Photochemistry and Photobiology, 2013, 89: 332–335

Fluorescence Quenching by Intercalation of a Pyrene Group Tethered toan N4-modified Cytosine in Duplex DNA

Rekha R. Avirah and Gary B. Schuster*School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USAReceived 4 June 2012, accepted 11 September 2012, DOI: 10.1111/j.1751-1097.2012.01243.x

ABSTRACT

A series of duplex DNA oligomers was prepared that contain apyrene chromophore linked by a trimethylene chain (-(CH2)3-)to N4 of a cytosine. The pyrene group stabilizes the DNA as evi-denced by an increase in melting temperature. The absorptionspectrum of the linked pyrene chromophore shows a tempera-ture-dependent shift and there is also a strong induced circulardichroism spectrum attributed to the pyrene group. The fluo-rescence of the pyrene chromophore is strongly quenched atroom temperature by linkage to the DNA, but it increasesabove the melting temperature. We attribute these observa-tions to intramolecular intercalation of the pyrene group at abase pair adjacent to its linkage site at cytosine.

INTRODUCTIONThere is widespread interest in using DNA as a supramolecular scaffoldto arrange functional components for nanoscale devices (1,2). Thisinterest is derived from the ease with which DNA can be modified andthe special ability of DNA to self-assemble into readily defined two-and three-dimensional structures (3). We have taken advantage of theseproperties of DNA to arrange monomers of conducting polymers in themajor groove of DNA and to form well-defined molecular wires fromtheir polymerization (4). The organizing and macromolecular scaffold-ing properties of DNA have also been exploited to assemble chromo-phores that enable vectorial energy transfer (5,6). These assembliesexhibit directional energy transfer among multiple chromophores resid-ing in well-defined positions on DNA (2,7,8). Because of pyrene’sunique spectroscopic and photophysical properties, pyrene-functional-ized oligonucleotides are of special interest (9). Herein, we describe aseries of DNA duplexes modified to contain a pyrene chromophorelinked through a trimethylene chain to N4 of a cytosine. Althoughexpected to reside in the major groove, the evidence suggests thatthe pyrene intercalates between neighboring base pairs resulting induplex stabilization and efficient fluorescence quenching.

MATERIALS AND METHODSAll reagents were used as received without further purification. 1-Pyrenepropyl-amine was prepared by a previously reported procedure (10). The PAC phospho-ramidates and O4-triazolyl-deoxyuridine phosphoramidite were obtainedfrom Glen Research and used as received. UV–Vis absorption spectra wererecorded on a Cary 1E spectrophotometer. Emission spectra were acquiredusing a Shimadzu RF-5301PC spectrofluorophotometer. Fluorescence

quantum yield was measured using quinine sulfate (Φ = 0.52) as the stan-dard in 0.05 M H2SO4 (11).

Preparation of pyrene modified DNA oligomers. DNA oligomerswere synthesized on an Expedite 8909 DNA synthesizer by the con-vertible nucleotide approach using the O4-triazolyl-deoxyuridine phos-phoramidite along with PAC phosphoramidites. The resin boundoligonucleotides were treated with about 500 lL of 500 lM 1-pyrenepro-pylamine (in DMF) for 24 h at 55°C. The supernatant was removed, andthe solid support was washed with acetonitrile and then treated with con-centrated aqueous ammonium hydroxide solution at room temperaturefor 12 h. The ammonium hydroxide solution was evaporated to drynesson a Speed Vac at 50°C, and samples were reconstituted with water andpurified by reverse phase HPLC using a Hitachi preparative HPLC sys-tem using Dynamax C18 column. A single peak in the chromatogramwas collected, desalted using Waters Sep Pak cartridges and analyzed byESI mass spectrometry, which showed the expected mass for the molecu-lar ion (see Supporting Information). The concentration of the modi-fied DNA oligomers was determined by its absorption at 260 nm; theextinction coefficient of pyrene amine at 260 nm was taken as2.25 9 104 M

�1cm�1 (12).Tm measurements. Samples for Tm measurement were prepared by

hybridizing 2 lM of the DNA strands in 10 mM sodium phosphate buffersolution (pH 7.0) containing 100 mM of NaCl. The thermal denaturationprofiles were recorded with a heating and cooling rate of 1°C min�1. UVmelting and annealing curves were recorded using a multicell block andtemperature controller on the spectrophotometer.

