Fluorescent probes provide the most powerful and direct means for studying the folding, function, and localization of biological macromolecules in living cells.(1,2) As compared to the extensive methodologies developed for proteins, relatively few imaging techniques are available for nucleic acids, and all of these are limited by their low sensitivity to alternatively folded structures, large perturbations to native systems, and/or inability to be applied in unmodified cells and organisms.(3) To address unresolved questions in DNA chemical biology, our group is developing probes for characterizing the structure, function, and dynamics of nucleic acids in vivo. Daily work in our lab relies heavily upon the rational design and synthesis of new organic compounds and their metal-containing complexes.(4,5,6) In some cases, novel synthetic methodologies are needed to realize our target molecules.(6,7,8,9,10) Photophysical and biophysical studies are used to characterize the new fluorophores and their ability to report alternatively folded nucleic acids structures.(11,12,13,14,15,16,17) We also use cell biology, fluorescence microscopy and flow cytometry as means to evaluate the efficacy and functional novelty of our compounds in cell cultures and in whole animals.(17,18,19) One key aim of this research has been the search for G-quadruplexes in human cells,(20) but the new tools developed in our laboratory are readily utilized in mainstream chemical genomics analyses as well. Six of our most important research discoveries and corresponding future goals are:

1. An improved metabolic label for detecting DNA synthesis in vivo:

Commonly-used metabolic labels for DNA, such as 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU), are toxic antimetabolites that cause DNA instability, apoptosis and cell cycle arrest.(21,22,23,24,25,26,27) When considering these reports, one might even speculate that DNA labeling and genome stability are inherently opposed by living systems. We recently discovered a new, relatively non-toxic means for delivering bioorthogonal functional groups into DNA using arabinosyl-modified nucleosides.(19) In this approach, DNA synthesis can be readily "birth dated" by adding F-ara-EdU to replicating cells, followed by azide-alkyne "click" reactions for DNA visualization in vivo.(19) This approach is more sensitive and less toxic than methods utilizing BrdU and EdU. F-ara-EdU should therefore replace these compounds in nearly every application that requires metabolic labeling of DNA - including approximately 1'000 academic research studies published each year. We are currently using F-ara-EdU to address long-standing questions in DNA biology that have been difficult or impossible to answer using existing technologies such as the "Immortal Strand Hypothesis." First proposed by Cairns in 1975,(28) this hypothesis states that stem cells can minimize mutations in their genomes by dividing their DNA asymmetrically. By retaining the same set of template DNA strands, a subset of stem cells might provide a true copy of genetic code that is protected from accumulated mutations due to DNA replication. Evidence for and against this controversial hypothesis have been reported,(29,30,31) but the immortal strand hypothesis has not been definitively proven or disproven due to the lack of non-perturbing techniques for tracking the flow of DNA in vivo.(33) We are currently reassessing this hypothesis by using F-ara-EdU to track embryonic chromosomes in vivo. Stem cells that continue to replicate, but retain a full set of chromosomes containing exactly 50% of an embryonic metabolic label would provide direct evidence for Cairns' theory. If this hypothesis holds true, our DNA birth dating approach using F-ara-EdU and a second label to detect new DNA synthesis, may even provide the first universal method for locating stem cells in whole animals.(19) F-ara-EdU is now commercially available from Sigma Aldrich

2. A novel approach for detecting DNA conformational changes:

Modified nucleobases can provide highly sensitive probes of DNA folding by serving as FRET acceptors for the proximal ensemble of unmodified residues that act as FRET donors. The resulting energy transfer efficiencies can be interpreted in terms of the folded state of the DNA or RNA molecule. In contrast to common FRET-based approaches that require two large, exogenous tags, our method requires only the addition of a single styryl or heteroaryl group to the oligonucleotide, and provides similar sensitivity as traditional FRET systems.(5,11,14) We are in the process of combining this approach with our metabolic labeling strategy described in Section 1 to generate nucleobase-derived fluorophores capable of detecting DNA conformational changes in vivo.

