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Spectroscopic characterization of i-motif forming c-myc derived sequences double-labeled with pyrene.

ABSTRACT: In current studies we use the oligonucleotides based on c-myc sequence: CCC CAC CCT CCC CAC CCT CCC C (cmyc22) and CCC CAC CCT CCC CAC CCT CCC CA (cmyc22A) functionalized by pyrene moieties at both termini. Results of the circular dichroism (CD), UV absorption melting experiments, and steady-state fluorescence measurements of pyrene-modified i-motifs as well as their unlabeled precursors are presented and discussed here. The pyrene labels have a remarkable influence on i-motif stability which was deduced from CD spectra and confirmed by UV melting experiments. Both probes emit fluorescence band of pyrene monomer with intensity decreasing upon pH lowering.

SUBMITTER: Dembska A

PROVIDER: S-EPMC3696180 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Spectroscopic characterization of i-motif forming c-myc derived sequences double-labeled with pyrene.

Dembska Anna A Rzepecka Patrycja P Juskowiak Bernard B

Journal of fluorescence 20130322 4

In current studies we use the oligonucleotides based on c-myc sequence: CCC CAC CCT CCC CAC CCT CCC C (cmyc22) and CCC CAC CCT CCC CAC CCT CCC CA (cmyc22A) functionalized by pyrene moieties at both termini. Results of the circular dichroism (CD), UV absorption melting experiments, and steady-state fluorescence measurements of pyrene-modified i-motifs as well as their unlabeled precursors are presented and discussed here. The pyrene labels have a remarkable influence on i-motif stability which wa ...[more]

PMID: 23519528

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Project description:Diverse life forms are driven by the evolution of gene regulatory programs including changes in regulator proteins and cis-regulatory elements. Alterations of cis-regulatory elements are likely to dominate the evolution of the gene regulatory networks, as they are subjected to smaller selective constraints compared with proteins and hence may evolve quickly to adapt the environment. Prior studies on cis-regulatory element evolution focus primarily on sequence substitutions of known transcription factor-binding motifs. However, evolutionary models for the dynamics of motif occurrence are relatively rare, and comprehensive characterization of the evolution of all possible motif sequences has not been pursued. In the present study, we propose an algorithm to estimate the strength of purifying selection of a motif sequence based on an evolutionary model capturing the birth and death of motif occurrences on promoters. We term this measure as the 'evolutionary retention coefficient', as it is related yet distinct from the canonical definition of selection coefficient in population genetics. Using this algorithm, we estimate and report the evolutionary retention coefficients of all possible 10-nucleotide sequences from the aligned promoter sequences of 27 748. orthologous gene families in 34 mammalian species. Intriguingly, the evolutionary retention coefficients of motifs are intimately associated with their functional relevance. Top-ranking motifs (sorted by evolutionary retention coefficients) are significantly enriched with transcription factor-binding sequences according to the curated knowledge from the TRANSFAC database and the ChIP-seq data generated from the ENCODE Consortium. Moreover, genes harbouring high-scoring motifs on their promoters retain significantly coherent expression profiles, and those genes are over-represented in the functional classes involved in gene regulation. The validation results reveal the dependencies between natural selection and functions of cis-regulatory elements and shed light on the evolution of gene regulatory networks.

Project description:The model-free analysis (MFA) was applied to measure the average rate constant (<k>) for pyrene excimer formation (PEF) in a series of pyrene-labeled dendrons referred to as Pyx-G(N), where x (= 2N) is the number of pyrenyl labels born by a dendron of generation N ranging from 1 to 6. <k> was measured in four different solvents, namely tetrahydrofuran (THF), toluene, N,N-dimethylformamide (DMF), and dimethylsulfoxide (DMSO). <k> was found to increase linearly with increasing local pyrene concentration ([Py]loc), where [Py]loc had been determined mathematically for the Pyx-G(N) dendrons. The slope of each straight line changed with the nature of the solvent and represented kdiff, the bimolecular rate constant for PEF. kdiff depended on the solvent viscosity (η) and the probability (p) for PEF upon encounter between an excited and a ground-state pyrene. In a same solvent, kdiff for the Pyx-G(N) dendrons was about 360 ± 30 times smaller than kdiff obtained for ethyl 4-(1-pyrene)butyrate (PyBE), a pyrene model compound similar to the pyrene derivative used to label the dendrons. The massive decrease in kdiff observed for the Pyx-G(N) samples reflected the massive loss in mobility experienced by the pyrenyl labels after being covalently attached onto a macromolecule compared to freely diffusing PyBE. Interestingly, the kdiff values obtained for the Pyx-G(N) dendrons and the PyBE model compound followed similar trends as a function of solvent, indicating that the difference in behavior between the kdiff values obtained in different solvents were merely due to the changes in the η and p values between the solvents. Normalizing the <k> values obtained with the Pyx-G(N) dendrons by the kdiff values obtained for PyBE in the same solvents accounted for changes in η and p, resulting in a master curve upon plotting <k>/(fdiff × kdiff) as a function of [Py]loc, where fdiff was introduced to account for some pyrene aggregation in the higher generation dendron (Py64-G(6)). This result demonstrates that <k> represents a direct measure of [Py]loc in pyrene-labeled macromolecules.

Dataset Information

Spectroscopic characterization of i-motif forming c-myc derived sequences double-labeled with pyrene.

Publications

Spectroscopic characterization of i-motif forming c-myc derived sequences double-labeled with pyrene.

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