Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA.

1.6 MB·English

by  Dierckx, Anke

Published online 29 January 2011 Nucleic Acids Research, 2011, Vol. 39, No. 10 4513–4524
doi:10.1093/nar/gkr010
Characterization of photophysical and
base-mimicking properties of a novel
fluorescent adenine analogue in DNA
Anke Dierckx1, Peter Dine´r2, Afaf H. El-Sagheer3,4, Joshi Dhruval Kumar1,
Tom Brown3, Morten Grøtli2 and L. Marcus Wilhelmsson1,*
1Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology,
2Department of Chemistry, Medicinal Chemistry, University of Gothenburg, S-41296 Gothenburg, Sweden,
3School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK and 4Department of
Science and Mathematics, Chemistry Branch, Faculty of Petroleum and Mining Engineering, Suez Canal
University, Suez 43721, Egypt
Received November 12, 2010; Revised January 3, 2011; Accepted January 4, 2011
ABSTRACT emissive molecules which are similar in shape to natural
nucleobases and are able to form one or more hydrogen
To increase the diversity of fluorescent base
bonds to a natural nucleobase in the complementary
analogues with improved properties, we here
strand. This means that these artificial bases do not rad-
present the straightforward click-chemistry-based
icallyalterthestructureofDNA,andthereforetheyhave
synthesis of a novel fluorescent adenine-analogue
major advantages compared to other dyes which are co-
triazole adenine (AT) and its photophysical charac-
valently tethered to the DNA outside the base-stack (e.g.
terization inside DNA. AT shows promising Cy-dyes, fluorescein, Alexa dyes or rhodamines).
properties compared to the widely used adenine Importantly, fluorescent base analogues allow experi-
analogue 2-aminopurine. Quantum yields reach ments to be performed while preserving the native struc-
>20% and >5% in single- and double-stranded ture of DNA by avoiding bulky external probes.
DNA, respectively, and show dependence on neigh- Furthermore, they are located rigidly in the DNA
bouring bases. Moreover, AT shows only a minor base-stack, in contrast to the covalently attached dyes,
andthisrestrictedmovementresultsinamorepredictable
destabilization of DNA duplexes, comparable to
orientation which can therefore yield more reliable fluor-
2-aminopurine, and circular dichroism investiga-
escenceresonanceenergytransfer(FRET)dataandfluor-
tionssuggestthatATonlycausesminimalstructural
escence anisotropy data. These probes have therefore
perturbations to normal B-DNA. Furthermore, we
become increasingly popular over the past decade for
find that AT shows favourable base-pairing
studyinginteractionsbetweenDNA/RNAandothermol-
properties with thymine and more surprisingly also ecules and to explore nucleic acid structure.
withnormaladenine.Inconclusion,ATshowsstrong Over the past years various classes of base analogues
potential as a new fluorescent adenine analogue have been developed, each with specific properties. With
formonitoringchangeswithinitsmicroenvironment exceptionofthetricycliccytosinefamilydescribedinmore
in DNA. detail below, virtually all these classes show significant
sensitivitytotheirmicroenvironment.Thepyrimidineana-
logues designed by Tor and co-workers (2–4), pyrrolo-dC
INTRODUCTION (5), the blue fluorescent triazole deoxycytidine analogues
Fluorescenceisaverypowerfulandcommonlyusedtech- (6) and the base discriminating fluorescent bases (BDF)
nique for the study of macromolecules, such as DNA, designed by Saito and co-workers (7–11) are just a few
RNA and proteins (1). The virtually non-fluorescent examples of this expanding class of molecules.
nature of the regular nucleic acid bases makes artificial Furthermore, the pteridine analogues of guanine (3-MI
fluorophoressuchasfluorescentbaseanaloguesimportant and 6-MI) (12–15) and adenine (6-MAP and DMAP)
and excellent tools in investigations of DNA or RNA (16) have also been studied thoroughly. Other interesting
systems. Fluorescent base analogues are significantly purine analogues are the adenine analogues A-3CPh and
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
(cid:2)TheAuthor(s)2011.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
by-nc/2.5),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
4514 NucleicAcidsResearch,2011,Vol.39,No.10
A-4CPh that show an increased emission inside RNA
(17,18) and the 7-deazapurines (19,20). Recently, our
group has developed and extensively characterized a
class of tricyclic cytosine analogues, tC, tCo and tC .
nitro
Surprisingly, neither tC nor tCo show sensitivity to their
environment in duplex DNA. Lately, we also presented
tCo and the non-emissive tC as the first nucleobase
nitro
FRET-pair, yielding accurate distance and orientation in-
formation in DNA (21–23). [for recent reviews on fluor-
escent (nucleic acid base) analogues, see references (24),
(25) and (1)].
Oneofthemostwidelyusedfluorescentbaseanalogues Figure 1. Structureof8-(1-pentyl-1H-1,2,3-triazole-4-yl)-20-deoxyadeno-
sine,AT, inwhichthe triazole ringwithn-pentyl,addedtothe normal
is the adenine analogue 2-aminopurine, which shows a
adenine structure, is shown in grey.
quantum yield of 0.68 in water. However, this decreases
dramatically in both single- (0.007–0.02) and double-
stranded DNA (0.0007–0.01) (26). Moreover, a destabil- compared to other fluorescent adenine analogues such as
ization of the duplex of 10(cid:2)C has been determined using the commercially available 2-aminopurine. In a previous
melting studies (27). Furthermore, 2-aminopurine is investigation, several adenosine derivatives of AT were
capable of forming base pairs with both thymine and studied and shown to exhibit a very high-quantum yield
cytosine and is, thus, not selective (28,29). Nonetheless, in THF and red-shifted absorption relative to the DNA
it has found use in countless studies as discussed further absorption band. One of the compounds, having an
below. isopentyl substituent in the triazole ring, showed a very
Fluorescent nucleic acid base analogues have found high-quantumyieldinbothTHF(0.62)andwater(>0.50)
many applications over the years, of which only a few (49). In this work, we characterize the 20-deoxyadenosine
will be highlighted here [for a broader overview see form of AT both in methanol and water. In addition, we
(1,24,30,31)]. The future perspective of personalized haveincorporated it into 10different10-meroligonucleo-
medicine is to allow treatment based on individual tides with various base-stacking environments in order to
genetic data. With this objective in mind, the detection perform a photophysical and structural characterization
of single nucleotide polymorphisms (SNP) has gained of AT in DNA. We find that AT causes only minor per-
increasing attention and fluorescent base analogues, such turbations to natural B-DNA. Furthermore, quantum
as the class of BDF analogues developed by Saito and yields for AT incorporated into single- and
co-workershavebeendesignedforthispurpose.Theprin- double-stranded DNA reach maximum values that far
ciple is based on these analogues showing a distinct dif- exceed corresponding values for 2-aminopurine.
ference in emissive signal depending on the opposite base Moreover, we have investigated the base-pairing specifi-
(7–10). Furthermore, many analogues are recognized as city of AT with all four natural DNA-bases. To our
substrates by DNA/RNA polymerases and have conse- surprise, we find that AT forms equally stable base pairs
quentlybeenusedinstudiesconcerningtheenzymemech- with thymine and adenine. In conclusion, AT is a very
anism,kinetics,substratespecificityandfidelity(5,32–38). promising new fluorescent base analogue, showing high
2-Aminopurine has been used in numerous studies on T4 sensitivity to its microenvironment.
