38A TGC) and 3'-d(CAC TAT ACG). Monomer 38: 2'-N-(pyren-1-yl)methyl-2'-amino-α-L-LNA-T. (Reproduced with permission from ref. 67. Copyright 2009 American Chemical Society).

Fig. 7

Formation of pyrene arrays in the minor groove using 2'-O-(pyren-1-yl)methyluridine 21 monomers. Upper: fluorescence emission spectra of duplexes between 19-mer 2'-O-methyluridine strands centrally modified with 1–4 sequential 21 monomers and complementary RNA (dsP1–dsP4, λ_ex = 350 nm). (Reproduced with permission from ref. 144 Copyright 2005 RSC) Lower: molecular modelling structure of dsP4. (Reproduced with permission from ref. 71. Copyright 2007 RSC Publishing).

Fig. 8

Formation of pyrene interstrand zipper arrays using 2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomer 36. Left: T_m-values and fluorescence characteristics of DNA duplexes modified with monomer 36. Arrows indicate −1 zipper orientation.Middle: molecular modelling structure of duplex with three −1 interstrand arrangements of monomer 36 (T_m = 77 °C). (Adapted from ref. 72. Copyright 2004 RSC Publishing). Right: fluorescence emission spectra illustrating stepwise self-assembly of higher order nucleic acid structures (using monomer 36). (Adapted with permission from ref. 74. Copyright 2005 Taylor & Francis).

Fig. 9

Left: illustration of pyrene array formation in the duplex core using 5'-(YΦ):3'-(AΦ)-units where Y denotes 2'-N-(pyren-1-yl)acetyl-2'-amino-α-L-LNA-T monomer 40 and monomer Φ is an abasic site analog. Right: molecular modelling structure of a 13-mer DNA duplex containing two separated 5'-(YΦ): 3'-(AΦ)-units. (Adapted with permission from ref. 79. Copyright 2008 American Chemical Society).

Fig. 10

Principle of hybridization probes. White and green droplets represent silent and emissive fluorophore.

Fig. 11

Pyrene-functionalized monomers utilized in hybridization probes. Py = pyren-1-yl.

Fig. 12

Principle of BDF probes.

Fig. 13

SNP discrimination using C5-[3-(1-pyrenecarboxamido)propynyl] DNA/LNA monomers. Fluorescence emission spectra of 5'-d(CG CAA GBG AAC GC-3'), where B = DNA monomer 4 (ON7) or LNA monomer 32 (ON11), against complementary or singly mismatched DNA. (Reproduced with permission from ref. 11. Copyright 2011 Wiley).

Fig. 14

Left: principle of SNP-discrimination by excimer-forming dual probes. Middle: fluorescence emission spectra of LNA-based dual probes end-functionalized with monomer 36 and complementary (red) or singly mismatched DNA target (black). Right: probe/target sequences; discrimination of SNPs in positions 1–12 (λ_em = 480 nm). (Adapted with permission from ref. 103. Copyright 2007 Wiley).

Fig. 15

Principle of mismatch detection using doubly modified 2'-N-(pyren-1-yl)acetyl-2'-amino-α-L-LNA 40. (Reproduced with permission from ref. 109. Copyright 2007 Wiley).

Fig. 16

A: General principle of a conventional MB. B–D: Different quencher-free MB designs. (Adapted with permission from ref. 112. Copyright 2008 RSC Publishing).

Fig. 17

Excimer-forming MBs. Left: Fluorescence emission spectra (λ_ex = 386 nm) of (a) MB alone, (b) MB + complementary DNA and, (c) MB + singly mismatched DNA. Right: Optical detection of complementary DNA. MB sequence: 5'-d(GC11 GAG AAG TTA GAA CCT ATG CTC 11GC). (Reproduced with permission from ref. 116. Copyright 2006 Elsevier Ltd.).

Fig. 18

Fig. 18. Fluorescence signalling of quadruplex formation. (Reproduced with permission from ref. 121. Copyright 2007 RSC Publishing).

Fig. 19

Non-nucleosidic monomers used for dsDNA-targeting.

Fig. 20

Left: illustration of the Invader LNA concept. Right: time course of steady-state fluorescence spectra upon addition of pre-annealed Invader LNA to pre-annealed isosequential dsDNA target (λ_ex = 335 nm, 20 °C). Invader LNA: 5'-d(GGT A38A TAT AGG C):3'-d(CCA TA38 ATA TCC G) using monomer 38. Isosequential dsDNA target: as for Invader LNA except 38 is placed by T. (Adapted from ref. 135. Copyright 2010 RSC Publishing).