DNA Structure/ Conformation

The structure and conformation of DNA are important considerations in studying the interactions of DNA with proteins and other DNA ligands. Techniques for determining DNA structure and conformation include X-ray crystallography, circular dichroism (CD), fluorescence, and UV-vis absorbance spectroscopy. With the exception of X-ray crystallography, all these techniques are supported by Olis instrumentation.

Circular Dichroism

DNA exhibits a distinctive CD spectrum in the 200-250 nm region that is particularly sensitive to DNA structure. CD spectra can be utilized to probe the DNA structure as perturbants such as increased temperature or DNA ligands are added. A typical experiment involves collection of spectra while the temperature is increasing or ligand is added to the sample.

All Olis CD models DSM 17, DSM 20, DSM 1000, and Protein Machine support CD measurements of DNA, and include accessories such as the CD 250 Peltier temperature controlled cell holder, Automatic Titrator for ligand binding, and Stopped-Flow for kinetic studies.

Fluorescence

Whether originating from the DNA molecule directly, or an external probe, fluorescence is a valuable technique to elucidate DNA structural information. Emission maxima and fluorescence intensity changes reveal information about the DNA structural integrity. Intercolating fluorescence probes exhibit enormous increases in fluorescence intensity upon binding DNA and are also useful probes of DNA structure. Fluorescence energy transfer (FRET) in which two probes with overlapping emission and excitation spectra are attached to the same DNA molecule. The energy transferred fro the donor dye to the acceptor dye is a function of the distance between the two. FRET probes can therefore be valuable tools in monitoring the structural changes undergone by an appropriately labeled DNA molecule. All Olis Fluorescence models DM 45, DM 245, SLM 8000, SPF-500, RSM 1000F, and Cary Fluorescence systems are ideally configured to support fluorescence studies of DNA.  The DSM 17, DSM 20, and DSM 1000 can also be configured for both CD and fluorescence studies.

Fluorescence Anisotropy

Fluorescence Anisotropy is a measure of the mobility of the fluorescence probe, can also be used to observe DNA structure and conformation. In this technique, the fluorescence probe is excited with vertically polarized light, the amount of polarization of the emission is related to the mobility of the fluorescence probe during the fluorescence lifetime. Fast moving probes will be completely depolarized (anisotropy  is zero) while slower probes, such as those bound to DNA, will remain partially polarized (non-zero anisotropy). The Olis Polarization Toolbox accessory adds anisotropy capability to any Olis fluorimeter (except the SLM series). Other useful accessories include the TLC 50 Peltier cell holder, 4-cell Peltier turret, Automatic Titrator, and Stopped-Flow.

UV Absorbance

UV absorbance spectra of DNA also reveal important information about DNA structure. While changes due to structural differences are typically subtle, Olis fitting algorithms in GlobalWorks can pull out even the smallest of spectral changes. The RSM 1000, Olis 14/17 and HPDA 8452 support the collection of UV-Vis absorbance spectra to probe DNA structure. Useful accessories include TLC 40 peltier cell holder and stopped-flow.

Client Publications
N Nagesh, R Busaglia, JM Dettler, EA Lewis. (2010). Studies on the Site and Mode of TMPyP4 Interactions with Bcl-2 Promoter Sequence G-Quadruplexes.
      Biophys J 98, 2628-2633
Achieved using the OLIS HPDA 8452

J Zhou, V Le, D Kalia, S Nakayama, C Mikek, E Lewis, HO Sintim. (2014). Diminazene or berenil, a classic duplex minor groove binder, binds to G-quadruplexes with low nanomolar dissociation constants and the amidine groups are also critical for G-quadruplex binding..
      Molecular Biosystems 10, 2724-2734
Achieved using the OLIS DSM 20

J Cui, P Waltman, VH Le, EA lewis. (2013). The Effect of Molecular Crowding on the Stability of Human c-MYC Promoter Sequence I-Motif at Neutral pH.
      Molecules 18, 12751-12767
Achieved using the OLIS HPDA 8452 and DSM 20

VR Machha, SB Jones, JR Waddle, VH Le, S Wellman, EA Lewis. (2013). Exploring the Energetics of Histone H1.1 and H1.4 Duplex DNA Interactions.
      Biophysical Chemistry 185C, 32-38
Achieved using the OLIS DSM 20

X-C Gou, J Liu, H -L Zhang. (2010). Monitoring human telomere DNA hybridization and G-quadruplex formation using gold nanorods.
      Analytica Chimica Acta 668, 208-214
Achieved using the OLIS DSM 1000

RD Gray, JO Trent, JB Chaires. (2014). Folding and Unfolding Pathways of the Human Telomeric G-Quadruplex.
      Journal of Molecular Biology 426, 1629-1650
Achieved using the OLIS RSM 1000

D Yadav and RD Sheardy. (2012). A single base permutation in any loop of a folded intramolecular quadruplex influences its structure and stability.
      J. Biophys. Chem. 3, 341-347
Achieved using the OLIS DSM 1000

A Sielaff, H Mackay, T Brown, M Lee. (2008). 2-Aminopurine/cytosine base pair containing oligonucleotides: Fluorescence spectroscopy studies on DNA?polyamide binding.
      BBRC 369, 630-634
Achieved using the OLIS DSM 20

C-Y Wei, J-H Wang, J Liu, L-H Wang. (2012). 4-(1H-Imidazo[4,5-f]-1,10-phenanthrolin-2-yl)phenol-based G-quadruplex DNA binding agents: telomerase inhibition, cytotoxicity and DNA-binding studies.
      Bioorganic and Medicinal Chemistry 21, 3379-3387
Achieved using the OLIS DSM 1000