DNA polymerase and Klenow fragment binding to
primer-template DNA as well as T7 RNA polymerase
interaction with the DNA promotor (39–44). Also, in
studies on the mechanism of other proteins and their MATERIALS AND METHODS
binding to RNA or DNA, fluorescent base analogues The synthesis of 8-(1-pentyl-1H-1,2,3-triazole-4-yl)-20-
have been proven to be highly useful. An excellent deoxyadenosine and of the triazolyl 20-deoxyadenosine
example of such an application for DNA is the role of
phosphoramidite monomer (see Scheme 1 in ‘Results’
3-MI in an investigation of the dissociation of the
section) as well as its incorporation into oligonucleotides
multimeric form of the HIV-1 integrase complex upon
and purification of the oligonucleotide strands are
DNA binding (45). In the RNA-context, protein binding
described in the Supplementary Data S1.
studies have also been performed using fluorescent base
analogues, for example, 2-aminopurine has been utilized
Sample preparation
instudiesofadenosinedeaminases(ADARs)whichplaya
role in RNA editing of eukaryotic pre-mRNA (46,47). All samples used in this work, unless stated otherwise,
Finally, fluorescent base analogues have recently entered were prepared in a sodium phosphate buffer, pH 7.5
the area of DNA nanotechnology, exemplified by the use with a total salt concentration of 500mM. The oligo-
of tCo in monitoring the local stability of self-assembling nucleotide concentration was determined by measuring
DNA hexagons (48). the absorption at 260nm. The extinction coefficients of
Herewereportthesynthesisandphotophysicalcharac- the modified oligonucleotide single strands (sequences
terizationofanewfluorescentadenineanalogueinDNA, listed in Table 1) were calculated by summation of the
8-(1-pentyl-1H-1,2,3-triazole-4-yl)-20-deoxyadenosine extinction coefficients of the natural oligonucleotides
(AT) (Figure 1), which shows promising features and of the AT-monomer. This sum was multiplied by 0.9
NucleicAcidsResearch,2011,Vol.39,No.10 4515
Table 1. DNA melting temperatures of the 10 AT-modified
DNA-duplexes (T AT), the corresponding unmodified duplexes (T A)
m m
and the difference in melting temperature between them ((cid:2)T )
m
DNA sequencea Neighbouring T AT T A (cid:2)T
m m m
bases=name ((cid:2)C)c ((cid:2)C)c ((cid:2)C)
of oligomerb
50-d(CGCACATATCG)-30 CA 43 52 (cid:3)9
50-d(CGCAGATGTCG)-30 GG 45 54 (cid:3)9
50-d(CGCAAATATCG)-30 AA 41 50 (cid:3)9
50-d(CGCATATATCG)-30 TA 39 47 (cid:3)8
50-d(CGCATATGTCG)-30 TG 42 50 (cid:3)8
50-d(CGCAGATATCG)-30 GA 43 50 (cid:3)7
50-d(CGCACATTTCG)-30 CT 45 52 (cid:3)7
50-d(CGCAAATCTCG)-30 AC 46 53 (cid:3)7
50-d(CGCACATCTCG)-30 CC 49 56 (cid:3)7
50-d(CGCAGATCTCG)-30 GC 49 54 (cid:3)5
aFor the unmodified strands AT is replaced by A.
bPurines neighbouring AT are shown in bold and pyrimidines are
shown in italic.
cSampleswerepreparedinphosphatebuffer(500mMNa+,pH7.5)ata
duplex concentration of 2.5mM.
M(cid:3)1cm(cid:3)1 and e =90400M(cid:3)1cm(cid:3)1. Extinction coeffi-
TG
cients of the unmodified oligonucleotides were calculated
in the same way.
UV melting experiments
Equalvolumesof5mMsolutionsofsingle-strandedoligo-
nucleotides in buffer were mixed at room temperature. In
order to achieve a correct and complete hybridization the
samples were heated to 85(cid:2)C, followed by cooling to 5(cid:2)C
at the rate of 1(cid:2)C/min. Next, data were recorded during
heatingofthesamplesto85(cid:2)C,followedbycoolingto5(cid:2)C
ata rate of 0.5(cid:2)C/min. The temperature was kept at85(cid:2)C
for 5min between heating and cooling. For unmodified
duplexes a temperature range of 15(cid:2)C to 92(cid:2)C was used
for recording those data. The melting curves were
obtained on a Varian Cary 4000 spectrophotometer with
Scheme 1. (a) Br /NaOAc-buffer, r.t., overnight, 81%. (b) Pd(PPh )
2 34
(4mol%), CuI (8mol%), TMS-acetylene, Amberlite IRA-67, THF, aprogrammablemulti-celltemperatureblockusinganab-
40(cid:2)C, 2h, 91%. (c) (i) DMTrCl, pyridine, overnight, r.t.; (ii) NH sorptionwavelengthof260nm.Meltingtemperaturespre-
3
(25% aq.), 2h 65%. (d) (i) Pentylbromide, NaN3, water, 140(cid:2)C, 1h; sentedinthisarticleareaveragesofthetemperaturevalues
(ii) CuI, ethyl acetate, 14h, r.t., 76%. (e) (i) TMSCl, pyridine, 2h;
at the maximum of the first derivative and at half
(ii) BzCl, 3h; (iii) Water, 15min then NH (aq.), 30min, 68%.
3 maximum of the melting curves and were measured at
(f) DIPEA (0.11ml, 0.64mmol), 2-Cyanoethyl-N,N-diisopropylchloro-
phosphoramidite, DCM, 1h, 91%. (g) Acetic acid, 30min, 34%. least twice.