3. The first means for controlling N7-metal coordination in DNA:

The N7 position of purine residues is arguably the most important metal-binding site in DNA and RNA molecules. N7-metal binding can have a profound impact on the biological and electronic properties of nucleic acids, but no previous methods have been available for directing metal ions to specific N7 sites in oligonucleotides. By adding a 2-pyridyl group to the C8-position of a guanine residue, a bidentate metal ligand 2PyG is created that can direct metal ions M to defined positions.(16) While 8-substituted guanosines can exhibit some preference for adopting a syn glycosidic bond, DNA folding forces 2PyG to adopt an anti conformation with only little loss in thermodynamic stability (ΔΔG < +1 kcal/mol) as compared to unmodified G-C base pairs.(5,11,14) This approach has the added benefit that changes in 2PyG fluorescence can be used as a direct readout of metal binding.(16) Future studies will utilize duplex DNAs containing two or more 2PyG residues on opposite strands to provide well-defined DNA-DNA interstrand cross-linking sites for metal ions like Pt and Ru that normally exhibit highly promiscuous DNA binding.

4. A new method for the synthesis of DNA-DNA interstrand crosslinks (ICLs):

Bis-chloroethylnitrosourea (BCNU) is a widely used chemotherapeutic drug that generates an ethylene bridge between N1 of deoxyguanosine (dG) and N3 of deoxycytidine (dC). Despite its importance in pharmacology and biochemistry, no synthesis of a homogenous DNA containing this adduct has ever been reported. With this goal, we developed a novel synthetic strategy that utilizes a O6-(2-chloroethyl)-guanine residue containing a photo-labile ortho-nitro-benzyloxycarbonyl (NBOC) group at the N2 position. NBOC stabilizes the normally reactive O6-chloroethyl-guanine by acting as an electron withdrawing group to the N1 position. The ICL precursor therefore remains stable during and after its synthetic incorporation into duplex DNA. NBOC can then be selectively removed by irradiation at 365 nm, and the resulting free amine at the N2 position electronically activates N1 for chloride displacement to give an N1,O6-ethanoguanine cyclic intermediate. This highly reactive species is opened by a cytosine residue in the opposite strand to generate a single ICL product in yields as high as 40%.(9)  In addition to NMR and X-ray crystallographic analyses, this ICL DNA will be used to characterize ICL repair pathways in tissues that exhibit resistance to BCNU treatment. Future studies will also be aimed at DNA-protein cross-linking and crystallization with O6-alkylguanine transferase,(34) as well as targeted mutagenesis in cells using triplex-forming oligonucleotides that contain our N2-NBOC-O6-chloroethyl-guanine ICL precursor.(35)

5. High-affinity fluorescent probes for G-quadruplex DNA:

While convincing evidence has demonstrated the existence of G-quadruplexes in single-cell organisms,(36,37,38,39) the presence of endogenous G-quadruplex structures in multi-cellular organisms remains uncertain.(40,41,42,43,44,45,46) To help address this question, we have developed high-affinity G-quadruplex ligands with dual functions: exhibiting turn-on photoluminescence upon DNA binding and the ability to regulate gene expression in living cells.(4,6,12,13,17,18) These orthogonal readouts are being used to address the potential relationships between G-quadruplex targeting and anti-cancer activities in vivo. Towards this goal, a new family of cationic phthalocyanines containing four guanidinium groups "GPcs" was synthesized (6) and found to exhibit good cellular uptake (18) and exceptionally high G-quadruplex affinity (Kd < 1 nM) with 1'000 to 10'000-fold lower affinities for duplex DNA in vitro.(17) Certain GPcs also inhibit cancer growth by light-dependent (phototoxicity) and light-independent (antimetastatic) pathways in vivo.(47) Unexpectedly, the antimetastatic activities of GPcs are not a result of G-quadruplex binding.(48) Nevertheless, our results provide an important new proof of principle for multifunctional anticancer agents where a single compound can facilitate the photodynamic treatment of a primary tumor while simultaneously inhibiting the formation of metastatic tumors throughout the body.(47)