B Babu, KA Brien, V Satam, A Plaunt, M Pressler, H Shen, S Alger, R Ogilvie, R Sjoholm, S Tzou, O Sweat, T Rice, H Mackay, M Lee. (2012). Synthesis and DNA-binding properties of 1,2,3-triazole-linked H-pin pyrrole- and imidazole-containing polyamides formed by the Huisgen reaction.
      Heterocyclic Communications 18, 79-85
Achieved using the OLIS DSM 20

Y Li, G Jia, C Wang, M Cheng, C Li. (2015). Higher-Order Human Telomeric G-Quadruplex DNA Metalloenzymes Enhance Enantioselectivity in the Diels?Alder Reaction.
      ChemBioChem DOI:?10.1002/cbic.201402692
Achieved using the OLIS DSM 1000

RD Gray and JB Chaires. (2008). Kinetics and Mechanism of K+- and Na+- Induced Folding of Models of Human Telomeric DNA into G-Quadruplex Structures.
      Nucleic Acids Res. 36, 4191-4203
Achieved using the OLIS RSM 1000

L Westrate, H Mackay, T Brown, B Nguyen, J Kluza, WD Wilson, M Lee, JA Hartley. (2009). Effects of the N-Terminal Acrylamido Group of Imadazole- and Pyrrole-Containing Polyamides on DNA Sequence Specificity and Binding Affinity.
      Biochemistry 48, 5679-5688
Achieved using the OLIS DSM 20

M Park, JW Toporowski, TC Bruice. (2006). Ribonucleic guanidine demonstrates an unexpected marked preference for complementary DNA rather than RNA.
      Bioorg. Chem. 14, 1743-1749
Achieved using the OLIS RSM 1000

YP Bhavsar-Jog, E Van Dornshuld, TA Brooks, Tschumper, RM Wadkins. (2014). Epigenetic modification, dehydration, and molecular crowding effects on the thermodynamics of i-motif structure formation from C-rich DNA.
      Biochemistry 53, 1586-1594
Achieved using the OLIS DSM 20

LA Gearheart, HJ Ploehn, CJ Murphy. (2001). Oligonucleotide Adsorption to Gold Nanoparticles: A Surface-Enhanced Raman Spectroscopy Study of Intrinsically Bent DNA.
      J. Phys. Chem. B 105, 12609-12615
Achieved using the OLIS DSM 1000

AT de Long. (2013). Effect of flanking bases on DNA specificity of EmBP-1.
      Biochemistry 52, 786-794
Achieved using the OLIS DSM 1000

SM Reilly, DF Lyons, SE Wingate, RT Wright, JC Correia, DM Jameson, RM Wadkins. (2014). Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs.
      Biophysical Journal 107, 1703-1711
Achieved using the OLIS DSM 20

SM Reilly, RK Morgan, TA Brooks, RM Wadkins. (2015). E!ect of Interior Loop Length on the Thermal Stability and pKa of i-Motif DNA.
      Biochemistry 54, 1364-1370
Achieved using the OLIS DSM 20

O Julien, JR Beadle, WC Magee, S Chatterjee, KY Hostetler, DH Evans, BD Sykes. (2011). Solution Structure of a DNA Duplex Containing the Potent Anti-Poxvirus Agent Cidofovir.
      JBC 133, 2264-2274
Achieved using the OLIS DSM 17

N Zhang, X Chu, M Fatalla, J Jayawickramarajah. (2013). Photonic DNA-Chromophore Nanowire Networks: Harnessing Multiple Supramolecular Assembly Modes.
      Langmuir 29, 10796-10806
Achieved using the OLIS DSM 1000

T Neumann, S Gajria, NF Bouxsein, L Jeager, M Tirrell. (2010). Structural Responses of DNA-DDAB Films to Varying Hydration and Temperature.
      JACS 132, 7025-7037
Achieved using the OLIS DSM 1000

M Garcia-Arriaga, G Hobley, JM Rivera. (2008). Isostructural Self-Assembly of 2?-Deoxyguanosine Derivatives in Aqueous and Organic Media.
      JACS 130, 10492-10493
Achieved using the OLIS DSM 10

BA Linkletter and TC Bruice. (2000). Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) modified oligonucleotides containing neutral urea linkages: effect of charge deletions on binding and fidelity.
      Bioorganic and Medicinal Chemistry 8, 1893-1901
Achieved using the OLIS RSM 1000

DJ Uribe, Y-J Shin, E Lau, SW Ebbinghaus, D Sun. (2013). Heterogeneous Nuclear Ribonucleoprotein K Binds to the Cytosine-Rich Sequence of the Hypoxia Inducible Factor 1 Alpha Proximal Promoter that forms a Stable i-Motif at Neutral pH.
      J Phys chem Biophys DOI: 10.4172/2161-0398.S5-001
Achieved using the OLIS DSM 20

MA Khan, H Yumak, DJ Goss. (2009). Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA: EFFECTS OF eIF4B AND POLY(A)-BINDING PROTEIN .
      J Biol Chem 284, 35461-35470
Achieved using the OLIS RSM 1000F

J Gidden, A Ferzoco, ES Baker, MT Bowers. (2004). Duplex Formation and the Onset of Helicity in Poly d(CG)n Oligonucleotides in a Solvent-Free Environment.
      JACS 126, 15132-15140
Achieved using the OLIS RSM 1000