Circular dichroism
to correct for base-stacking interactions. The extinction
coefficient of the highly soluble AT was determined by Circular dichroism (CD) spectra were recorded on a
dissolving 1.4mg of compound in 50ml of methanol. An Chirascan CD spectrophotometer at 25(cid:2)C. Spectra of all
absorption shift of 4nm was observed compared to AT in samples were recorded between 200 and 350nm and cor-
Milli-Q water. The extinction coefficient of AT in water rected for background contributions. Spectra of solutions
was therefore calculated using the absorption value at containing 2.5mM DNA duplexes, prepared as described
264nminmethanol(9400M(cid:3)1cm(cid:3)1).Theextinctioncoef- above,wererecordedanddatawereaveragedoveratleast
ficients that were used for the different bases at 260nm five measurements at a scan rate of 2nm/s.
were e =9300M(cid:3)1cm(cid:3)1, e =7400M(cid:3)1cm(cid:3)1,
e =11800TM(cid:3)1cm(cid:3)1, e =15300CM(cid:3)1cm(cid:3)1 and Steady-state fluorescence measurements
G A
e =9400M(cid:3)1cm(cid:3)1. Using these values, the following Quantumyields(F)oftheAT-monomerandthedifferent
AT f
extinction coefficients were obtained for the modified AT-modified oligonucleotides were determined relative to
oligonucleotides: e =95900M(cid:3)1cm(cid:3)1, e =86500 thequantumyieldofquininesulphate(F =0.55)in0.5M
GA CT f
M(cid:3)1cm(cid:3)1, e =88800M(cid:3)1cm(cid:3)1, e =91900M(cid:3)1cm(cid:3)1, H SO at 25(cid:2)C (50). Samples containing duplex DNA
GC CA 2 4
e =92700M(cid:3)1cm(cid:3)1, e =84800M(cid:3)1cm(cid:3)1, e = were prepared as described above. The monomer or
GG CC TA
93600M(cid:3)1cm(cid:3)1, e =99000M(cid:3)1cm(cid:3)1, e =91900 samples containing single-stranded oligonucleotides were
AA AC
4516 NucleicAcidsResearch,2011,Vol.39,No.10
settoanabsorptionof(cid:4)0.03attheexcitationwavelength. three-exponential expressions with the program Fluofit
Spectra were recorded on a SPEX fluorolog 3 Pro v.4 (PicoQuant GmbH).
spectrofluorimeter (JY Horiba). For the duplexes and
single-stranded oligonucleotides an excitation wavelength Computation of the internal wobble of AT in DNA
of 300nm was used and emission spectra were recorded
Duplex samples of strands AA and TA (Table 1) were
between 305 and 550nm. The monomer was excited at
prepared as described above in phosphate buffer
282nm and the emission was monitored between 290 (500mM Na+, pH 7.5) containing 65% (w/v) ((cid:3)/(cid:3) &
and 700nm. For the reference, quinine sulphate, water
20)and77.2%(w/v)sucrose((cid:3)/(cid:3) &58).Anexcessof
emission was recorded between 305 and 700nm. water
12% of the unmodified strand was used. Samples were
Steady-state excitation anisotropy spectra were annealed by heating to 75(cid:2)C for 20min, followed by
recorded on a SPEX fluorolog 3 spectrofluorimeter (JY
slowcoolingtoroomtemperature.Steady-stateexcitation
Horiba) using Glan polarizers and emission was
anisotropy measurements were performed as described
measured at 355nm using excitation wavelengths from
above for which spectra were averaged over 22 repeats
280 to 340nm. The polarized excitation spectra of the
and were corrected for background contributions.
AT-monomer were recorded between 240 and 350nm in
Internal dye motion was computed using an in-house
a H O:ethylene glycol (1:2 mixture) glass at (cid:3)100(cid:2)C,
2 designedMatlabprogram(S.Preus,personalcommunica-
monitoring the emission at 352nm. The fluorescence an- tion) based on calculations carried out by Barkley and
isotropy was calculated as: Zimm (51).
I (cid:3)GI
r¼ VV VH
I +2GI
VV VH RESULTS
where the instrumental correction factor Synthesis of AT phosphoramidite monomer
G¼ IHV Starting from commercially available 20-deoxyadenosine
I 1, 8-bromoadenosine 2 was easily obtained using
HH
bromine in a sodium acetate buffer (Scheme 1) as previ-
andI istheexcitationspectrumforwhichthesubscripts
XY ously described (52). The TMS-protected alkyne 3 was
X and Y denote polarization directions of the excitation
installed in the 8-position in good yield (91%) via a
and emission light, respectively, and H and V refers to
standard Sonogashira coupling protocol (53). The
horizontal and vertical, respectively. For an immobile 50-hydroxygroupwasprotectedusing4,40-dimethoxytrityl
fluorophore, such as AT in H2O/ethylene glycol glass, chloride in dry pyridine, followed by an in situ
the fundamental anisotropy (r0i) for a certain transition deprotection of the TMS-group using aqueous ammonia
(i) is related to the angle between the absorbing and the (25%) yielding compound 4 in 65%. In the next step, the
emitting transition moments as follows: pentyl azide was generated in situ through the reaction
between pentyl bromide and sodium azide, followed by
r ¼1(cid:2)3cos2(cid:2) (cid:3)1(cid:3): the addition of compound 4 and copper iodide, yielding
0i 5 i
the cycloaddition product 5 in 76%yield. Asmall sample
of compound 5 was treated with acetic acid to generate
sufficient amount of 8 for measuring its fluorescence
Time-resolved fluorescence measurements quantum yield and anisotropy. The exocyclic amino
Fluorescence lifetimes were measured using group of compound 5 was protected with a benzoyl
time-correlated single-photon counting. The excitation group. In the last step, compound 7 was prepared using
pulse was generated with a Tsunami Ti:sapphire laser DIPEA and 2-cyanoethyl-N,N-diisopropylchloropho-
(Spectra-physics) that was pumped by a Millenia Pro X sphoramidite in dichloromethane (91%).