6. Design and synthesis of planar telomestatin analogs:

The natural product telomestatin is one of the most potent and specific G-quadruplex ligands reported to date.(49) It is also a preclinical anti-cancer drug candidate that possesses better drug-like properties than cationic G-quadruplex ligands like GPcs. Telomestatin has a good, but not ideal, shape for binding G-quadruplex structures due to the presence of a single thiazoline unit that makes telomestatin a non-planar molecule. We have developed a computation-assisted de novo design of planar telomestatin analogs containing 8 variable azole units (oxazole, thiazole, imidazole, or selenazole). While the design of these compounds was relatively straightforward, their synthesis proved to be highly challenging. Only recently did we succeed in preparing the first example of a macrocyclic octazole [oxazole-thiazole]4. To reach this goal, the development of novel synthetic methodology was ultimately needed. Manuscripts describing this new synthetic approach,(10) as well as the G-quadruplex affinity and anti-cancer activities of [oxazole-thiazole]4 are currently in preparation.



Many key questions regarding the structure-function relationships exhibited by RNA and DNA molecules remain unanswered. For example, there are subnuclear organizations such as nucleoli, telomeres, centromeres, and repair/recombination foci that are known to contain single-stranded DNAs with sequences that can adopt hairpin, cruciform, triplex, G-quadruplex, H-motif, and i-motif structures in vitro; but very little is known about the possible presence or function of such structures in vivo. In recent years, a flurry of investigations have addressed the potential biological relevance of G-quadruplex structures, but the presence of functional G-quadruplex structures in multi-cellular organisms remains controversial.(40,41,42,43,44,45,46) This is due, in part, to the common misconception that DNA and RNA are passive information carriers with relatively little structural or functional complexity. A more informed skeptic, however, might argue that evolution will select against G-quadruplex DNA structures in higher organisms due to the need for differential gene expression in variable tissue types.(50)

The possible presence and function of non-canonical DNA structures in multi-cellular organisms will remain a controversial topic until unbiased methods become available for determining structures and dynamics of nucleic acids in vivo. In addition to addressing fundamental questions about G-quadruplexes and other unusual DNA structures, future research in our group will be focused on tracking the movement of parental and progeny virus genomes in vivo, cataloging the cellular factors required for nuclear entry and replication of viral genomes, and re-evaluating Cairns' immortal strand hypothesis using arabinosyl-modified nucleosides. In addition to increasing our basic knowledge about DNA chemistry and biology, these studies will reveal new opportunities for therapeutic intervention, and may even provide the first universal method for locating stem cells in whole animals.



1.         Giepmans, B.N., Adams, S.R., Ellisman, M.H. and Tsien, R.Y. (2006) The fluorescent toolbox for assessing protein location and function. Science, 312, 217-224.

2.         Luedtke, N.W., Dexter, R.J., Fried, D.B. and Schepartz, A. (2007) Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH. Nat Chem Biol, 3, 779-784.

3.         http://www.vectorlabs.com/data/brochures/MBB.pdf

4.         Alzeer, J. and Luedtke, N.W. (2009) pH-mediated fluorescence and G-quadruplex binding of amido phthalocyanines. Biochemistry, 49, 4339-4348.

5.         Dumas, A. and Luedtke, N.W. (2011) Highly fluorescent guanosine mimics for folding and energy transfer studies. Nucleic Acids Res, 39, 6825-6834.

6.         Alzeer, J., Roth, P.J. and Luedtke, N.W. (2009) An efficient two-step synthesis of metal-free phthalocyanines using a Zn(II) template. Chem Commun, 1970-1971.

7.         Seyfried, M.S., Lauber, B.S. and Luedtke, N.W. (2009) Multiple-turnover isotopic labeling of Fmoc- and Boc-protected amino acids with oxygen isotopes. Org Lett, 12, 104-106.

8.         Mata, G. and Luedtke, N.W. (2012) Stereoselective N-glycosylation of 2-deoxy-thioribosides for fluorescent nucleoside synthesis. J. Org. Chem. In press.

9.         Hentschel, S., Alzeer, J., Anglov, T., Schärer, O.D., and Luedtke, N.W. (2012) Synthesis of DNA interstrand crosslinks using a photocaged nucleobase. Angew Chemie Intl Ed, 3466-3469.

10.        Seyfried, M.S., Alzeer, J., Roth, F., and Luedtke, N.W. (2012) Design and synthesis of a planar telomestatin analog. In preparation.

