Presentation Title

Os(phen)2dppz3+ : an osmium metallointercalator that selectively oxidizes guanine oxidation products

Faculty Mentor

Eric Stemp

Start Date

18-11-2017 12:30 PM

End Date

18-11-2017 1:30 PM

Location

BSC-Ursa Minor 135

Session

Poster 2

Type of Presentation

Poster

Subject Area

physical_mathematical_sciences

Abstract

8-oxoguanine is a common oxidation product in DNA and can lead to missense mutations. The metallointercalator Ru(phen)2dppz2+ is a useful luminescent probe for DNA that has also found use as a guanine-selective oxidizing agent via the flash-quench technique. Here, we show that its osmium analogue can selectively oxidize 8-oxoguanine in double-stranded DNA. With a 3+/2+ couple of 1.15 V, Os(phen)2dppz3+ should be able to oxidize 8-oxo-G (~0.7 V) without oxidizing guanine (~1.3V). MALDI mass spectrometry data confirms that oxidizing guanine using Ru(NH3)63+ and Ru(phen)2dppz2+ produces 8-oxo-G. In plasmid DNA where 8-oxo-G has been incorporated as above, further flash-quench treatment with Os(phen)2dppz2+ and Co(NH3)5Cl2+ leads to crosslinking with histone protein in gel shift experiments and in the chloroform extraction assay. Furthermore, in gel shift experiments with a duplex of the oligonucleotide 5’-ATATGATAT8GATATGATAT -3’ (8 = 8-oxo-G), flash-quench treatment with Ru(phen)2dppz2+ in the presence of histone produces a band of intermediate mobility (presumably 1:1 crosslink) and well-shifted material. In contrast, analogous treatment with Os(phen)2dppz2+ produces only the band of intermediate mobility, consistent with the presence of only a single site that is oxidizable by the osmium complex. Lastly, control experiments indicate that with a duplex of the oligonucleotide 5’-ATATGATATGGATATGATAT -3’ flash quench treatment with Ru(phen)2dppz2+ in the presence of histone produces well-shifted material, whereas treatment with Os(phen)2dppz2+ does not produce crosslinked material, as expected since the osmium (III) complex formed should not be capable of guanine oxidation. Taken together, these results show that Os(phen)2dppz2+ is a promising selective oxidant of 8-oxoguanine in double stranded DNA.

Summary of research results to be presented

8-oxoguanine is a major product of the flash-quench technique Os(phen)2dppz3+ formed using the flash quench technique is promising as a selective oxidant for 8-oxo-G in duplex DNA.

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Nov 18th, 12:30 PM Nov 18th, 1:30 PM

Os(phen)2dppz3+ : an osmium metallointercalator that selectively oxidizes guanine oxidation products

BSC-Ursa Minor 135

8-oxoguanine is a common oxidation product in DNA and can lead to missense mutations. The metallointercalator Ru(phen)2dppz2+ is a useful luminescent probe for DNA that has also found use as a guanine-selective oxidizing agent via the flash-quench technique. Here, we show that its osmium analogue can selectively oxidize 8-oxoguanine in double-stranded DNA. With a 3+/2+ couple of 1.15 V, Os(phen)2dppz3+ should be able to oxidize 8-oxo-G (~0.7 V) without oxidizing guanine (~1.3V). MALDI mass spectrometry data confirms that oxidizing guanine using Ru(NH3)63+ and Ru(phen)2dppz2+ produces 8-oxo-G. In plasmid DNA where 8-oxo-G has been incorporated as above, further flash-quench treatment with Os(phen)2dppz2+ and Co(NH3)5Cl2+ leads to crosslinking with histone protein in gel shift experiments and in the chloroform extraction assay. Furthermore, in gel shift experiments with a duplex of the oligonucleotide 5’-ATATGATAT8GATATGATAT -3’ (8 = 8-oxo-G), flash-quench treatment with Ru(phen)2dppz2+ in the presence of histone produces a band of intermediate mobility (presumably 1:1 crosslink) and well-shifted material. In contrast, analogous treatment with Os(phen)2dppz2+ produces only the band of intermediate mobility, consistent with the presence of only a single site that is oxidizable by the osmium complex. Lastly, control experiments indicate that with a duplex of the oligonucleotide 5’-ATATGATATGGATATGATAT -3’ flash quench treatment with Ru(phen)2dppz2+ in the presence of histone produces well-shifted material, whereas treatment with Os(phen)2dppz2+ does not produce crosslinked material, as expected since the osmium (III) complex formed should not be capable of guanine oxidation. Taken together, these results show that Os(phen)2dppz2+ is a promising selective oxidant of 8-oxoguanine in double stranded DNA.