Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'-phosphate

Abstract

The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl N,N-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-m4)-d(T)15 and 5'-(pyr-m4)-d(T)15-d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The Tm values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 degrees C, respectively. The pyrene-induced stabilization corresponds to a free energy change (delta delta G degrees) of -2.6 kcal/mol.

2‐(1‐Ethynylpyrene)‐Adenosine as a Folding Probe for RNA—Pyrene in or out

“…[24,25] This effect is generally strong in all compounds that exhibit intercalation of pyrene into the base stack. [30][31][32] Additionally, intercalation usually imposes significant disturbance to the original system; this leads to a change of the local RNA structure and an alteration of the conformational dynamics of the unmodified system. This has to be taken into consideration in cases in which the conformational and structural dynamics of a compound are monitored.…”

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions

“…pyrene) can exhibit various fluorescent characteristics (e.g. excimer formation and FRET) by interactions with another fluorophore; (iii) the introduction of lipophilic moieties into oligonucleotides may lead to increased transport of the nucleotides across cell membranes, as pointed out by Mann et al (11). Papers show that an excimer fluorescence from pyrene can be induced in a dilute solution upon hybridization between a target nucleotide and its two probe nucleotides (12)(13)(14).…”

Base-Sequence Dependence of Emission Lifetimes for D141018-30-6NA Oligomers and Duplexes Covalently Labeled with Pyrene: Relative Electron-Transfer Quenching Efficiencies of A, G, C, and T Nucleosides toward Pyrene

“…21 A number of studies have involved pyrene linked covalently to oligodeoxyribonucleotides and to oligoribonucleotides. [22][23][24][25][26][27][28][29] In general these studies have found that the amount of pyrene emission from the linked assemblies is very sensitive to the environment surrounding the pyrene label. One such study has concluded that 5'-linked pyrene labels are ideal for probing the binding and dynamics of RNA substrate^.^^ A frequent finding in these studies is that the pyrene-labeled duplexes emit more strongly than do the corresponding labeled oligomers.…”

“…[1][2][3][4][30][31][32][33][34][35][36][37][38] Similarly, a number of oligomers and duplexes have been constructed with covalently attached pyrene labels, yet little systematic work has been done comparing how flanking bases affect the emission properties of these pyrene labels. 2,[22][23][24][25][26][27]29,30,39,40 Finally, a number of labeling studies relied solely on continuous emission measurements and did not time-resolve the pyrene label's emission kinetics.…”