11.        Dumas, A. and Luedtke, N.W. (2010) Cation-mediated energy transfer in G-quadruplexes revealed by an internal fluorescent probe. J Am Chem Soc, 132, 18004-18007.

12.        Qin, H., Ren, J., Wang, J., Luedtke, N.W. and Wang, E. (2010) G-quadruplex-modulated fluorescence detection of potassium in the presence of a 3500-fold excess of sodium ions. Anal Chem, 82, 8356-8360.

13.        Ren, J., Qin, H., Wang, J., Luedtke, N.W. and Wang, E. (2011) Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence. Anal Bioanal Chem, 399, 2763-2770.

14.        Dumas, A. and Luedtke, N.W. (2011) Fluorescence Properties of 8-(2-Pyridyl)guanine "2PyG" as Compared to 2-Aminopurine in DNA. Chembiochem, 12, 2044-2051.

15.        Huber, S.M., Seyfried, M.S., Linden, A., and Luedtke, N.W. (2012) Excitonic luminescence of hemiporphyrazines. Inorg Chem, 51, 7032–7038.

16.        Dumas, A., and Luedtke, N.W. (2012) Site-specific control of N7-metal coordination in DNA using a fluorescent purine derivative. Chem Eur J, 18, 245-254.

17.        Alzeer, J., Vummidi, B.R., Roth, P.J. and Luedtke, N.W. (2009) Guanidinium-modified phthalocyanines as high-affinity G-quadruplex fluorescent probes and transcriptional regulators. Angew Chem Int Ed, 48, 9362-9365.

18.        Membrino, A., Paramasivam, M., Cogoi, S., Alzeer, J., Luedtke, N.W. and Xodo, L.E. (2010) Cellular uptake and binding of guanidine-modified phthalocyanines to KRAS/HRAS G-quadruplexes. Chem Commun, 46, 625-627.

19.        Neef, A., and Luedtke, N.W. (2011). Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proc Natl Acad Sci USA, 108, 20404-20409.

20.        Luedtke, N.W. (2009) Targeting G-Quadruplex DNA with Small Molecules. Chimia, 63, 134-139.

21.        Diermeier-Daucher, S., Clarke, S.T., Hill, D., Vollmann-Zwerenz, A., Bradford, J.A. and Brockhoff, G. (2009) Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A, 75A, 535-546.

22.        Cassiman, J.J., De Clercq, E. and van den Berghe, H. (1983) Induction of sister-chromatid exchange by 5-substituted 2'-deoxyuridines. Mutat Res, 117, 317-327.

23.        Danenberg, P.V., Bhatt, R.S., Kundu, N.G., Danenberg, K. and Heidelberger, C. (1981) Interaction of 5-ethynyl-2'-deoxyuridylate with thymidylate synthetase. J Med Chem, 24, 1537-1540.

24.        De Clercq, E., Descamps, J., Balzarini, J., Giziewicz, J., Barr, P.J. and Robins, M.J. (1983) Nucleic acid related compounds. 40. Synthesis and biological activity of 5-alkynyluracil nucleosides. J Med Chem, 26, 661-666.

25.        Meneni, S., Ott, I., Sergeant, C.D., Sniady, A., Gust, R. and Dembinski, R. (2007) 5-Alkynyl-2'-deoxyuridines: Chromatography-free synthesis and cytotoxicity evaluation against human breast cancer cells. Bioorg Med Chem, 15, 3082-3088.

26.        Cappella, P., Gasparri, F., Pulici, M. and Moll, J. (2008) A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A, 73A, 626-636.

27.        Taupin, P. (2007) BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation. Brain Res Rev, 53, 198-214.

28.        Cairns, J. (1975) Mutation selection and the natural history of cancer. Nature, 255, 197-200.

29.        Karpowicz, P., Morshead, C., Kam, A., Jervis, E., Ramunas, J., Cheng, V. and van der Kooy, D. (2005) Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro. J Cell Biol, 170, 721-732.

30.        Kiel, M.J., He, S., Ashkenazi, R., Gentry, S.N., Teta, M., Kushner, J.A., Jackson, T.L. and Morrison, S.J. (2007) Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU. Nature, 449, 238-242.

