Nucleic acid Basics

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

2. 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)

3. 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

4. A Dinucleotide 5’ end phosphodiester 3’ end

5. 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.

6. The 2’OH as a catalyst 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

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

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

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

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

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

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

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

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

15. Factors That Influence TmProperties 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.

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

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

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

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

20. 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).

21. Fluorescence in situ hybridizationFISH

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

23. Control probe HER-2 probe