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:
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
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
F-ara-EdU is now
commercially available from Sigma
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.
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