laser(Spectra-Physics).TheTsunamioutputwastunedto
900nm and frequency-tripled to 300nm. Samples were Spectroscopic characterization of the AT monomer
excited with a repetition rate of 80MHz, a 1-2 ps pulse The absorption and emission spectra of AT, where the
width, IRF 70 ps (FWHM) and a time-window of 10ns. triazole-modified A-nucleobase constitutes the chromo-
However, for the AT monomer and the single stranded phoric/fluorophoric unit of the nucleoside, in Milli-Q
samples AA and GC (Table 1) the frequency was water are shown in Figure 2. Their shapes are virtually
reduced to 4MHz by a pulse picker (Spectra physics, identical to the corresponding spectra recorded for AT in
Model 3985) and a time window of 20ns was used. The methanol (data not shown). In water, the lowest energy
emission was monitored at 355nm and photons were col- absorption band is centred at 282nm with an extinction
lected by a microchannel-plate photomultiplier tube coefficientof16500M(cid:3)1cm(cid:3)1andtheemissionmaximum
(MCP-PMT R3809U-50; Hamamatsu). These counts is located at 353nm. The absorption spectrum recorded
were fed into a multichannel analyzer with 4096 for AT in methanolshows a redshift of 4nm compared to
channels (Lifespec, Edinburgh Analytical Instruments), in water whereas a blueshift of 7nm was observed for the
where a minimum of 10000 counts were recorded in the corresponding emission spectrum of AT. Moreover, AT
top channel. The intensity data were convoluted with the shows a very high quantum yield both in water (0.61)
instrument response function and fitted to one-, two- or and in methanol (0.49). Furthermore, a lifetime of 1.8ns
NucleicAcidsResearch,2011,Vol.39,No.10 4517
Figure 2. Absorption (dashed line) and emission (solid line) spectrum Figure 4. CD spectra of all 10 DNA duplexes containing the AT
of the AT monomer in water. analogue. Duplexes are denoted by the bases neighbouring AT and
consist of the modified strands GA (black), CT (red), GC (dark
blue), CA (brown), GG (purple), CC (orange), TA (light blue), AA
(grey), AC (pink) and TG (green) hybridized to the complementary
natural DNA strand. Spectra were recorded in phosphate buffer
(500mM Na+, pH 7.5) at 25(cid:2)C at a duplex concentration of 2.5mM.
combinations of purines and pyrimidines and their prox-
imity both 30 and 50 to AT. The melting temperatures of
unmodified and modified duplexes prepared by mixing
with their unmodified complementary DNA strands are
presented in Table 1. Comparison between the melting
temperatures of the modified and the corresponding un-
modified duplexes shows an average drop in melting tem-
perature of 8(cid:2)C, and thus a destabilization of double
stranded DNA upon incorporation of AT. Further
analysis of the changes in T suggests that sequences
m
with purines 30 of AT have the largest destabilizing effect
whereas pyrimidines at the 30-side of AT generally have a
Figure 3. Excitation anisotropy spectrum (r, solid line) of the AT nu-
0 smaller effect.
cleoside in a H O/ethylene glycol glass (1:2 mixture) at (cid:3)100(cid:2)C. The
2
isotropic absorption (A , dashed line) is shown as a comparison and
iso
was measured in Milli-Q water. Circular dichroism (CD). The CD spectra that were
recorded for the duplexes, consisting of a modified
was recorded for the AT monomer in water. A small strand containing one AT (Table 1) and its unmodified
residual could be observed, which is most probably a complementary sequence are presented in Figure 4.
result of small deviations in the instrument response Analysis of the spectra between 200 and 300nm shows a
function. high resemblance to the CD signature of a natural
The fundamental fluorescence anisotropy of AT (r , B-DNA-helix, which is characterized by a positive band
0
Figure 3) was measured in a H O/ethylene glycol glass at275nm,anegativebandat240nm,abandwhichisless
2
(1:2 mixture). A constant value of r =0.38 can be negativeorpositiveat220nmandanarrownegativeband
0
observed between 280 and 320nm, which approaches the intheregionbetween220and190nm,precededbyalarge
theoretical maximum value of 0.4 and suggests that positive peak at 180–190nm. Some modified duplexes
the emission and absorption transition moments are vir- show an almost identical CD-spectrum to their natural
tually parallel in this region. The constant r value in this DNA (CT, CA, TG, CC and AA) (Supplementary Data
0
region also indicates a single transition dipole moment in S2), whereas others show more distinct differences
this absorption band. between the corresponding spectra (strands GA, GC,
GG, TA and AC) (Supplementary Data S2). Sequences
GA, GC and GG, having a guanine flanking AT at the
Structure and stability of DNA duplexes containing AT
50-side, show the most perturbed CD signals. As an illus-
UV duplex melting experiments. The 10 modified and tration of this variation, one of the latter spectra (GG) is
normal single-stranded DNA sequences that were used shown in Figure 5 and compared to the CD spectrum of
inthisstudyarelistedinTable1.Toexaminetheinfluence thecorrespondingunmodifiedhelixandtotheabsorption
ofvariousbasessurroundingthemodifiedadenineAT,the differencebetweenthemodifiedandnaturalduplexofthis
neighbouring positions are varied in these 10 sequences. sequence. The resulting differential absorption band cor-
Sequences were designed in order to evaluate relates well to the regions ((cid:4)210, 230 and 295nm) where
4518 NucleicAcidsResearch,2011,Vol.39,No.10
the CD-spectra of natural and modified duplexes differ When a mismatched guanine or cytosine is positioned
most significantly (Figure 5). opposite to AT, further destabilization of the modified
duplexes by an average of 8(cid:2)C can be observed.
Basepairingspecificity. ToinvestigatetheabilityofATto Surprisingly, the positioning of an adenine opposite of
form base pairs with bases other than thymine, we chose AT results in virtually no change in melting temperatures
three of the modified duplexes and replaced the base (<1(cid:2)C) compared to the duplexes with the thymine-AT
opposite AT by a guanine, cytosine and adenine, respect-
base pair. In contrast, an adenine–adenine mismatch in
ively(Table2).Thesethreesequencecontextswerechosen
the three unmodified duplexes leads to an average drop
so that the influence of neighbouring bases could also be in melting temperature of 14(cid:2)C in comparison to the
explored: AT is surrounded either by (i) two purines,
natural adenine–thymine pair (data not shown). This
(ii) two pyrimidines or (iii) a purine and a pyrimidine. suggests that AT is capable of forming equally stable
The melting temperatures and the quantum yields that
base pairs with thymine and adenine.
were obtained for these mismatched duplexes are listed
The quantum yields of the duplexes containing a
in Table 2. For comparison, the data recorded for the
guanine-AT mismatch increase between 13 and 36% in
modified matched duplexes are also shown. The neigh-
comparison to the normal matched modified duplexes
bouring bases did not significantly influence the depres-
and the corresponding values for cytosine-AT mismatches
sion in melting temperature caused by the mismatches.
are0and36%.Fortheadenine-ATbasepairadecreasein
quantum yield between 0 and 27% is observed. However,
we calculated up to 16% of the duplexes to be denatured
atthetemperatureatwhichthesemeasurementswereper-
formed(25(cid:2)C).Takingintoaccountthisandexperimental
errors when measuring low-quantum yields, the changes
are fairly low and should not be over-interpreted.
