DNA TBIC Meeting - April 3, 2012 By Navin Sabharwal, Yatsunyk - - PowerPoint PPT Presentation

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DNA TBIC Meeting - April 3, 2012 By Navin Sabharwal, Yatsunyk - - PowerPoint PPT Presentation

Oct 31, 2023 148 likes •325 views

Investigating the Interactions between a Platinum Metalloporphyrin and Quadruplex DNA TBIC Meeting - April 3, 2012 By Navin Sabharwal, Yatsunyk Laboratory Swarthmore College But first, some background 2 This structure has novel

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By Navin Sabharwal, Yatsunyk Laboratory Swarthmore College

Investigating the Interactions between a Platinum Metalloporphyrin and Quadruplex DNA

TBIC Meeting - April 3, 2012

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But first, some background…

Rosalind Franklin’s Photo 51 Canonical Watson-Crick Duplex DNA

“This structure has novel features which are of considerable biological interest” -Watson and Crick

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Novel DNA Structures

Triplex DNA Quadruplex DNA

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G-Quadruplex (GQ) DNA

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Diversity of GQ DNA

(A) Parallel G-Tetrad Core (B) (3+1) G-Tetrad Core (C) Antiparallel G- Tetrad Core (up-up-down-down) (D) Antiparallel G-Tetrad Core (up-down- up-down) (E) Diagonal Loop (F) Edgewise Loop (G) Double-chain Reversal Loop

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Why study GQ DNA?

  • Telomeres are found at the end of chromosomes.
  • Beads-on-a-string model for GQ presence in telomeric DNA sequences.
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Regulation of oncogene transcription

Model for hindrance of oncogene transcription through GQ formation. C-myc, an oncogene which regulates expression in 15%

  • f genes. This oncogene is

guanine rich and can consequently form GQ DNA

Transcriptional activation Altered activation

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Ligand binding to GQ DNA

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 GQ structures have a large π

surface

 Twice that of duplex DNA  Four co-planar bases instead

  • f 2

 Small molecules tend to have

large π surfaces

 Maximize π- π interactions  Porphyrins

 GQ DNA has a strong negative

charge

 Cationic ligands will bind tightly  Non-specific

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Fluorescent Resonance Energy Transfer (FRET)

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First derivative curve

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My ligand – PtT4

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Pt2+

Pt(II)5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H23H-porphine (PtT4)

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Preliminary FRET Results

Melting curves for 2 to 20 eq PtT4 added are indicative of exceptional GQ stabilizing

F21D 2 eq 5 eq 8-20 eq

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Reduced Concentration FRET Results

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Melting curves for 0 to 3 eq PtT4 added suggest excellent GQ stabilization even at low concentrations.

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Reduced Concentration FRET results (cont.)

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  • Eq. PtT4 Added

Tm (°C)

Δ Tm (rel. to F21d) (°C)

0 (F21D) 44 0.5 72.9 28.9 1 72.9 28.9 1.5 73.4 29.4 2 74.5 30.5 2.5 75.9 31.9 3 76.3 32.3

Results indicate excellent GQ stabilization at low PtT4 concentrations.

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Competition Studies

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Addition of duplex calf thymus (CT) DNA to F21D to evaluate PtT4’s selectivity for GQ DNA

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Competition Studies (continued)

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PtT4 is mildly selective for GQ DNA

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Acknowledgements

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 All members of the Yatsunyk lab

 Jack Nicoludis  Vienna Tran  Steven Barrett  Cole Harbeck

 Kaplinsky Lab

 RT-PCR machine  Christina Rabeler

 Professor Yatsunyk