Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39
A new system for fluorescent 5'-end labelling of oligonucleotides with cyanine dyes is proposed. Reaction of primary amine with pyrylium salt was used for the efficient conjugation of monomethyne pyrylium cyanine dye with 5'-aminoalkyl oligonucleotide with simultaneous conversion of the «b...
Saved in:
| Published in: | Биополимеры и клетка |
|---|---|
| Date: | 1998 |
| Main Authors: | , , , |
| Format: | Article |
| Language: | English |
| Published: |
Інститут молекулярної біології і генетики НАН України
1998
|
| Subjects: | |
| Online Access: | https://nasplib.isofts.kiev.ua/handle/123456789/154137 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Journal Title: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Cite this: | Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 / S.M. Yarmoluk, A.M. Kostenko, O.S. Kornushyna, I.Y. Dubey // Биополимеры и клетка. — 1998. — Т. 14, № 1. — С. 82-86. — Бібліогр.: 16 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-154137 |
|---|---|
| record_format |
dspace |
| spelling |
Yarmoluk, S.M. Kostenko, A.M. Kornushyna, O.S. Dubey, I.Y. 2019-06-15T08:49:57Z 2019-06-15T08:49:57Z 1998 Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 / S.M. Yarmoluk, A.M. Kostenko, O.S. Kornushyna, I.Y. Dubey // Биополимеры и клетка. — 1998. — Т. 14, № 1. — С. 82-86. — Бібліогр.: 16 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0004BD https://nasplib.isofts.kiev.ua/handle/123456789/154137 A new system for fluorescent 5'-end labelling of oligonucleotides with cyanine dyes is proposed. Reaction of primary amine with pyrylium salt was used for the efficient conjugation of monomethyne pyrylium cyanine dye with 5'-aminoalkyl oligonucleotide with simultaneous conversion of the «brightless» pyrylium dye Cyan 39 into «bright» pyridinium one Cyan 40. Запропоновано новий підхід для флюоресцентного мічення олігонуклеотидів монометиновими пірилієвими барвниками. Реакція пірилісвих солей з первинними амінами була використана для кон'югації ціанінових барвників з 5'-алкіламіно-олігонуклеотидами і перетворення нефлюоресцентного пірилієвого барвника у флюоресцентний піридинісвий – Cyan 40. Предложен новый подход для флюоресцентного мечения олигонуклсотидов монометиновыми пирилиевыми красителями. Реакция пирилиевых солей с первичными аминами была использована для конъюгации цианиновых красителей с 5'-алкилами-ноолигонуклеотидами и превращения нефлюоресцентного пирилиевого красителя во флюоресцентный пиридиниевый – Cyan 40. The authors gratefully acknowledge the assistance of Ms. Vladislava Kovalska in spectral characterization of dye-oligonucleotide conjugates. We thank Dr. Yury Kovtun for providing us with the sample of cyanine dye Cyan 39. en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Методы Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 Взаємодія ціанінових барвників з нуклеїновими кислотами. 4. Ефективне 5'-флюоресцентне мічення олігонуклеотидів монометиновим пірилієвим барвником Cyan 39 Взаимодействие цианиновых красителей с нуклеиновыми кислотами. 4. Эффективное 5'-флюоресцентное мечение олигонуклсотидов монометиновым пирилиевым красителем Cyan 39 Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 |
| spellingShingle |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 Yarmoluk, S.M. Kostenko, A.M. Kornushyna, O.S. Dubey, I.Y. Методы |
| title_short |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 |
| title_full |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 |
| title_fullStr |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 |
| title_full_unstemmed |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 |
| title_sort |
interaction of cyanine dyes with nucleic acids. 4. efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, cyan 39 |
| author |
Yarmoluk, S.M. Kostenko, A.M. Kornushyna, O.S. Dubey, I.Y. |
| author_facet |
Yarmoluk, S.M. Kostenko, A.M. Kornushyna, O.S. Dubey, I.Y. |
| topic |
Методы |
| topic_facet |
Методы |
| publishDate |
1998 |
| language |
English |
| container_title |
Биополимеры и клетка |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Взаємодія ціанінових барвників з нуклеїновими кислотами. 4. Ефективне 5'-флюоресцентне мічення олігонуклеотидів монометиновим пірилієвим барвником Cyan 39 Взаимодействие цианиновых красителей с нуклеиновыми кислотами. 4. Эффективное 5'-флюоресцентное мечение олигонуклсотидов монометиновым пирилиевым красителем Cyan 39 |
| description |
A new system for fluorescent 5'-end labelling of oligonucleotides with cyanine dyes is proposed. Reaction of primary amine with pyrylium salt was used for the efficient conjugation of monomethyne pyrylium cyanine dye with 5'-aminoalkyl oligonucleotide with simultaneous conversion of the «brightless» pyrylium dye Cyan 39 into «bright» pyridinium one Cyan 40.