Furthermore, when comparing the spectral shape and
emission maxima as well as CD-spectra of the various
mismatched duplexes to that of the corresponding
matched duplexes, virtually no changes can be observed
(data not shown).
Computation of the internal wobble of AT in DNA. To
estimate the internal motion of AT inside the base stack
ofDNA,anisotropymeasurementswererecordedfortwo
of the modified duplexes, AA and TA (Table 1), which
were prepared in phosphate buffer (500mM Na+, pH
Figure 5. CDspectraofthemodifiedGG-duplex(solidline)andofthe 7.5) of high viscosity (65 and 77.2% sucrose w/v). The
corresponding natural duplex (dashed line). The difference in absorp- high viscosity hinders motion of the DNA helices in
tionspectraofATandA(dottedline)wasobtainedbysubtractingthe between the moment of excitation and emission. In
absorption spectra of equimolar solutions of the natural GG-duplex standard phosphate buffer (500mM Na+, pH 7.5) anisot-
from the modified GG-duplex. Spectra were recorded in phosphate
buffer (500mM Na+, pH 7.5) at 25(cid:2)C at a duplex concentration of ropyvaluesof0.30and0.21wererecordedforsamplesAA
2.5mM. and TA, respectively. When performing the same
Table 2. Comparison of the melting temperatures and quantum yields of duplexes containing a mismatched base opposite of AT with its
matched counterparts
Neighbouring Base T AT T AT (cid:2)T (cid:3) b,c (cid:3)ATb,c
m mismatch m m f mismatch f
bases=name of opposite ((cid:3)(cid:2)C)b ((cid:3)(cid:2)C)b ((cid:3)(cid:2)C)
oligonucleotidea ATa
GA G 36 (cid:3)7 0.009 0.008
C 36 43 (cid:3)7 0.010
A 43 0 0.008
CT G 36 (cid:3)9 0.006 0.005
C 36 45 (cid:3)9 0.005
A 46 1 0.004
CA G 36 (cid:3)7 0.015 0.011
C 36 43 (cid:3)7 0.015
A 42 (cid:3)1 0.008
aOligonucleotidesarenamedusingthetwobasesneighbouringAT.FullsequencescanbefoundinTable1.ThebaseoppositeATintheduplexesis
replaced by the three other bases that normally do not base-pair with adenine (G, C, A).
bSamples were prepared in phosphate buffer (500mM Na+, pH 7.5) at a duplex concentration of 2.5mM.
cFluorescencequantumyieldsweredeterminedrelativetothereferencequininesulphatein0.5MH SO ((cid:3) =0.55)andareaveragesofatleasttwo
2 4 f
independent measurements.
NucleicAcidsResearch,2011,Vol.39,No.10 4519
measurements in high-viscosity buffer, a limiting anisot- Table 3. Wavelengths of emission maxima and quantum yields of the
ropy of on average 0.34 was measured for both samples, ten AT-modified single strands
AA and TA. The difference between the values measured
Oligonucleotidea Em (nm) Fb
at 65 and 77.2% sucrose (w/v) was within experimental max f
error.Thelimitinganisotropyrecordedhereislowerthan AA 353 0.21
the fundamental anisotropy that was recorded for the AT CA 351 0.090
monomerinavitrifiedmatrix(0.38).Thedifferenceinthe TA 352 0.076
GC 358 0.042
anisotropy in DNA in viscous solution and the monomer
AC 357 0.031
inavitrifiedmatrixisaresult ofmotionsofATinsidethe
CC 355 0.021
base-stack and calculations (51) suggest an internal GA 353 0.018
wobble of 16 degrees. CT 358 0.014
TG 355 0.006
GG 355 0.005
Photophysical characterization of AT in DNA context aOligonucleotides are named by the two bases neighbouring AT. Full
sequences can be found in Table 1.
FluorescencepropertiesofATinsingle-strandedDNA. The bFluorescencequantumyieldsweredeterminedrelativetothereference
fluorescencepropertiesofATuponincorporationintothe quinine sulpfate in 0.5M H2SO4 ((cid:3)f=0.55) at an excitation wave-
lengthof300nmandareaveragesofthreeindependentmeasurements.
10 single-stranded oligonucleotides (listed in Table 1) are
Samples were prepared in phosphate buffer (500mM Na+, pH 7.5).
shown in Table 3. These data show no large shift in the
emission maxima (351–358nm) in comparison to the free
AT-monomer (354nm). Furthermore, when comparing Table 4. Wavelength of emission maxima and quantum yields of the
the emission spectra of the various modified oligonucleo- ten AT-modified double-strands
tides to that of the monomer AT and the corresponding
modified duplex, only minimal changes in the spectral Oligonucleotidesa Emmax (nm) (cid:3)fb
envelope can be observed (data not shown).
AA 350 0.050
The fluorescence quantum yields for the 10 modified TA 353 0.016
oligonucleotides range between 0.005 and 0.21 (Table 3). CA 354 0.011
TheAAsequenceshowsthehighestquantumyield(0.21). AC 349 0.009
GA 351 0.008
In general, with exception of GA, the highest quantum
CT 351 0.005
yields are observed for the sequences with an adenine
GC 351 0.005
flanking AT at the 30-side (AA, CA, TA). Lower CC 352 0.004
quantum yields are observed for a cytosine 30 of AT TG 351 0.003
(GC, AC, CC) and these values decrease further for GG 354 0.003
having a thymine (CT) and finally a guanine 30 of AT aOligonucleotides are named by the two bases neighbouring AT. Full
(TG, GG). Not surprisingly, the lowest quantum yield sequences can be found in Table 1.
(0.005) was found for the sequence with two guanines bFluorescencequantumyieldsweredeterminedrelativetothereference
(GG) neighbouring AT. The average quantum yield quinine sulphate in 0.5M H2SO4 ((cid:3)f=0.55) and are averages of at
least two independent measurements. Samples were prepared in phos-
(0.051) is approximately 12 times lower for the modified
phate buffer (500mM Na+, pH 7.5) at a duplex concentration of
oligonucleotides in comparison to the free AT-monomer 2.5mM.
