Hybridization Diagnostic tools Nucleic acid Basics PCR Electrophoresis DNA-Protein interactions Chromatin Gene expression

Six Nucleosides Cytidine (base: cytosine) 5-methyl Cytidine (base: 5-methy cytosine) Guanosine (base: guanine) Thymidine (base: thymine) thymidine is deoxynucleotide Uridine (bsae: uracil) Adenosine (base: adenine)

Features of Nucleosides 1’ carbon forms a glycosidic linkage to a base (adenine is shown here 5’ oxygen forms a phosphoester bond. 4’ 1’ 5’ 2’ 3’ 3’ oxygen forms a phosphoester bond. 2’ carbon is connected to: - H in DNA - OH in RNA In RNA the OH may function as a catalyst in some reactions. Cytidine

A Dinucleotide 5’ end phosphodiester 3’ end

Single Stranded Nucleic Acids In cells, RNAs are the most abundant single stranded nucleic acids secondary structure is largely in the form of “hairpin loops”. tertiary structures are important for catalysis.

The 2’OH as a catalyst Cytidine 1’ carbon forms a glycosidic linkage to a base (adenine is shown here 5’ oxygen forms a phosphoester bond. 4’ 1’ 5’ 2’ 3’ 3’ oxygen forms a phosphoester bond. 2’ carbon is connected to: - H in DNA - OH in RNA In RNA the OH may function as a catalyst in some reactions. Cytidine

Single Stranded Nucleic Acids Tertiary structures are important for interactions with proteins and can be manipulated to produce designer drugs: Interference RNAs Aptamers.

RNA inhibitors of clotting factor IXa Rusconi et al, 2002 Nature 419:90-94

RNA inhibitor of clotting factor IXa and its antidote Rusconi et al, 2002 Nature 419:90-94

Single Stranded Nucleic Acids Single stranded DNAs are important in clinical and scientific investigations. Probes and primers are synthetic single stranded DNAs

Double Stranded Polynucleotides G:C Three H-bonds A:T Two H-bonds

Important Forces H-bonds stabilize Negative charges on phosphates destabilize Base-base stacking interactions stabilize (bases at the ends lack this stabilizing force)

Hybridization Diagnostic tools Nucleic acid Basics PCR Electrophoresis DNA-Protein interactions Chromatin Gene expression

DNA “Melting” The DNA strands separate when heated Strand separation occurs over a narrow temperature range. The midpoint is Tm, the “melting temperature”.

Factors That Influence Tm Properties of the helix Base composition: C:G rich is more stable than A:T rich Mismatches: Sequences with perfect complementarity are more stable than those with mismatches. Length of the helix Very short helicies are less stable that moderately long ones.

Factors That Influence Tm Properties of the solution Ionic conditions Solutons with high ionic strength will stabilize. Extremes of pH Chemicals that disrupt H-bonds Urea, formamide, formaldehyde

Factors That Influence Tm Properties of cells Helix-destabilizing proteins These proteins play physiologically important roles in a number of cellular processes.

Separated Strands Can Rehybridize - Duplex formation is a bimolecular reaction: thermodynamically favored - Hair-pin helix formation is a monomolecular reaction: kinetically favored

Hybridization: Conditions are important Concentration is important Hydridization is a bimolecular reaction. A high concentration of DNA will favor duplex formation.

Hybridization: Conditions are important Temperature is important Slow cooling will favor the formation of DNA duplexes. Fast cooling will favor the formation of hair-pin loops, which may prevent duplex formation. The temperature must be near the Tm if high stringency is desired (formation of duplexes with perfect complementarity).

Fluorescence in situ hybridization FISH

Biopsy from a patient with breast cancer showing HER-2 amplification Control probe HER-2 probe

Control probe HER-2 probe