RESULTS

Synthesis of oligonucleotides

The preparation of the oligonucleotides containing N4-modified cyto-sine, shown in Fig. 1, was accomplished in two steps. Standardmachine-based phosphoramidite synthesis was used to prepareDNA strands containing triazolyl-substituted uracils (13–15). Thesubsequent reaction of these modified strands with 1-(3-aminopro-pyl) pyrene gives the CPy-modified oligomers DNA(1-4) in goodyield. These oligomers were purified by reverse phase HPLC andcharacterized by mass spectrometry (see Supporting Information).In these CPy-containing oligomers, the nucleobase in the 3′-posi-tion adjacent to the modified base are G, C, A and T for DNA(1-4),respectively. As expected, stable duplexes are formed when thepyrene-containing oligomers are combined with their appropriatecomplementary strands.

The melting temperatures (Tm) of the CPy-modified duplexeswere determined by monitoring the intensity of the 260 nmabsorption band characteristic of DNA as a function of temperature.The results are shown in Table 1. The introduction of a single pyrene-modified cytosine results in a significant increase in Tm comparedwith the corresponding unmodified DNA duplex. This signalsthe stabilization of the duplex structure by the CPy modification.

*Corresponding author email: schuster@gatech.edu (Gary B. Schuster)© 2012 Wiley Periodicals, Inc.Photochemistry and Photobiology © 2012 The American Society of Photobiology 0031-8655/13

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For example, the Tm of the duplex formed from DNA-1, whichhas G at the position 3′-to the Py-modified site, is 7°C higherthan that of the corresponding unmodified duplex. (see Support-ing Information).

Absorption and fluorescence properties

The UV absorption spectra for each of the CPy-containing duplexeswere determined in 10 mM phosphate buffer solution. The data areshown in Fig. 2A. For comparison, the absorption spectrum of thepyrene-containing dinucleotide ACPy (see Fig. 1) was recordedunder identical conditions. The UV spectrum of ACPy shows char-acteristic bands at 329 and 344 nm. Interestingly, the pyrene absorp-tion bands of the pyrene-modified duplexes are redshifted by ca10 nm. This shift in absorption maximum is indicative of a stronginteraction between the pyrene chromophore and the DNA (16).The pyrene absorption is also redshifted in the single strands DNA(1-4), (see Supporting Information).

The fluorescence emission spectra of the CPy-modified duplexwere determined in buffer solution and compared with that of ACPy.The results are shown in Table 1 and Fig. 2B. Clearly, incorpora-tion of the pyrene chromophore in DNA duplex results in strong flu-orescence quenching when compared with ACPy. The lattercompound shows structured emission bands at 378, 397 and418 nm with an emission quantum yield (Φem) = 0.046 ± 0.002.There are significant differences in Φem among the different duplexoligomers. For example, DNA-4, which has a T in the 3′-positon,has a Φem that is ca three times greater than DNA-2 or DNA-3where a C or A occupies this position, respectively. The redshift inthe absorption and the quenching of the fluorescence of the pyrenemodified strands suggest that the pyrene chromophore is interca-lated between base pairs in the duplex oligomers (17). The data for

the pyrene chromophore in single strands (see Supporting Informa-tion) indicate that in these cases the pyrene is also sandwiched bynucleobases.

Temperature dependent absorption and fluorescence studies

Examination of the temperature dependence of the absorptionand emission properties of the CPy-modified duplexes supportsan intercalative mode of binding for the pyrene chromophore.For example, increasing the temperature in buffer solution from20 to 80°C for the duplex formed from DNA-1 causes a signifi-cant change to the absorption band at 354 nm, see Fig. 3A. Theabsorption change is relatively modest between 20 and 60°C, butat ca 70°C there is a large decrease in intensity of the 354 nmband accompanied by an increase in the intensity of the absorp-tion bands at ca 331 and 346 nm. An isosbestic point is apparentin Fig. 3A at 351 nm which shows that the concentrations of thetwo absorbing species are linearly related. It is significant that aninflection point in the change of absorption behavior for thisduplex occurs approximately at its Tm.

Temperature dependent changes are also observed in the fluo-rescence spectrum from the duplex containing DNA-1, seeFig. 3B. The fluorescence intensity of the pyrene chromophore(kex = 351 nm, the isosbestic point) increases slowly as the tem-perature is increased from 20 to 60°C and then it increases dra-matically between 60 and 70°C, which corresponds to themelting temperature of the duplex. It is unusual that the fluores-cence intensity of a chromophore increases with increasing tem-perature unless coupled with a positional or environmentalchange. In this case this observation signals the reduction ofquenching caused by intercalation. Similar observations weremade for the duplexes formed from DNA(2-4).

