Interaction of cyanine dyes with nucleic acids . 4 . Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye , Cyan 39

Introduction. Oligonucleotides labelled with fluores­ cent reporter groups are widely used as hybridization probes, primers for automated DNA sequencing, re­ search tools for the studies on structure and dynamics of nucleic acids and proteins, e tc [1—3]. Cyanine dyes have excellent properties of the most sensitive nucleic acid fluorescence probes currently available. They have large extinction coefficients (about 10 M 1 cm"), fluorescence covering a wide spectral range (500—750 nm), and high nucleic acid binding cons­ tants 13]. Monomethyne benzolhiazole and -oxazole cyanines are most suitable to develop new homogeneous detection systems for nucleic acids [4 ]. These dyes are nonfluorescent in free state becoming strongly fluorescent when bound to nucleic acids (> 1000-fold fluorescence increase) [5, 6] . No separation of the excess of fluorescent probe is therefore essential for the detection based on this principle. Recently, Pitner et al. [7] reported on covalent monomethyne cyanine dye-oiigonucleotide conjugates for the detection of specific DNA sequences. NHydroxysuccinimide ester of Thiazole Orange (TO)

Introduction.Oligonucleotides labelled with fluores cent reporter groups are widely used as hybridization probes, primers for automated DNA sequencing, re search tools for the studies on structure and dynamics of nucleic acids and proteins, etc [1][2][3].Cyanine dyes have excellent properties of the most sensitive nucleic acid fluorescence probes currently available.They have large extinction coefficients (about 10 5 M 1 cm" 1 ), fluorescence covering a wide spectral range (500-750 nm), and high nucleic acid binding cons tants 13].
Monomethyne benzolhiazole and -oxazole cyanines are most suitable to develop new homogeneous detection systems for nucleic acids [4 ].These dyes are nonfluorescent in free state becoming strongly fluorescent when bound to nucleic acids (> 1000-fold fluorescence increase) [5,6].No separation of the excess of fluorescent probe is therefore essential for the detection based on this principle.
Recently, Pitner et al. [7] reported on covalent monomethyne cyanine dye-oiigonucleotide conjugates for the detection of specific DNA sequences.N-Hydroxysuccinimide ester of Thiazole Orange (TO) Y.

DUBEY, 1998
dye was used for the conjugation of cyanine with 5'-end amino-modified oligonucleotides [7].Before some synthetic procedures for the covalent labelling of biomolecules with cyanine dyes were described.Func tional groups in proposed cyanine reagents were sulfhydryl [8 J, isotiocyanate [9 ] and succinimidyl esters [10].
In this paper we propose a new efficient proce dure for the fluorescent labelling of oligonucleotides with cyanine dyes.
Materials and Methods.Oligonucleotide synthesis was performed on Applied Biosystems Model 380B DNA synthesizer by standard phosphoramidite me thod using reagents and solvents from Milligen/Biosearch (USA).5 , -Aminoalkyl linker was in troduced by reaction of 5'-deblocked oligonucleotide on polymer support with carbonyldiimidazole followed by hexamethylenediamine, according to [11].Functionalized oligonucleotides were purified by electro phoresis in 20 % denaturing polyacrylamide gel.

