1. Biochemistry Lecture 6

2. Functions of Nucleotides and Nucleic Acids Nucleotide Functions: –Energy for metabolism (ATP) –Enzyme cofactors (NAD + ) –Signal transduction (cAMP) Nucleic Acid Functions: –Storage of genetic info (DNA) –Transmission of genetic info (mRNA) –Processing of genetic information (ribozymes) –Protein synthesis (tRNA and rRNA)

3. Discovery of DNA Structure One of the most important discoveries in biology Why is this important – " This structure has novel features which are of considerable biological interest “ --- Watson and Crick, Nature, 1953 Good illustration of science in action: –Missteps in the path to a discovery –Value of knowledge –Value of collaboration –Cost of sharing your data too early

4. Covalent Structure of DNA (1868-1935) Friedrich Miescher isolates “nuclein” from cell nuclei Hydrolysis of nuclein: –phosphate –pentose –and a nucleobase Chemical analysis: –phosphodiester linkages –pentose is ribofuranoside Structure of DNA: 1929 (Levene and London) Structure of DNA: 1935 (Levene and Tipson)

6. Road to the Double Helix Franklin and Wilkins: –“Cross” means helix –“Diamonds” mean that the phosphate- sugar backbone is outside – Calculated helical parameters Watson and Crick: – Missing layer means alternating pattern (major & minor groove) – Hydrogen bonding: A pairs with T G pairs with C Double helix fits the data! Watson, Crick, and Wilkins shared 1962 Nobel Prize Franklin died in 1958

10. Other forms of DNA

11. NucleotideNucleosideNucleobase

12. Pyrimidine Nucleobases

13. Purine Nucleobases

14.  N-Glycosidic Bond

15. Polynucleotides

16. Hydrolysis of RNA

17. Hydrogen Bonding!

18. The Central Dogma

19. DNA Replication “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Watson and Crick, in their Nature paper,1953

23. Using DNA Structure

24. Why detect Transcription Factor targets? Transcription factors are medically relevant –~10% of human genes –Crucial roles in development and cell life cycle –Misregulation and mutation cause disease –Critically, most cancers involve TF overactivity Darnell, Nature Reviews Cancer 2, 740 (2002)

25. Traditional methods for Transcription Factor detection Expression Microarrays Gel Shift Assays The challenge: Most of these methods are indirect, slow (hours), or can’t differentiate active and inactive protein. Western Blots

26. Bio-mimicry is a powerful motivation Velcro: inspired by burrs Conformation Switching Probes Marvin J S et al. PNAS 1997;94:4366- 4371

27. Optical Conformation Switching TF Switch Sensors

28. Rationally Tuning TF Sensors % switches open KS = 10 KS = 1 KS = 0.1 KS = 0.01 KS = 0.001 KS = [ ] KS [target] KD (1+ KS) + KS [target] % switches open = KD = [ ] Target [M]

29. TF Beacon Actual Performance

30. Quantitative Detection in 4 easy steps

32. Thermal Denaturation

33. Molecular Mechanisms of Spontaneous Mutagenesis Deamination Very slow reactions Large number of residues The net effect is significant: 100 C  U events /day in a mammalian cell Depurination N-glycosidic bond is hydrolyzed Significant for purines: 10,000 purines lost/day in a mammalian cell Cells have mechanisms to correct most of these modifications.

35. UV Absorption of Nucleobases

36. Pyrimidine Dimers from UV http://highered.mcgraw-hill.com/olc/dl/120082/micro18.swf

38. DNA Technologies

40. DNA Cloning

43. Restriction Enzymes

46. PCR Polymerase Chain Reaction

48. Antibiotic Selection Antibiotics, such as penicillin and ampicillin, kill bacteria Plasmids can carry genes that give host bacterium a resistance against antibiotics Allows growth (selection) of bacteria that have taken up the plasmid

49. Site-Directed Mutagenesis

50. Expression of Cloned Genes

51. Protein Purification

52. Eukaryotic Gene Expression in Bacteria An eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium Eukaryotic genes have –Exons: coding regions –Introns: noncoding regions Introns in eukaryouric gene pose problems Bacteria cannot splice introns out mRNA is intron-free genetic material

54. cDNA

55. DNA Electrophoresis

56. DNA Sequencing

58. Shotgun Sequencing

59. Electrochemical Sequencing http://www.youtube.com/watch?v=yVf2295JqUg

62. DNA Fingerprinting

63. DNA Microarrays: Applications DNA Microarrays allow simultaneous screening of many thousands of genes: high-throughput screening genome wide genotyping –Which genes are present in this individual? tissue-specific gene expression –Which genes are used to make proteins? mutational analysis –Which genes have been mutated?

64. DNA Microarrays: Design Two fundamental approaches One-color array –Patented and commerialized by Affymetrix –Photolitographic synthesis of probe DNA on the chip –Targets are biotin labeled –Bound targets detected using streptavidin-fluorofore complex –Widely used in industry Two-color array –Developed by Stanford University, 1996 –Probes sometimes pipetted on the chip –Targets linked to either green or red fluorescent labels –Used often in academia