Tetraplex DNA transitions within the human c-myc promoter detected by multivariate curve resolution of fluorescence resonance energy transfer.
The nuclease hypersensitive element (NHE) III(I) of the c-myc promoter regulates the expression of oncogene c-myc and hence is an important anti-cancer target. Paranemic secondary structure formation within the promoter has been implicated in mechanistic regulation models. Here, it is shown that two monomeric tetraplexes form within the c-myc promoter, which coexist in solution. The development and application of a new experimental approach for detection of conformation transitions in nucleic acids [which exploits the sensitivity of fluorescence resonance energy transfer (FRET) for theoretical spectral resolution by multivariate curve resolution-alternating least-squares (MCR-ALS) method] has been used for this study. The pK(a) for tetraplex transitions are centered around 5.9 +/- 0.2 (between two intercalation topologies) and 6.8 +/- 0.1 (tetraplex to random coil). The presence of two tetraplexes has been further confirmed by S1 nuclease digestion. Finally, it is established that MCR-ALS analysis of FRET at different temperatures, pH, and salt concentrations allows resolution of pure species. Results are discussed in the light of recent observations implicating paranemic DNA motifs within the c-myc NHE in regulation of the oncogene. This method has several advantages over other methods vis-à-vis, high sensitivity and linear detection over a wide concentration range and, particularly, potential applications in intracellular probing.
Kumar
Praveen
P
Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi 110007, India.
Verma
Anjali
A
Maiti
Souvik
S
Gargallo
Raimundo
R
Chowdhury
Shantanu
S
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D031541:Fluorescence Resonance Energy Transfer; D016259:Genes, myc; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015999:Multivariate Analysis; D011401:Promoter Regions, Genetic
D017931:DNA Primers; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D004247:DNA