G-quadruplex formation at human DAT1 gene promoter: Effect of cytosine methylation.

The dopamine transporter gene (DAT1), a recognized genetic risk factor for attention deficit hyperactivity disorder (ADHD) is principally responsible for the regulation of dopamine synaptic levels and serves as a key target in many psychostimulants drugs. DAT1 gene methylation has been considered an epigenetic marker in ADHD. The identification of G-rich sequence motifs potential to form G-quadruplexes is correlated with functionally important genomic regions. Herein, biophysical and biochemical techniques are employed to investigate the structural polymorphism along with the effect of cytosine methylation on a 26-nt G-rich sequence present in the promoter region of the DAT1 gene. The gel electrophoresis, circular dichroism spectroscopy, and UV-thermal melting data are well correlated and conclude the formation of a parallel (bimolecular), as well as antiparallel (tetramolecular) G-quadruplex in Na+ solution. Interestingly, the existence of uni-, bi-, tri-, and tetramolecular quadruplex structures in K+ solution exhibited only the parallel type G-quadruplex. The results demonstrate that in presence of either cation (Na+ or K+) the cytosine methylation reserved the structural topologies unaltered. However, methylation lowers the thermal stability of G-quadruplexes and the duplex structures, as well. These findings provide insights to understand the regulatory mechanisms underlying the formation of the G-quadruplex structure induced by DNA methylation.

Alkali cation-mediated topology displayed by an exonic G-rich sequence of TRPA1 gene.

G-rich sequences are intrinsic parts of the genome, widespread in promoters, telomeres, or other regulatory regions. The in vivo existence and biological significance have established the functional aspect of G-quadruplex structures and thus have developed immense interest in exploring their therapeutic aspects. Herein, using biophysical methods, we examined the structural status and comprehensive cation-dependence of a 17-bp G-rich genomic sequence (SKGT17) located in the coding region of the human TRPA1 gene, known to be associated with various neurovascular, cardiovascular, and respiratory conditions. TRPA1 is primarily seen as a therapeutic target for the development of novel analgesics. Bioinformatics analysis has suggested that 17-bp quadruplex motif is a binding site for transcription factor 'Sp1'. The formation and recognition of SKGT17 G-quadruplex might impact its regulatory functioning. Biophysical studies confirmed that the presence of alkali metal ions facilitated the formation of G-quadruplex in parallel topology. Native gel further substantiated the formation of a biomolecular species. Circular dichroism (CD), UV-thermal melting (Tm), and CD melting confirmed the formation of parallel G-quadruplex with metal ion-dependent stability. The stability of the G-quadruplex formed is found to be significantly high in the presence of K+ ions than that of other ions. Intriguingly, we have also established that this segment of the TRAP1 gene favors G-quadruplex formation over its participation in the corresponding duplex formation under K+ ions conditions. This study attempts to explain the rationale for the stabilization of G-quadruplex in the presence of alkali metal ions and may add to a better understanding and insights into DNA-metal ions interactions.

Structural switching/polymorphism by sequential base substitution at quasi-palindromic SNP site (G → A) in LCR of human ß-globin gene cluster.

The human ß-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression contains five tissue-specific DNase I-hypersensitive sites (HSs). A single nucleotide polymorphism (SNP) (A â†’ G) present in HS4 region of locus control region (LCR), have shown a notable association between the G allele and the occurrence of ß-thalassemia. This SNP site exhibiting a hairpin - duplex equilibrium manifested in A â†’ B like DNA transition has previously been reported from this laboratory. Since, DNA is a dynamic and adaptable molecule, so any change of a single base within a primary DNA sequence can produce major biological consequences commonly manifested in genetic disorders such as sickle cell anemia and ß-thalassemia. Herein, the differential behavior of sequential single base substitutions G â†’ A on the quasi-palindromic sequence (d-TGGGGGCCCCA; HPG11) has been explored. A combination of native gel electrophoresis, circular dichroism (CD), and UV-thermal denaturation (Tm) techniques have been used to investigate the structural polymorphism associated with various variants of HPG11 i.e. HPG11A2 to HPG11A5. The CD spectra confirmed that all the HPG11 variants exhibit a hairpin - duplex equilibrium. Oligomer concentration dependence on CD spectra has been correlated with A â†’ B DNA conformational transition. However, as revealed in gel electrophoresis, HPG11A2 â†’ A5 exhibit the formation of a tetramolecular structure (four-way junction) at higher oligomer concentration. UV-melting studies also supported the melting of hairpin, duplex and four-way junction structure. This polymorphism pattern may possibly be significant for DNA-protein recognition, in the process of regulation of LCR in the ß-globin gene.

Sequence-specific recognition of a coding segment of human DACH1 gene via short pyrimidine/purine oligonucleotides.

With growing in vivo evidence of the roles of triplexes in biological processes, oligonucleotide-directed targeting of double-helical DNA for selective modulation of gene functions has become imperative in their therapeutic aspects. This study comprises a comparative investigation of 17-mer Py- and Pu-TFO for the formation of an intermolecular triplex with a 27-bp genomic homopurine-homopyrimidine track present in the transcriptional element of the human DACH1 gene. The biochemical and biophysical studies have revealed that triplex formation takes place only with Py-TFO and not with its Pu-counterpart. Non-denaturating gel electrophoresis indicated the formation of an intermolecular triplex in Py-motif with an increasing amount of Py-TFO, whereas no such interaction was observed for the Pu-counterpart. UV-thermal melting (T m), circular dichroism (CD) and thermal difference spectra (TDS) studies confirmed the pyrimidine motif triplex formation, which was observed to be significantly pH-dependent and stable at acidic pH (5.2) in the presence of 100 mM Na+ ions. Contrarily, Pu-TFO was not found to bind to the target predominantly, owing to its self-association properties. Further studies have revealed that the GA-rich Pu-TFO adopts a homoduplex structure leading to a limit in its availability for triplex formation. These results may add to our understanding of sequence-specific gene targeting and give insight into designing more specific TFOs depending on genomic targets.