Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Abstract

Dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition of his outstanding contributions to the area of nucleic acid chemistry.Herein, a novel fluorescent nucleotide analogue, perylene-2′-amino-α-L-LNA, has been prepared and studied within synthetic oligonucleotides of different sequences. The phosphoramidite reagent was synthesized in 85% overall yield starting from 2′-amino-α-L-LNA nucleoside. Incorporation efficiency of the resulting perylene-2′-amino-α-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards complementary DNA and RNA targets, batochromically shifted excitation/emission wavelengths with respect to the often applied polyaromatic hydrocarbon pyrene, high fluorescent quantum yields and very low target detection limits (5-10 nM). Fluorescence of single stranded LNA/DNA mixmer oligonucleotide having two incorporations of monomers T* was quenched (quantum yield Φ F = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Φ F = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues sequences containing monomers T* at 5′-and 3′-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose that a covalent link between two T* monomers in the double-labeled probe provides a remarkable degree of rigidity in the double helix which enforces positioning of the bulky perylene moieties in the nonpolar groove resulting in reduced fluorescence quenching.

Rapid genotyping using pyrene−perylene locked nucleic acid complexes

“…Previously investigated (pyrene-1-yl)methyl 2'-amino-LNA (monomer M 1 ) was incorporated into oligonucleotides as a reference for the new FRET donor M 2,41 monomers M 3 and M 4 were prepared as previously described. 42,43 For initial evaluation of pyrene-perylene FRET and SNP diagnostic potential, monomers M 1 -M 4 were incorporated in a model dual-probe FRET system ON1-ON4 and covalently linked double-labeled analogs ON5-ON8 adopted from previous research on PAH-LNA monomers ( Table 1). 29,30 The model FRET probes were prepared using automated phosphoramidite DNA synthesis and first characterized by their melting temperatures with complementary and mismatched DNA/RNA targets (T m ; Tables S1-S6).…”

Synthesis and evaluation of a photoresponsive quencher for fluorescent hybridization probes

“…The design, synthesis, and biological evaluation of modified nucleotides and oligonucleotides have attracted substantial attention in recent years. [1][2][3][4][5][6][7][8] Applications of those analogues include homogeneous DNA sequencing techniques 9 for molecular diagnostics as the basis of personalized medicine, 10,11 single nucleotide polymorphism (SNP), [12][13][14][15][16][17] as well as for the identification and quantification of nucleic acids. [18][19][20][21] Fluorescence and fluorescence quenching form the basis of the majority of detection systems, such as specific displacement hybridization probes, [22][23][24] Molecular Beacons (MBs), 25 Scorpion, 9 and TaqMan probes.…”

Branched DNA nanostructures efficiently stabilised and monitored by novel pyrene–perylene 2′-α-l-amino-LNA FRET pairs

“…5 Among various fluorescent nucleotide analogues, polyaromatic hydrocarbons (PAHs) attached to 2 0 -amino-and the isomeric 2 0 -a-Lamino locked nucleic acids (LNA) have proven to be promising units for DNA nanotechnology. [6][7][8] In the present work we prepared a series of branched DNA nanostructures containing two novel internally positioned pyrene-perylene 2 0 -a-L-amino-LNA FRET pairs leading simultaneously to stabilization and efficient fluorescent sensing of DNA nano-assemblies (Fig. 1).…”

“…1). We report the synthesis of oligonucleotides containing pyrene and perylene 2 0 -a-L-amino-LNA nucleotides and 5 0 -alkyne modifications, their further branching by microwave-assisted CuAAC click reactions through three types of 2-and 3-way junctions (6)(7)(8), and comprehensive spectroscopic characterization of the resulting fluorescent nanostructures.…”

“…Based on our previous research data, [6][7][8][9] we selected perylenemethyl and pyrenecarbonyl 2 0 -a-L-amino-LNA monomers M 1 -M 3 for the novel interstrand communication FRET systems (Fig. 1).…”