31.        Lansdorp, P.M. (2007) Immortal strands? Give me a break. Cell, 129, 1244-1247.

32.        Williams, S.E., Beronja, S., Pasolli, H.A. and Fuchs, E. (2011) Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature, 470, 353-358.

33.        Tajbakhsh, S. (2008) Stem cell identity and template DNA strand segregation. Curr Opin Cell Biol, 20, 716-722.

34.        Margison, G.P. and Santibanez-Koref, M.F. (2002) O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy. Bioessays, 24, 255-266.

35.        Chin, J.Y. and Glazer, P.M. (2009) Repair of DNA lesions associated with triplex-forming oligonucleotides. Mol Carcinog, 48, 389-399.

36.        Schaffitzel, C., Berger, I., Postberg, J., Hanes, J., Lipps, H.J. and Pluckthun, A. (2001) In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc Natl Acad Sci U S A, 98, 8572-8577.

37.        Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D. and Lipps, H.J. (2005) Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat Struct Mol Biol, 12, 847-854.

38.        Cahoon, L.A. and Seifert, H.S. (2009) An alternative DNA structure is necessary for pilin antigenic variation in Neisseria gonorrhoeae. Science, 325, 764-767.

39.        Smith, J.S., Chen, Q., Yatsunyk, L.A., Nicoludis, J.M., Garcia, M.S., Kranaster, R., Balasubramanian, S., Monchaud, D., Teulade-Fichou, M.P., Abramowitz, L. et al. (2010) Rudimentary G-quadruplex-based telomere capping in Saccharomyces cerevisiae. Nat Struct Mol Biol, 18, 478-485.

40.        Sen, D. and Gilbert, W. (1988) Formation of parallel 4-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature, 334, 364-366.

41.        Hanakahi, L.A., Sun, H. and Maizels, N. (1999) High affinity interactions of nucleolin with G-G-paired rDNA. J Biol Chem, 274, 15908-15912.

42.        Siddiqui-Jain, A., Grand, C.L., Bearss, D.J. and Hurley, L.H. (2002) Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc Natl Acad Sci U S A, 99, 11593-11598.

43.        Maizels, N. (2006) Dynamic roles for G4 DNA in the biology of eukaryotic cells. Nat Struct Mol Biol, 13, 1055-1059.

44.        Balasubramanian, S., Hurley, L.H. and Neidle, S. (2011) Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat Rev Drug Discov, 10, 261-275.

45.        Eddy, J., Vallur, A.C., Varma, S., Liu, H., Reinhold, W.C., Pommier, Y. and Maizels, N. (2011) G4 motifs correlate with promoter-proximal transcriptional pausing in human genes. Nucleic Acids Res, 39, 4975-4983.

46.        Chen, C.Y., Wang, Q., Liu, J.Q., Hao, Y.H. and Tan, Z. (2011) Contribution of telomere G-quadruplex stabilization to the inhibition of telomerase-mediated telomere extension by chemical ligands. J Am Chem Soc, 133, 15036-15044.

47.        Vummidi, B.R., Noreen, F., Moelling, K., and Luedtke, N.W. (2012) A photodynamic agent with anti-metastatic activities in vivo. In review.

48.        Walser, T.C., Rifat, S., Ma, X., Kundu, N., Ward, C., Goloubeva, O., Johnson, M.G., Medina, J.C., Collins, T.L. and Fulton, A.M. (2006) Antagonism of CXCR3 inhibits lung metastasis in a murine model of metastatic breast cancer. Cancer Res, 66, 7701-7707.

49.        Shin-ya, K., Wierzba, K., Matsuo, K., Ohtani, T., Yamada, Y., Furihata, K., Hayakawa, Y. and Seto, H. (2001) Telomestatin, a novel telomerase inhibitor from Streptomyces anulatus. J Am Chem Soc, 123, 1262-1263.

50.        Giri, B., Smaldino, P.J., Thys, R.G., Creacy, S.D., Routh, E.D., Hantgan, R.R., Lattmann, S., Nagamine, Y., Akman, S.A. and Vaughn, J.P. (2011) G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA. Nucleic Acids Res, 39, 7161-7178.