Code Sequences

DNA-1 5’GCGTACPyGACGTATCGA3’

DNA-2 5’GCGTACPyCACGTATCGA3’

DNA-3 5’GCGTACPyAACGTATCGA3’

DNA-4 5’GCGTACPyTACGTATCGA3’

ACPy 5’ACPy3’

Figure 1. Structure of pyrene linked cytidine (CPy) and the sequences ofDNA(1-4).

Table 1. Melting temperature and fluorescence quantum yields of theduplex DNA(1-4) in 10 mM NaPi-buffer, pH 7.0, 100 mM NaCl.

Tm (°C)* Φ†

DNA-1 70 (+7) 0.0009 ± 0.00005DNA-2 68 (+3) 0.0003 ± 0.00002DNA-3 66 (+4) 0.0003 ± 0.00003DNA-4 65 (+3) 0.001 ± 0.0005ACPy - 0.046 ± 0.002

*The number in parentheses is the difference in Tm between the CPy-modifiedduplex and the corresponding unmodified Watson/Crick duplex. †Averageof more than three experiments. kex = 340 nm.

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DNA-1 DNA-2 DNA-3 DNA-4ACPy

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A

Figure 2. (A) Absorption and (B) fluorescence spectra of duplex DNA(1-4) and model system ACPy in 10 mM NaPi-buffer, pH 7.0, 100 mM

NaCl. kex = 351 nm for DNA(1-4) and 340 nm for ACPy.

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Figure 3. Temperature-dependent (A) absorption (2 lM) and (B) fluores-cence (2 lM) spectra of duplex DNA-1 in 10 mM NaPi-buffer, pH 7.0,100 mM NaCl. kex = 351 nm.

Photochemistry and Photobiology, 2013, 89 333

Circular dichroism studies

Circular dichroism spectra of the duplex formed from DNA-1 areshown in Fig. 4. In addition to the CD signals at 254 and 280 nmthat are typical of B-form DNA there is a readily apparent inducedCD signal in the region between 310 and 365 nm corresponding tothe pyrene chromophore absorption. Pyrene is achiral and theobservation of an induced CD signal is strongly indicative of inter-calation of the pyrene chromophore in the helical duplex (18).

The electronic transition of the pyrene chromophoreassigned to the absorption in the 310–365 nm region is shortaxis polarized (19). The negative sign of the ICD observed forDNA-1 can be attributed to the intercalative binding of thepyrene chromophore wherein the pyrene short axis is alignedperpendicularly to the helical axis. Increasing the temperatureresults in a decrease in the intensity of the ICD signal, andabove Tm the ICD signal is completely changed indicating thatthe stacking interaction between pyrene and the base pairs is dis-rupted and the pyrene is outside the base pair pocket. The ICDsignal for the pyrene chromophore above 65°C is weak anddominated by noise. The CD signal at 280 nm, where the DNAbases are the dominant absorbers, decreases as the temperatureis increased.

DISCUSSIONThe substituents on oligonucleotides containing N4-modifiedcytosine are usually expected to reside within the major grooveof the duplex structure. This allows the modified nucleobase tomaintain the normal Watson–Crick hydrogen bonding contactswith guanine, thereby maintaining the B-form DNA structure.Theoretical modeling studies on DNA oligomers having thismodified base have shown that the substituent group is indeedplaced in the sterically less demanding major groove (13). Theadoption of the antirotamer conformation in these cases,although thermodynamically less favorable, is compensated bybase pairing with guanine. However, it is clearly possible that aflat, hydrophobic substituent tethered to N4 of cytosine by a suf-ficiently flexible chain may intercalate between adjacent basepairs. The resulting steric destabilization of the duplex may beoffset by relief of hydrophobic interactions of the chromo-phore and by stabilizing p-stacking interactions with adjacentnucleobases.

The hydrophobic surface area of the pyrene chromophore is approx-imately 184 Å2, which is nearly equivalent to that of a Watson/Crickbase pair (223 Å2) (20,21). Consequently, the pyrene chromophorereadily p-stacks with nucleobases and appears to intercalate intoduplex DNA. Intercalation results in increased thermal stability of

the duplex, a redshift in the pyrene absorption and quenching ofthe pyrene fluorescence as a result of photoinduced electron trans-fer (18,22). Modified nucleotides have been reported in whichpyrene chromophores are tethered to the C5-position of a pyrimidineor the C8-position of a purine. In these instances, intercalation isobserved when a sufficiently long and flexible tether is used (9).The evidence reported above indicates that the pyrene chromo-phore linked through a trimethylene chain to the N4-modifiedcytosine is intercalated at an adjacent base pair in duplex DNA.