(0.61).
Average lifetimes of AT incorporated into the 10
modified oligonucleotides listed in Table 1 range
between 0.9 and 2.0ns. Fluorescence intensity decays single-stranded oligonucleotides, sequence AA shows the
were fitted to three lifetimes for all samples, except for highestquantumyield.Furthermore, theobservationthat
strands AA and GC, which showed a mono-exponential anadenine30 ofAT(AA,TA,CA)yieldsthehighestfluor-
decay. For all strands, except for GC ((cid:4)=2.0ns), the escence (with exception of GA) and a guanine 30 of AT
average lifetime of the incorporated AT is lower than the (TG,GG)yieldsthelowestquantumyieldisfoundalsofor
lifetime of the AT monomer in water (1.8ns). the duplex case. As for the single strands, sequence GG
shows the lowest quantum yield in the duplex. The
Fluorescence properties of AT in double-stranded average quantum yield of AT incorporated into duplexes
DNA. The fluorescence properties of the AT-monomer (0.011) is reduced (cid:4)5-fold in comparison to the average
when incorporated into duplex DNA, consisting of quantum yield of the single strands (0.051).
the modified sequences (listed in Table 1) with their Average lifetimes of AT incorporated into the 10
unmodified complement are shown in Table 4. The modified duplexes vary between 0.2 and 1.2ns. All fluor-
emission maxima for the modified duplexes escence intensity decays for these samples were fitted to
(349–354nm) are virtually not shifted in comparison to three fluorescence lifetimes. For all duplexes, the average
the AT-monomer (354nm) and the modified single lifetimes of AT are lower than the lifetime of the AT
strands (351–358nm). monomer in water. Also when compared to the corres-
Thefluorescencequantumyieldsthatweremeasuredfor ponding lifetimes of AT in single-stranded oligonucleo-
the 10 modified duplexes range between 0.050 and 0.003 tides, these lifetime values are lower except for strands
(Table 4). Analogous to the quantum yields for the GG, TG and GA.
4520 NucleicAcidsResearch,2011,Vol.39,No.10
DISCUSSION 0.34whichwasrecordedforATinduplexesAAandTAin
viscous sucrose solutions. The difference compared to the
In a recent study, we characterized a novel series of 8-
fundamental anisotropy of AT in a vitrified matrix (0.38)
substituted adenosine analogues. That investigation
revealed promising features for triazole adenine (AT) wascalculatedtobeduetoaninternalwobbleofATinthe
DNA duplex of 16(cid:2). This value is higher than the
suchasahigh-quantumyieldandared-shiftedabsorption
estimated wobble for the natural canonical bases ((cid:4)5(cid:2)),
band, allowing specific excitation outside the
but lower than the corresponding value for the
DNA-absorption band (49). In the study presented here
we therefore decided to synthesize the 20-deoxyribose de- intercalating dye ethidium bromide (21(cid:2)) (51).
rivative of AT in order to perform a comprehensive Furthermore, melting experiments were performed to
photophysical and structural characterization when AT examine the base pairing specificity of AT. Three of the
is incorporated into DNA. modified sequences (GA, CT, CA) were annealed with
strands containing an adenine, guanine or cytosine
Structure and stability of DNA duplexes containing AT oppositeofATinsteadofathymine.Meltingtemperatures
recorded for AT-cytosine/guanine mismatches reveal an
To investigate the influence on B-DNA stability after in-
average drop in melting temperatures of 16(cid:2)C, almost
corporationofAT,weperformedUV-meltingexperiments
twice as large compared to an AT-thymine match (8(cid:2)C).
on the 10 different 10-mer duplexes. These sequences
The AT-cytosine/guanine mismatch melting temperatures
contain one AT flanked by varying combinations of
areinlinewiththeaveragedestabilization(14(cid:2)C)recorded
purines and pyrimidines as direct 30 and 50 neighbours.
for a single-base mismatch of adenine–adenine in these
The resulting melting data show a moderate destabiliza-
duplexes compared to their natural matching counter-
tion of duplexes containing AT by an average drop in
parts. It is therefore reasonable to assume that AT
melting temperature of 8(cid:2)C compared to the natural
exhibits some hydrogen-bonding with thymine but shows
duplexes. The decrease in stability is dependent on the
bases surrounding AT. A more thorough investigation virtually no base-pairing with guanine or cytosine.
shows that duplexes containing a pyrimidine 30 of AT Surprisingly, the same mismatch experiment performed
for an AT-adenine mismatch showed on average no
are the most stable. This is most likely due to a better
stacking between AT and the surrounding bases when it further destabilization of the duplexes (8(cid:2)C) compared
is flanked by a pyrimidine as a 30 neighbour. The gen- to the AT-thymine case. This suggests that AT is able to
form equally strong basepairs withthymine and adenine.
eral destabilization is most probably caused by steric
To the best of our knowledge, no similar findings have
clashes of the substituent triazole ring and pentyl chain
in the 8-position of AT with the phosphate backbone been previously reported for other adenine analogues.
and sugar moieties. This steric clash would account for a The putative AT.A base pair in Figure 6 would have a
considerableenergycostifthebaseistobeaccommodated similar overall shape to a Watson–Crick base pair and
in the duplex in its anti conformation. would have good stacking interactions with surrounding
Similar observations have been made for 8-methoxy base pairs and, thus, constitutes a plausible structure.
and 8-bromo substituted adenine, which showed an Protonation of N(1) of adenine would provide a second
average destabilization of 6(cid:2)C in 11-mer DNA duplexes hydrogen bond. Additionally a possible third weakly
(1.0M NaCl, pH 7, 0.010M phosphate, 0.001M EDTA) stabilizing C-H—N hydrogen bond to H(2) on adenine
(54). It has also been shown previously that many may be formed (58). It should be mentioned that the
8-substituted purines show a preference for the syn con- pKa of N(1) of adenine is approximately 4.0. However,
formation(55,56). However,calculations suggesteda syn/ in double-stranded DNA, if it is or has the possibility of
anti equilibrium to be present in DNA helices for other beinginvolvedinH-bonding,itcanberaisedmuchhigher.
analogues (57). The preferred orientation of the modified Thus, the proposed base pair structure is presently specu-
base around the glycosidic bond in duplex DNA depends lative and future high-resolution structural studies will be
on the hydrogen bonding preferences, which can change
depending on the bulky substituent and can improve the
relative stability of Hoogsteen pairs. Stabilization of the
syn orientation compared to the anti orientation helps to
explain the mutagenicity of bulky DNA adducts. In the
caseoftheAT(anti).T basepair thetriazole moietywould
have strong unfavourable steric and electrostatic inter-
actions with the adjacent phosphodiester backbone,
whereas in the AT(syn) orientation there is the possibility
of a third weakly stabilizing C-H—O hydrogen bond to
thymine (58). In both versions of the AT.T base pair, the
pentyl group will project into the aqueous environment
and this could lead to entropic destabilization.