Spectroscopic measurements.
The absorption spectra were recorded on «Specord UV-VIS» spec trophotometer («Karl Zeiss Jena», Germany).Fluo rescence spectra were obtained with fluorescence spectrophotometer Hitachi Model 850 (Japan).Fluo rescence was excited with 150W Xe-lamp emission and measurements were carried out in thermostatable quartz cell (0.5 * 0.5 cm).All spectra were corrected by multiplying fluorescence intensities measured over an interval of 5 nm by proper correction factor for corresponding wavelengths.In corrected spectra fluo rescence intensity values were proportional to a num bers of photons per unit of wavelength interval.
Results and Discussion.Fluorescent reporter gro ups can be covalently attached to oligonucleotides by a wide variety of methods [1-3, 12, 13].The most popular of them are based on the reaction of 5'aminoalkyl oligonucleotides with reagents bearing amine-specific reactive groups, usually isothiocyanate or N-hydroxysuccinimide dye derivatives.We have found a new approach to oligonucleotide labelling utilizing pyrylium cyanine dyes.
Some new fluorescent cyanine dyes for nucleic acid staining have been recently proposed in our laboratory.Monomethyne cyanine dye Cyan 40 ( ab U max 434 nm, em A max 475 nm) has characteristics of suitable fluorescent dye for the quantification of nucleic acids: 1) low intrinsic fluorescence, 2) great fluorescence enhancement upon binding of dye to nucleic acids and 3) high fluorescence quantum yield for nucleic acid-dye complexes.At the same time, monomethyne pyrylium cyanine dye Cyan 39 ( abs A max 470 nm, em A max 490 nm) has structure like Cyan 40, but low fluorescence intensity upon binding to nucleic acids [14] (Fig. 1).
Pyrylium salts are known to react with primary amines giving corresponding pyridinium cations via formal O-N substitution [15].Thus, in the reaction of 5'-aminoalkyl functionalized oligonucleotide with pyrylium cation oligonucleotide labelling could be achieved with simultaneous conversion of pyrylium dye into fluorescent pyridinium one.Proposed label ling procedure allows to prepare oligonucleotides mo dified with pyridinium dye Cyan 40 starting from pyrylium heterocycle of Cyan 39 (Fig. 2).In our approach fluorescent oligonucleotide probe is obtained from non-fluorescent predecessor.From this point of view, some analogy can be found in the labelling of biomolecules with non-fluorescent bromobimane transforming into fluorescent residue after reaction with thiol or thiophosphate group [3 ].
One of the main problems was that pyridinium cations like Cyan 40 are susceptible to nucleophilic displacement of the N-substituent [15].Therefore, the labelling conditions should have been carefully selected, since basic pH was essential for the reaction of aminoalkyl group with pyrylium cation, whereas at higher pH the alkaline hydrolysis of coupling product was observed as important yield-decreasing side reac tion.We have found optimum labelling conditions in model reaction of Cyan 39 with aminocaproic acid which were then used for oligonucleotides.5'-Aminohexyl pentadecathymidylate was prepared by intro ducing aminoalkyl linker group into oligonucleotide during solid phase synthesis by carbonyldiimidazole method [11].The highest yield of functionalized oligonucleotide labelling with Cyan 39 was achieved for the reaction in 0.1 M sodium bicarbona te/carbonate buffer (pH 9.5).With equimolar amount of reagents the completion of model reaction could be determined by UV-Vis spectra which showed that as the reaction progressed the absorption maximum of Cyan 39 (470 nm) declined almost to zero and the absorption at 434 nm increased correspondingly.But since the excess of dye (ca. 10 eq.) was used to increase the overall yield of oligonucleotide conjugate, the labelling reaction had to be monitored by HPLC.
It should be noted that in Dye-T, 5 conjugate dye absorption maximum was shifted from 434 to 446 nm due to the interaction of cyanine residue with oligo nucleotide.The same effect was observed for the interaction of free Cyan 40 with nucleic acids in the solution |14].UV/Vis spectrum of conjugate clearly confirmed the presence of cyanine dye residue by specific absorbance at 446 nm.Detailed study of spectro-fluorescent properties of conjugates will be published elsewhere.
It the end of reaction oligonucleotide material was precipitated by 2 % LiC10 4 in acetone and chromatographed on C, g reverse phase column with yield about 60 % (Fig. 3).Purified oligonucleotide conjugate was shown to be homogeneous by both HPLC arid PAGE.In the reverse phase chroma tography, the conjugate had higher retention time than starting oligonucleotide due to the hydrophobic character of dye residue.The labelled oligonucleotide migrated in the polyacrylamide gel slower than nonmodified oligomer and was easily identified by its greenish fluorescence under long wavelength (365 nm) UV light.UV/Vis spectrum of conjugate clearly confirmed the presence of cyanine dye residue by specific absorbance at 446 nm.
Cyan 40-labelled oligonucleotides could be used as hybridization probes for the detection of nucleic acids by common techniques.At the same time, the preliminary experiments to study the changes of fluorescence upon interaction of labelled oligonuc leotide with complementary nucleic acids in the solu tion were also carried out using the simplest model systems.The fluorescence of T 15 -Cyan 40 probe was measured before and after hybridization to the comp lementary poly (r A) and poly(dA) chains.After the formation of duplexes the fluorescence emission at 475 nm increased 1.9 and 2.1 times, respectively (Fig. 4).This increase of fluorescence for dye conjugate was much lower than could be observed for the interaction free Cyan 40 with double-stranded nucleic acids [14 ].
The obtained data are in full agreement with Pitner's results for TO-oligonucleotide conjugates de monstrated that the fluorescence of resulting probetarget double-stranded complexes increased 3.5-5fold in comparison with that of single-stranded TO conjugate before hybridization [71, whereas the inc rease of fluorescence of non-conjugated TO upon interaction with double-stranded nucleic acids was 3 orders of magnitude higher [6,7].At least two hypothesis can be presented to explain the insig nificant increase in fluorescence of Cyan 40 (or Thiazole Orange) when it was conjugated to oligo nucleotide.The first explanation would be that the ratio of 1 dye per 15 b.p. upon formation of duplex is insufficient to reach a maximum of fluorescence emission observed at the ratio 1 dye/2 b. p. for Cyan 40 [14 J.Then, it is possible that conjugated dye does not intercalate into formed oligonucleotide-target dup lex, probably due to some steric factors, and simply «sticks» to the end nucleotides as cationic molecule.In this case, the choice of appropriate linker cons truction could allow the efficient intercalation.Studies of these problems on various model systems are in progress.
Thus, we propose a new oligonucleotide labelling system that does not require any preparation of active intermediates as a convenient, efficient and flexible alternative for known methods of nucleic acids label ling and detection.This method also seems to be perspective for the development of homogeneous de tection systems for nucleic acids.