Запропоновано новий підхід для флюоресцентного мічення олігонуклеотидів монометиновими пірилієвими барвниками. Реакція пірилісвих солей з первинними амінами була використана для кон'югації ціанінових барвників з 5'-алкіламіно-олігонуклеотидами і перетворення нефлюоресцентного пірилієвого барвника у флюоресцентний піридинісвий – Cyan 40.
Предложен новый подход для флюоресцентного мечения олигонуклсотидов монометиновыми пирилиевыми красителями. Реакция пирилиевых солей с первичными аминами была использована для конъюгации цианиновых красителей с 5'-алкилами-ноолигонуклеотидами и превращения нефлюоресцентного пирилиевого красителя во флюоресцентный пиридиниевый – Cyan 40.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154137 |
| citation_txt |
Interaction of cyanine dyes with nucleic acids. 4. Efficient 5-fluorescent labelling of oligonucleotides with monomethyne pyrylium cyanine dye, Cyan 39 / S.M. Yarmoluk, A.M. Kostenko, O.S. Kornushyna, I.Y. Dubey // Биополимеры и клетка. — 1998. — Т. 14, № 1. — С. 82-86. — Бібліогр.: 16 назв. — англ. |
| work_keys_str_mv |
AT yarmoluksm interactionofcyaninedyeswithnucleicacids4efficient5fluorescentlabellingofoligonucleotideswithmonomethynepyryliumcyaninedyecyan39 AT kostenkoam interactionofcyaninedyeswithnucleicacids4efficient5fluorescentlabellingofoligonucleotideswithmonomethynepyryliumcyaninedyecyan39 AT kornushynaos interactionofcyaninedyeswithnucleicacids4efficient5fluorescentlabellingofoligonucleotideswithmonomethynepyryliumcyaninedyecyan39 AT dubeyiy interactionofcyaninedyeswithnucleicacids4efficient5fluorescentlabellingofoligonucleotideswithmonomethynepyryliumcyaninedyecyan39 AT yarmoluksm vzaêmodíâcíanínovihbarvnikívznukleínovimikislotami4efektivne5flûorescentnemíčennâolígonukleotidívmonometinovimpírilíêvimbarvnikomcyan39 AT kostenkoam vzaêmodíâcíanínovihbarvnikívznukleínovimikislotami4efektivne5flûorescentnemíčennâolígonukleotidívmonometinovimpírilíêvimbarvnikomcyan39 AT kornushynaos vzaêmodíâcíanínovihbarvnikívznukleínovimikislotami4efektivne5flûorescentnemíčennâolígonukleotidívmonometinovimpírilíêvimbarvnikomcyan39 AT dubeyiy vzaêmodíâcíanínovihbarvnikívznukleínovimikislotami4efektivne5flûorescentnemíčennâolígonukleotidívmonometinovimpírilíêvimbarvnikomcyan39 AT yarmoluksm vzaimodeistviecianinovyhkrasiteleisnukleinovymikislotami4éffektivnoe5flûorescentnoemečenieoligonuklsotidovmonometinovympirilievymkrasitelemcyan39 AT kostenkoam vzaimodeistviecianinovyhkrasiteleisnukleinovymikislotami4éffektivnoe5flûorescentnoemečenieoligonuklsotidovmonometinovympirilievymkrasitelemcyan39 AT kornushynaos vzaimodeistviecianinovyhkrasiteleisnukleinovymikislotami4éffektivnoe5flûorescentnoemečenieoligonuklsotidovmonometinovympirilievymkrasitelemcyan39 AT dubeyiy vzaimodeistviecianinovyhkrasiteleisnukleinovymikislotami4éffektivnoe5flûorescentnoemečenieoligonuklsotidovmonometinovympirilievymkrasitelemcyan39 |
| first_indexed |
2025-11-24T11:38:37Z |
| last_indexed |
2025-11-24T11:38:37Z |
| _version_ |
1850845755590311936 |
| fulltext |
ISSN 0233-7657. Биополимеры и клетка. 1998. Т. 14. № 1
МЕТОДЫ
Interaction of cyanine dyes with nucleic acids.