Despite this possible change in conformation, AT still
shows base pairing capacity to thymine and seems to be
Figure 6. Putative stabilized AT.A base pair. Only the sugar attached
stacked reasonably well in the DNA duplex in that case. toATisshownanditstriazoleandpentylchainarecolouredgrey.R ,
1
Thiscanbeconcludedfromalimitinganisotropyvalueof R and R represent the rest of the DNA structure.
2 3
NucleicAcidsResearch,2011,Vol.39,No.10 4521
necessary to accurately determine the base pairing minor differences in CD-signal can be detected where the
properties of AT. regular DNA bases absorb (60,63).
As was mentioned above, bulky DNA adducts can in-
fluence the hydrogen bonding preferences of the base, re-
sulting in potential mispairing involving the Hoogsteen Fluorescence properties of AT in single- and
face. Deviations in the base pairing specificity have also double-stranded DNA
beenfoundforotherbaseanaloguesduetotheintroduced
The emission maximum and the spectral envelope of AT
modifications to the natural base. The adenine analogue
arevirtuallyunaffectedbyincorporationintooligonucleo-
2-aminopurine is, for example, able to form base pairs
tides.Ontheotherhand,asreportedforvirtuallyallbase
both with thymine and cytosine (28,59). Furthermore,
analogues (5,15,16,24,26), incorporation of AT in oligo-
the base discriminating fluorescent analogue BPP can
nucleotides causes a significant drop in fluorescence
formstable basepairs bothwithadenine andguanine (7).
quantum yield. Quantum yield values for AT in
The drop in melting temperature caused by AT (8(cid:2)C) is
single-stranded DNA (0.005–0.21) are on average 12
not dramatic when compared to most other fluorescent
times lower than those recorded for the AT monomer in
base analogues which have been incorporated into
water (0.61). These values are reduced approximately
DNA. For example, earlier observations for decamers
another five times in duplexes (0.003–0.050).
containing the fluorescent analogue 2-aminopurine have
Furthermore, the average lifetimes of AT in both single-
shown a drop in stability of 10(cid:2)C (0.1M KCl, 0.1mM
(0.9–1.5ns; except GC: <(cid:4)>=2ns) and double-stranded
EDTA, 21mM DNA duplexes) (27). In another study
DNA (0.2–1.2ns) are also reduced compared to the
involving 14-mer duplexes a decrease of 6(cid:2)C in melting
lifetimeoftheATmonomerinwater(1.8ns).Withexcep-
temperature was observed after incorporation of
tionofthesingle-strandedsamplesAAandGC,showinga
2-aminopurine in place of adenine (0.015M sodium
mono-exponentialdecay,threelifetimeswererecordedfor
citrate, pH 7.25, 50mM DNA duplexes) (60). However, AT incorporated in all double- and single-stranded
in a recent study, a decrease in melting temperature of
samples. These changes in photophysical properties
merely 3.5(cid:2)C was recorded for 13-mer duplexes (10mM when going from monomer AT to DNA-incorporated
Na2HPO4/NaH2PO4, 150mM NaCl, pH6.5, 11.9mM AT may be explained by different levels of stacking and,
DNA duplexes) (61). Besides 2-aminopurine, other
thus, electronic interaction with other bases in DNA. For
adenine analogues such as 6MAP and DMAP on
both the single- and double-stranded samples an increase
averagehaveshown aslight(2.4(cid:2)C) tomoderatedestabil-
in the non-radiative rate constant (k ) as well as a
ization (4.6(cid:2)C) in 21-mer duplexes (10mM Tris, pH 7.5, decrease in the radiative rate constant (nkr ) of AT can be
f
with 10mM NaCl), respectively (16). Furthermore, the
observed compared to its free monomeric form (data not
pteridine guanine analogue 3-MI shows an average drop shown). First of all, the absorption of AT is most likely
in melting temperature (8.3(cid:2)C) comparable to a
decreasedinDNAcomparedtothefreemonomer,asseen
single-base mismatch under similar conditions (15).
forallnatural bases,due tostackinginteractions withthe
Importantly, the CD-spectra recorded for the different
surroundingbases.Asaresulttheradiativerateconstant,
duplexes containing AT show a general signature for
k, is lowered and gets influenced by the reduction of the
regular B-form DNA. This means that the AT-modifica- exftinction coefficient since they are related through the
tiondoesnotperturbtheoverallB-formoftheDNA.Five Strickler–Berg equation (64). This corresponds well with
ofthemodifiedduplexes(CT,CA,TG,CCandAA)show the drop in quantum yield and decrease in k (except for
f
an almost identical CD-spectrum to their corresponding strand AC) which we observed for the duplex samples
natural duplexes. Surprisingly, no change in CD signal compared to the single strands. Second, several conform-
was found for the duplexes AA and CT in the region ations of AT might be present in DNA. Calculations
where only the AT absorption bands are situated. suggest that an increase in the dihedral angle between
Similar findings have been reported in the case of tCo the triazole ring and the adenine moiety (!), starting
and currently we have no satisfactory explanation for from a planar conformation (!=0), results in a
this phenomenon (23). Duplexes CA, TG and CC show blueshifted absorption and decreased oscillator strength
slight differences which correspond well to the AT- ab- of the lowest electronic transition (49) (B. Albinsson,
sorption bands. In contrast, the other duplexes show personal communication). It was previously suggested by
more distinct differences compared to the CD-signature looking at the orbital diagrams of the model compound
of their corresponding natural duplexes (GA, GC, GG, 9-methyl-8-(1H-1,2,3-triazol-4-yl)adenine that the single
TA and AC). For the duplexes GA, GC and GG, the bond character of the bond between the adenine and
CD is most perturbed, which may indicate that the triazole moiety of AT gains bond order in the excited
B-DNA structure is more distorted when AT is flanked state, resulting in a planar excited state (49). However,
by a guanine at the 50-side. As expected, the deviations in DNA AT might be forced to remain in a twisted
inCDsignalscorrespondwelltotheabsorptiondifference geometry in the excited state which also results in
of AT and A in duplex DNA. Comparable results were changes in oscillator strength compared to the monomer