The thermal stabilization and spectral shift of the characteristicpyrene absorption bands for the duplexes formed from DNA(1-4)are findings typical of those resulting from the stacking interac-tion between the pyrene and nucleobases. In particular, the elec-tronic interaction between the p-electrons of the intercalatedpyrene and the adjacent bases causes a reduction of the pyreneHOMO-LUMO gap and thereby the redshift in its absorption(23). That intercalation is responsible for the observed shift issupported by the hyperchromism and the blueshift in the absorp-tion upon unstacking at high temperatures where the duplexmelts. Similarly, the quenching of pyrene fluorescence in theCPy-modified duplexes is indicative of intercalation and theobservation that the pyrene fluorescence intensity increases aboveTm also strongly supports intercalative binding. Finally, theinduced CD spectrum of the pyrene chromophore, and its lossabove Tm further substantiates intercalation.

The intercalation of the pyrene chromophore on the N4-modifiedcytosine was unexpected. However, it is clear now that a pyreneattached to the N4-position of the cytosine by a trimethylene linkerhas sufficient length and flexibility to enable intercalation. Theincreased stability of the CPy-containing duplexes is likely due tothe reduction in hydrophobic interactions of the pyrene in thewater-rich major groove. This may provide the distinction betweenpyrene and 2,5-bis(2-thienyl)pyrrole modified systems, whichresides in the major groove; the latter is less hydrophobic thanpyrene and remaining in the major groove is less unfavorable.

In summary, a pyrene chromophore linked through a trimeth-ylene chain to N4-modified cytosine undergoes intramolecularintercalative binding in duplex DNA. This is unusual since thenonbase paired substituent of cytosine projects in the majorgroove. Intercalation has been confirmed through an increasedTm, a redshift in the chromophore absorption, fluorescencequenching and an induced CD signal of pyrene (18).

Acknowledgements—This work was supported by the Vasser WoolleyFoundation, for which we are grateful. Dr. Wen Chen of GeorgiaInstitute of Technology assisted with the preparation of some graphicalmaterial. Dr. Joshy Joseph assisted with the preparation of the DNAsamples. We are grateful for their help.

SUPPLEMENTARY MATERIALSESI mass spectra of the DNA strands, absorption and emissionspectra and fluorescence quantum yields of single stranded oligo-nucleotides, thermal melting profiles of modified and unmodifiedDNA(1-4) and temperature-dependent absorption and emissionspectra of DNA-4 can be found at DOI: 10.1562/2006-xxxxxx.s1.

SUPPORTING INFORMATIONAdditional Supporting Information may be found in the onlineversion of this article:

-30

-15

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250 300 350 400 300 325 350 375 400-4

-3

-2

-1

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1

40 oC50 oC60 oC65 oC70oC

80 oC

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, mde

g

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A B

, mde

gWavelength, nm

Figure 4. Temperature-dependent circular dichroism (10 lM) spectra ofduplex DNA-1 in 10 mM NaPi-buffer, pH 7.0, 100 mM NaCl.

334 Rekha R. Avirah and Gary B. Schuster

Figure S1. ESI spectrum of DNA-1.Figure S2. ESI spectrum of DNA-2.Figure S3. ESI spectrum of DNA-3.Figure S4. ESI spectrum of DNA-4.Figure S5. The melting behaviors of (A) modified and (B)

unmodified DNA 1-4.Figure S6. (A) Absorption and (B) fluorescence spectra of

single strand DNA1-4 in 10 mM sodium phosphate buffer, pH7.0, 100 mM NaCl. Λex = 350 nm.

Figure S7. Temperature dependent (A) absorption (2 lM) and(B) fluorescence (2 lM) spectra of duplex DNA-4 in 10 mM

sodium phosphate buffer, pH 7.0, 100 mM NaCl. Λex = 351 nm.Table S1. Fluorescence quantum yield of single stranded oli-

gonucleotides 1-4 in 10 mM phosphate buffer, pH 7.0, 100 mM

NaCl.Please note: Wiley-Blackwell are not responsible for the con-

tent or functionality of any supporting materials supplied by theauthors. Any queries (other than missing material) should bedirected to the corresponding author for the article.

REFERENCES

1. Bandy, T. J., A. Brewer, J. R. Burns, G. Marth, T. Nguyen and E. Stulz(2011) DNA as supramolecular scaffold for functional molecules: Pro-gress in DNA nanotechnology. Chem. Soc. Rev. 40, 138–148.

2. Ruiz-Carretero, A., P. G. A. Janssen, A. Kaeser and A. P. H. J.Schenning (2011) DNA-templated assembly of dyes and extendedpi-conjugated systems. Chem. Commun. 47, 4340–4347.

3. Su, W., V. Bonnard and G. A. Burley (2011) DNA-templated pho-tonic arrays and assemblies: Design principles and future opportuni-ties. Chem. Eur. J. 17, 7982–7991.