4. Efficient 5-fluorescent labelling of
oligonucleotides with monomethyne pyrylium
cyanine dye, Cyan 39
Sergiy M. Yarmoluk, Alexander M. Kostenko, Olga S. Kornushyna, Igor Y. Dubey1
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150 Zaboiotnogo str., 252143 , Kyiv, Ukraine
^Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine
1 Murmanska str., 253660 , Kyiv, Ukraine
A new system for fluorescent 5'-end labelling of oligonucleotides with cyanine dyes is proposed. Reaction
of primary amine with pyrylium salt was used for the efficient conjugation of monomethyne pyrylium
cyanine dye with 5'-aminoalkyl oligonucleotide with simultaneous conversion of the «brightless» pyrylium
dye Cyan 39 into «bright» pyridinium one Cyan 40.
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 105
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 cyani-
nes 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)
( § ) S ML YAJRMOLUK, A. M. K.OSTENKO, O. S. K O R N U S H Y N A ,
I. Y. D U B E Y , 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 Milli-
gen/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]. Func-
tionalized oligonucleotides were purified by electro
phoresis in 20 % denaturing polyacrylamide gel.
Reverse phase HPLC was carried out on Beckman
«Gold System» chromatograph using Ultraprep C I 8
(21 x 1,5 cm, «Весктап», USA) column with a linear
gradient of acetonitrile (0—30 %) in 0.1 M TEAB
buffer (pH 6.5) at flow rate 1 ml/min.
Spectroscopic measurements. The absorption
82
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.
The dye-oligonucleotide I polynucleotide complexes
were prepared by adding 10-fold excess of poly
nucleotides to 3 1 0 ъ M dye conjugate in ТЕ buffer
(10 mM Tris-HCl, 0.5 raM EDTA, pH 7). Working
solutions were then heated to 60 °С and equilibrated
at 20 °С for at least 24 h. Extinction coefficient of T t 5
at 260 nm calculated according to [16] was
1.22-105 iM-'cm"1,
Genaral procedure for oligonucleotide 5'-labelling.
5-NH2-(CH 2 ) 6 -NH-COO-(Tp) M T (5 0 0 ^ , 4 0 p i n o l )
was dissolved in 60 /Л of 0.1 M N a H C 0 3 - N a 2 C 0 3
(3:1) buffer (pH 9.5). 15 ii\ of Cyan 39 solution in
DMSO (10 mmol/ml, 150 nmol) was added to oligo
nucleotide with agitation. The reaction mixture was
incubated at 50 °С for 12 h in the dark. The progress
of reaction was monitored by HPLC. Oligonucleotide
material was precipitated by 2 % LiC104 in acetone
and 5'-labelled oligonucleotide was isolated by re
verse phase HPLC Yield of purified conjugate was
3.1 OD 2 6 0 (62 % ) . Structure and purity of product
were confirmed by spectroscopic measurements,
HPLC and PAGE.
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
( a b U m a x 434 nm, e m A m a x 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 ( a b sAm a x
470 nm, e m A m a x 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
83
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'-Amino-
hexyl 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 % LiC104 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 non-
modified 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
20 00 24.00 28.00 32.00 Minutes
Fig. 3. HPLC profiles of reaction mixture of oligonucleotide labelling
with Cyan 39 and absorbance spectra of components: 1 — 5 ' - N H 2 -
( C H 2 ) b - N H - C O O - ( T p ) 1 4 T ; 2 — 5 - D y e - ( C H 2 ) 6 - N H - C O O -
( T p ) , 4 T < a b s A m a x 446 nm)
84
INTERACTION O F CYANINE DYES WITH NUCLEIC ACID
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 1 5 -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 of 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 probe-
target double-stranded complexes increased 3.5—5-
fold 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
Ш. ( : Г Г '
440 500 560
Wavelength, mn
Fig. 4. Fluorescence spectra of free Cyan 40: J — 5 ' - D y e ~ ( C H 2 ) 6 -
N H - C O O - ( T p ) | 4 T ; 2 — and duplexes of oligonucleotide-dye con
jugate with poly(dA) (3) and poly(rA) (4)
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.
Acknowledgement. The authors gratefully ac
knowledge the assistance of Ms. Vladislava Kovalska
in spectral characterization of dye-oligonucleotide co
njugates. We thank Dr. Yury Kovtun for providing us
with the sample of cyanine dye Cyan 39.
С. M. Ярмолюк, О. M. Костенко, О. С. Корнюшина, I. Я. Дубей
Взаємодія ціанінових барвників з нуклеїновими кислотами.
4. Ефективне 5'-флюоресцентне мічення олігонуклеотидів
монометиновим пірилієвим барвником Cyan 39
Резюме
Запропоновано новий підхід для флюоресцентного мічення
олігонуклеотидів монометиновими пірилієвими барвниками.