foundfortheCDspectraofduplexescontainingthefluor- AT, corresponding to different geometries of AT around
escent analogue tC (62). Also the adenine analogue, the dihedral bond. Again, since oscillator strength is pro-
2-aminopurine, shows a particular significant CD-band portional to the radiative rate constant (k) of the excited
f
>300nm when incorporated into duplex DNA, whereas state through the Strickler–Berg equation (64), these
4522 NucleicAcidsResearch,2011,Vol.39,No.10
different emitting species may also explain the multiple CONCLUSION
fluorescence lifetimes observed. Finally, changes in
We have performed a comprehensive photophysical and
photophysical properties can also be explained by in-
structuralcharacterizationofthe20-deoxyribosederivative
creases in knr upon incorporation of AT in oligonucleo- ofATincorporated intoDNAandinitsmonomericform
tides as a result of, for example, photoinduced electron and have found very promising properties of AT
transfer from neighbouring bases, as will be further
compared to the widely used commercially available
described below. 2-aminopurine. AT causes only minimal structural per-
Not only we do notice a general decrease in quantum turbations to B-DNA. Furthermore, AT has a very
yield upon incorporation of AT into DNA, but also sig-
high-quantum yield as a monomer in water (0.61) as
nificantvariationsinfluorescenceareobservedwhenATis
well as in methanol (0.49). Inside DNA, the quantum
placed in various sequence contexts. This is a common
yield reaches values >20% in certain single strands and
property of many fluorescent base analogues (15,16,65–
5% in double strands, 10 to 5 times higher, respectively,
67). Sequence AA, with two adenines neighbouring AT,
than corresponding values for 2-aminopurine. These
shows the highest quantum yield value both for single-
promising features should lead to future applications of
(0.21) and double-stranded DNA (0.05). Quantum yield AT in studies concerning the structure, dynamics and
valuesdecreaseforhavinganadenine(CA,TA,exception: interactions of nucleic acids.
GA),acytosine(GC,AC,CC)orathymine(CT)flanking
AT on the 30-side. The lowest quantum yield values are
detected in the case where AT is flanked with two SUPPLEMENTARY DATA
guanines (GG; 0.005 ss; 0.003 ds) or with a guanine at
Supplementary Data are available at NAR Online.
the 30-side (TG; 0.006 ss; 0.003 ds). This is not surprising,
since guanine has the lowest oxidation potential of all
natural bases (68) and therefore might transfer an
ACKNOWLEDGEMENTS
electron into the neighbouring AT resulting in a fast
relaxation to the ground state. This kind of quenching Joakim Ka¨rnbratt, Søren Preus and Dr Francois-
Alexandre Miannay are gratefully acknowledged for
pattern has been seen before both for the base
their input concerning the photophysical properties of
discriminating analogue BPP and 2-aminopurine when
AT and for technical assistance.
flanked by a guanine (7,66). However, sequences that
have a guanine neighbouring AT at the 50-side (GC and
GA) show significantly higher quantum yields (0.042 and
FUNDING
0.018, respectively). This corresponds to previous obser-
vations made for the fluorescent base analogue tCo, Swedish Research Council (to L.M.W.); Olle Engkvist
namely that both proximity to a guanine and structural Byggma¨stare Foundation (to L.M.W. and M.G.);
differences areimportantforquenching (23).Thesestruc- European Community’s Seventh Framework Programme
tural differences probably include parameters such as entitled READNA (FP7/2007-2013; grant
relative orientation and stacking of AT and neighbouring HEALTH-F4-2008-201418 to A.H.E.-S. and T.B.).
guanines. Funding for open access charge: Swedish Research
Finally, it is important to note that the maximum Council.
quantum yield recorded for AT in single strands (0.21,
Conflict of interest statement. None declared.
AA) is approximately 10 times higher than the maximum
value measured for the widely used dye 2-aminopurine.
Furthermore, the maximum quantum yield of AT when
REFERENCES
incorporated into duplexes is approximately five times
higher than the corresponding value for 2-aminopurine. 1.Sinkeldam,R.W., Greco,N.J. and Tor,Y. (2010) Fluorescent
It should also be noted that good lines of evidence analogs of biomolecular building blocks: design, properties, and
applications. Chem. Rev., 110, 2579–2619.
suggest that these values for 2-aminopurine are overesti-
2.Greco,N.J. and Tor,Y. (2005) Simple fluorescent pyrimidine
mations since in some cases 2-aminopurine was analogues detect the presence of DNA abasic sites.
incorporated at the ends of the oligonucleotide (26). J. Am. Chem. Soc., 127, 10784–10785.
Moreover, the maximum quantum yield values for AT in 3.Srivatsan,S.G., Greco,N.J. and Tor,Y. (2008) A highly emissive
fluorescent nucleoside that signals the activity of toxic
single-stranded DNA reported here are also five and two
ribosome-inactivating proteins. Angew. Chem., Int. Ed. Engl., 47,
times higher, respectively, than for the pteridine adenine 6661–6665.
analogues 6MAP (0.041) and DMAP (0.11) under similar 4.Srivatsan,S.G., Weizman,H. and Tor,Y. (2008) A highly
conditions (16). In duplexes, the fluorescence of 6MAP fluorescent nucleoside analog based on thieno 3,4-d pyrimidine
senses mismatched pairing. Org. Biomol. Chem., 6, 1334–1338.
and DMAP is quenched by an additional 2% compared
5.Berry,D.A., Jung,K.Y., Wise,D.S., Sercel,A.D., Pearson,W.H.,
tothemonomerfluorescencequantumyield.Furthermore
Mackie,H., Randolph,J.B. and Somers,R.L. (2004) Pyrrolo-dC
thebrightness((cid:4)(cid:3) (cid:5)e)ofATinduplexDNAisapproxi- and pyrrolo-C: fluorescent analogs of cytidine and
f
mately three times higher than for 2-aminopurine and es- 20-deoxycytidine for the study of oligonucleotides.
Tetrahedron Lett., 45, 2457–2461.
sentially the same as for 6MAP when comparing with
6.Dodd,D.W., Swanick,K.N., Price,J.T., Brazeau,A.L.,
extinction in the lowest energy absorption band
Ferguson,M.J., Jones,N.D. and Hudson,R.H.E. (2010) Blue
(16,26,63). fluorescent deoxycytidine analogues: convergent synthesis,