Limits...
Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

Huang WC, Tseng TY, Chen YT, Chang CC, Wang ZF, Wang CL, Hsu TN, Li PT, Chen CT, Lin JJ, Lou PJ, Chang TC - Nucleic Acids Res. (2015)

Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.

Show MeSH

Related in: MedlinePlus

(A) CD spectra of 4 μM mt6363 and (B) NMR spectra of 100 μM mt6363 annealed in 10 mM Tris buffer, after the addition of 150 mM K+ for 1 h, and overnight. (C) UV shadowing of gel assays of (i) LD12 + o-BMVC-12C-P, (ii) mt6363 + o-BMVC-12C-P, (iii) LD12 + mt6363 + o-BMVC-12C-P, (iv) LD12 + o-BMVC-6C-P, (v) mt6363 + o-BMVC-6C-P, (vi) LD12 + mt6363 + o-BMVC-6C-P (750 pmol per well). (D) The melting curves monitored at 265 nm of 4 μM mt6363 and their complexes with 8 μM o-BMVC-6C-P and o-BMVC-12C-P in 20 mM K+ solution.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4666356&req=5

Figure 4: (A) CD spectra of 4 μM mt6363 and (B) NMR spectra of 100 μM mt6363 annealed in 10 mM Tris buffer, after the addition of 150 mM K+ for 1 h, and overnight. (C) UV shadowing of gel assays of (i) LD12 + o-BMVC-12C-P, (ii) mt6363 + o-BMVC-12C-P, (iii) LD12 + mt6363 + o-BMVC-12C-P, (iv) LD12 + o-BMVC-6C-P, (v) mt6363 + o-BMVC-6C-P, (vi) LD12 + mt6363 + o-BMVC-6C-P (750 pmol per well). (D) The melting curves monitored at 265 nm of 4 μM mt6363 and their complexes with 8 μM o-BMVC-6C-P and o-BMVC-12C-P in 20 mM K+ solution.

Mentions: CD spectra (Figure 4A) and imino proton NMR spectra (Figure 4B) illustrate the G4 formation of the mt6363 sequence in the presence of K+. The aromatic and methyl NMR spectra can also be found in Supplementary Figure S7. The mt6363 sequence is located at positions 10182–10207 in the reversed mtDNA of the ND3 gene. Specifically, there are 24 imino proton NMR signals of mt6363 in the region of 10–12.5 ppm, indicating that two different G4s co-exist with 3-G in vitro. Competition gel analysis (23) was also conducted to determine whether o-BMVC-12C-P and o-BMVC-6C-P preferentially bind with mt6363 G4 or the LD12 duplex (Figure 4C). The relative quantities of duplex remaining in the mixture and the considerable weakening of pre-stained duplex suggest that these G4 ligands have a higher binding preference for mt6363 G4 than for the LD12 duplex. We further investigated the ability of these ligands to stabilize the G4s of mt6363. Figure 4D presents the CD melting curves of mt6363 at 265 nm in the absence and presence of o-BMVC-12C-P and o-BMVC-6C-P. The melting temperature (Tm) of G4 increased by ∼30°C upon interacting with these compounds, indicating that these G4 ligands are indeed able to stabilize the G4s formed by mt6363. It appears that these o-BMVC derivatives are all good G4 ligands, but not necessarily anti-cancer agents.


Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

Huang WC, Tseng TY, Chen YT, Chang CC, Wang ZF, Wang CL, Hsu TN, Li PT, Chen CT, Lin JJ, Lou PJ, Chang TC - Nucleic Acids Res. (2015)

(A) CD spectra of 4 μM mt6363 and (B) NMR spectra of 100 μM mt6363 annealed in 10 mM Tris buffer, after the addition of 150 mM K+ for 1 h, and overnight. (C) UV shadowing of gel assays of (i) LD12 + o-BMVC-12C-P, (ii) mt6363 + o-BMVC-12C-P, (iii) LD12 + mt6363 + o-BMVC-12C-P, (iv) LD12 + o-BMVC-6C-P, (v) mt6363 + o-BMVC-6C-P, (vi) LD12 + mt6363 + o-BMVC-6C-P (750 pmol per well). (D) The melting curves monitored at 265 nm of 4 μM mt6363 and their complexes with 8 μM o-BMVC-6C-P and o-BMVC-12C-P in 20 mM K+ solution.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4666356&req=5

Figure 4: (A) CD spectra of 4 μM mt6363 and (B) NMR spectra of 100 μM mt6363 annealed in 10 mM Tris buffer, after the addition of 150 mM K+ for 1 h, and overnight. (C) UV shadowing of gel assays of (i) LD12 + o-BMVC-12C-P, (ii) mt6363 + o-BMVC-12C-P, (iii) LD12 + mt6363 + o-BMVC-12C-P, (iv) LD12 + o-BMVC-6C-P, (v) mt6363 + o-BMVC-6C-P, (vi) LD12 + mt6363 + o-BMVC-6C-P (750 pmol per well). (D) The melting curves monitored at 265 nm of 4 μM mt6363 and their complexes with 8 μM o-BMVC-6C-P and o-BMVC-12C-P in 20 mM K+ solution.
Mentions: CD spectra (Figure 4A) and imino proton NMR spectra (Figure 4B) illustrate the G4 formation of the mt6363 sequence in the presence of K+. The aromatic and methyl NMR spectra can also be found in Supplementary Figure S7. The mt6363 sequence is located at positions 10182–10207 in the reversed mtDNA of the ND3 gene. Specifically, there are 24 imino proton NMR signals of mt6363 in the region of 10–12.5 ppm, indicating that two different G4s co-exist with 3-G in vitro. Competition gel analysis (23) was also conducted to determine whether o-BMVC-12C-P and o-BMVC-6C-P preferentially bind with mt6363 G4 or the LD12 duplex (Figure 4C). The relative quantities of duplex remaining in the mixture and the considerable weakening of pre-stained duplex suggest that these G4 ligands have a higher binding preference for mt6363 G4 than for the LD12 duplex. We further investigated the ability of these ligands to stabilize the G4s of mt6363. Figure 4D presents the CD melting curves of mt6363 at 265 nm in the absence and presence of o-BMVC-12C-P and o-BMVC-6C-P. The melting temperature (Tm) of G4 increased by ∼30°C upon interacting with these compounds, indicating that these G4 ligands are indeed able to stabilize the G4s formed by mt6363. It appears that these o-BMVC derivatives are all good G4 ligands, but not necessarily anti-cancer agents.

Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.

Show MeSH
Related in: MedlinePlus