Реакція пірилісвих солей з первинними амінами була викори
стана для кон'югації ціанінових барвників з 5'-алкіламіно-
олігонуклеотидами і перетворення нефлюоресцентного піри-
лієвого барвника у флюоресцентний піридинісвий — Cyan 40.
С. Н. Ярмолюк, О. М. Костенко, О. С. Корнюіиина,
И Я. Дубей
Взаимодействие цианиновых красителей с нуклеиновыми
кислотами. 4. Эффективное 5'-флюоресцентное мечение
олигонуклсотидов монометиновым пирилиевым
красителем Cyan 39
Резюме
Предложен новый подход для флюоресцентного мечения олиго
нуклсотидов монометиновыми пирилиевыми красителями.
Реакция пирилиевых солей с первичными аминами была исполь
зована для конъюгации цианиновых красителей с 5' -алкилами-
ноолигонуклеотидами и превращения нефлкюресцентного пи-
рилиевого красителя во флюоресцентный пиридиниевый —
Cyan 40.
REFERENCES
1. English U., Gauss D. / / . Chemically modified oligonucleotides
as probes and inhibitors / / Angew. Chem. Int. Ed. Engl.—
1991 .—30.—P. 6 1 3 - 6 2 9 .
2. DNA Probes / Eds G. H. Keller, M. Manak.—New York:
MacMiilan press, 1993 — 6 5 6 p.
3 . Haughland R. P. Handbook of fluorescent probes and research
chemicals / / Molecular Probes.—Eugene: OR, 1996.—679 p.
4. Schwartz H. E.t Vlfelder K. J. II Anal. Chem.—1992 .—64.—
P. 1737—1740.
5. Lee G. L., Chen С.-/У., Chiu L A. Thiazole Orange: a new
dye for reticulocyte analysis / / Cytometry.—1986.—7.—
P. 508—517 .
6. Rye H. S.t Quesada M. A., Peck K. et al. High-sensitive
two-color detection of double-stranded DNA with a confocal
85
YARMOLUK S. M. ET AJL.
fluorescence gel scanner using ethidium homodimer and thia-
zole orange / / Nucl. Acids R e s . ~ 1 9 9 1 . ~ 1 9 . - - P . 3 2 7 — 3 3 3 .
7. Pitner J. В., Mize P. D., Linn C. P. Synthesis and fluores
cence properties of covalent thiazole orange-oligonucleotide
conjugates / / The 3rd Int. Symp. on Functional Dyes (July
16—21, 1995).—Santa Cruz, 1995 .—P. 52 .
8. Ernst L. A.t Gupta R. K., Mujumdar R. B. et al Cyanine dye
labeling reagents for sulfhydryl groups / / Cytometry.—
1989 .—10.—P. 3—10 .
9. Mujumdar R. В., Ernst L. A., Mujumdar S. R. et al Cyanine
dye labeling reagents containing isothiocyanate groups / /
Ib id .—1989.—10.—P. 11 — 19.
10. Southwick P. L., Ernst L. A., Tauriello E. W. et al Cyanine
dye labeling reagents — carboxymethylindocyaninc succinimi-
dyl esters / / Ib id .—1990 .—11. — P. 418—430 .
11. Wachter L.f Jablonski J., Ramachandran K. L A simple and
efficient procedure for the synthesis of 5'-aminoalkyl oligo
nucleotides / / Nucl. Acids R e s . — 1 9 8 6 . — 1 4 . - P . 7985—7994 .
12. Goodchild J. Conjugates of oligonucleotides and modified
oligonucleotides: a review of their synthesis and properties / /
Bioconjugate C h e m . — 1 9 9 0 . — 1 . — P . 165—187.
13. Protocols for oligonucleotide conjugates / / Meth. Мої. Biol.—
New York: Humana press, 1994 .—377 p.
14. Yarmoluk S. M., Kovalska V. B*, Kovtun Yu. P. Interaction of
cyanine dyes with nucleic acids. 5. Towards model of «Half
interaction* of monomethyne cyanine dyes into double-stran
ded nucleic acids / / Biopolymers and Cel l .—1998.—14,
N 5.—(in press).
15. Katritzky A. R. Conversions of primary amino groups into other
functionality mediated by pyrylium cations / / Tetrahedron.—
1980 .—36 .—P. 6 7 9 — 6 9 0 .
16. Handbook of biochemistry and molecular biology / Ed. G.
Fasman.—Boca Raton: CRC, 1975 .—P. 175.
Received 05.05 .97
http://Res.~1991.~19.--P
|