4. Chen, W., G. Guler, E. Kuruvilla, G. B. Schuster, H. C. Chiu and E.Riedo (2010) Development of self-organizing, self-directing molecu-lar nanowires: Synthesis and characterization of conjoined DNA-2,5-Bis(2-thienyl)pyrrole oligomers. Macromolecules 43, 4032–4040.

5. Malinovskii, V. L., D. Wenger and R. Haner (2010) Nucleic acid-guidedassembly of aromatic chromophores. Chem. Soc. Rev. 39, 410–422.

6. Schwartz, E., S. Le Gac, J. J. L. M. Cornelissen, R. J. M. Nolte andA. E. Rowan (2010) Macromolecular multi-chromophoric scaffold-ing. Chem. Soc. Rev. 39, 1576–1599.

7. Kashida, H., T. Takatsu, K. Sekiguchi and H. Asanuma (2010) Anefficient fluorescence resonance energy transfer (FRET) betweenpyrene and perylene assembled in a DNA duplex and its potentialfor discriminating single-base changes. Chem. Eur. J. 16, 2479–2486.

8. Varghese, R. and H. A. Wagenknecht (2009) DNA as a supramolec-ular framework for the helical arrangements of chromophores:Towards photoactive DNA-based nanomaterials. Chem. Commun.,261, 5–2624.

9. Ostergaard, M. E. and P. J. Hrdlicka (2011) Pyrene-functionalizedoligonucleotides and locked nucleic acids (LNAs): Tools for funda-mental research, diagnostics, and nanotechnology. Chem. Soc. Rev.40, 5771–5788.

10. Dale, T. J. and J. Rebek (2006) Fluorescent sensors for organophos-phorus nerve agent mimics. J. Am. Chem. Soc. 128, 4500–4501.

11. Brouwer, A. M. (2011) Standards for photoluminescence quantumyield measurements in solution (IUPAC Technical Report). PureAppl. Chem. 83, 2213–2228.

12. Friedel, R. A. and M. Orchin (1951) Ultraviolet Spectra of AromaticCompounds. Wiley, NY.

13. Macmillan, A. M. and G. L. Verdine (1990) Synthesis of function-ally tethered oligodeoxynucleotides by the convertible nucleosideapproach. J. Org. Chem. 55, 5931–5933.

14. Macmillan, A. M. and G. L. Verdine (1991) Engineering tetheredDNA-molecules by the convertible nucleoside approach. Tetrahedron47, 2603–2616.

15. Xu, Y. Z., Q. G. Zheng and P. F. Swann (1992) Synthesis of DNAcontaining modified bases by postsynthetic substitution - synthesisof oligomers containing 4-substituted thymine - O4-alkylthymine,5-methylcytosine, N4-(dimethylamino)-5-methylcytosine, and 4-thio-thymine. J. Org. Chem. 57, 3839–3845.

16. Long, E. C. and J. K. Barton (1990) On demonstrating DNA interca-lation. Acc. Chem. Res. 23, 273–279.

17. Kumar, C. V. and E. H. Asuncion (1993) DNA-binding studies andsite-selective fluorescence sensitization of an anthryl probe. J. Am.Chem. Soc. 115, 8547–8553.

18. Nakamura, M., Y. Fukunaga, K. Sasa, Y. Ohtoshi, K. Kanaori, H.Hayashi, H. Nakano and K. Yamana (2005) Pyrene is highlyemissive when attached to the RNA duplex but not to the DNAduplex: The structural basis of this difference. Nucleic Acids Res. 33,5887–5895.

19. Kepinska, D., G. J. Blanchard, P. Krysinski, J. Stolarski, K.Kijewska and M. Mazur (2011) Pyrene-loaded polypyrrole microves-sels. Langmuir 27, 12720–12729.

20. Filichev, V. V. and E. B. Pederson (2009) DNA-conjugated organicchromophores in DNA stacking interactions. Wiley EncyclopediaChem. Biol. 1, 493–524.

21. Matray, T. J. and E. T. Kool (1998) Selective and stable base pair-ing without hydrogen bonds. J. Am. Chem. Soc. 120, 6191–6192.

22. Mann, J. S., Y. Shibata and T. Meehan (1992) Synthesis and proper-ties of an oligodeoxynucleotide modified with a pyrene derivative atthe 5′-phosphate. Bioconjug. Chem. 3, 554–558.

23. Duff, M. R., V. K. Mudhivarthi and C. V. Kumar (2009) Rationaldesign of anthracene-based DNA binders. J. Phys. Chem. B 113,1710–1721.

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