Unraveling the Kinetics of Spare-Tire DNA G-Quadruplex Folding.

The folding of DNA G-quadruplexes (G4) is essential to regulate expression of oncogenes and involves polymorphic long-lived intermediate states. G4 formation requires four G-tracts, but human gene-promoters often contain multiple G-tracts that act as spare-tires. These additional G-tracts are highly conserved and add multiple layers of functional complexity, as they are crucial to maintain G4 function after oxidative damage. Herein, we unravel the folding dynamics of the G4 sequence containing five G-tracts from cMYC, the major proliferation-driving oncogene. We devise a general method to induce folding at constant experimental conditions using a photochemical trapping strategy. Our data dissect the individual kinetics and thermodynamics of the spare-tire mechanism of cMYC-G4.

Parallel reaction pathways accelerate folding of a guanine quadruplex.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Photo-Tethers for the (Multi-)Cyclic, Conformational Caging of Long Oligonucleotides.

Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne-modified photolabile nucleosides into DNA sequences, followed by a CuI -catalyzed alkyne-azide cycloaddition with bis-azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full-length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo-tether strategy presented here provides a robust and versatile tool for the light-activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.

Sensitive detection of cancer cells using light-mediated apta-PCR.

Apta-PCR is an ultrasensitive assay in which aptamers are exploited not only as biomolecular recognition elements, but also as reporter labels for amplification via real-time PCR. This methodology has been successfully applied to the detection of proteins, achieving limits of detection in the picomolar range. The introduction of caged aptamers that bear photo-labile groups, so called cages, at strategic positions so that their tertiary structure and thus their binding properties can be controlled by light, facilitates a more robust and attractive assay in terms of sample conservation and reusability. In this work, we report for the first time the use of caged aptamers for cell detection in an apta-PCR assay. Specifically, a sandwich format is used combining the capture of B-cells by an antibody with the specific detection of Burkitt's lymphoma cancer cells by a caged aptamer, acting as a reporter probe. Elution of the aptamer bound to the cancer cells is performed by light and the number of cells is then correlated with the amount of eluted caged aptamer using real-time PCR analysis. The reported technique shows an excellent sensitivity, achieving detection of as few as 77 cells, and due to the inherent robustness of the assay, this detection platform can be reused for further analyses, demonstrating potential applicability in proteomics and clinical diagnostics.

Three-dimensional protein networks assembled by two-photon activation.

Spatial and temporal control over chemical and biological processes plays a key role in life and material sciences. Here we synthesized a two-photon-activatable glutathione (GSH) to trigger the interaction with glutathione S-transferase (GST) by light at superior spatiotemporal resolution. The compound shows fast and well-confined photoconversion into the bioactive GSH, which is free to interact with GST-tagged proteins. The GSH/GST interaction can be phototriggered, changing its affinity over several orders of magnitude into the nanomolar range. Multiplexed three-dimensional (3D) protein networks are simultaneously generated in situ through two-photon fs-pulsed laser-scanning excitation. The two-photon activation facilitates the three-dimensional assembly of protein structures in real time at hitherto unseen resolution in time and space, thus opening up new applications far beyond the presented examples.

Caged glutathione - triggering protein interaction by light.

Light, GSH, action! Glutathione (GSH) fulfills a universal role as redox factor, scavenger of reactive oxygen species, and as an essential substrate in the conjugation, detoxification, and reduction reactions catalyzed by glutathione S-transferase (GST). A photoactivatable glutathione allows the GSH-GST network to be triggered by light. GST fusion proteins can be assembled in situ at variable density and structures by laser-scanning activation.

The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates.

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.