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Chemistry 2100 Lecture 12. Purine/Pyrimidine Bases.

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Presentation on theme: "Chemistry 2100 Lecture 12. Purine/Pyrimidine Bases."— Presentation transcript:

1 Chemistry 2100 Lecture 12

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3 Purine/Pyrimidine Bases

4 -N-glycoside adenosine [a nucleoside] adenosine monophosphate [a nucleotide] 1' 2'3' 5' 4' adenine ribose 9

5 (- H2O)(- H2O)

6 (- H2O)(- H2O)

7 (- H2O)(- H2O)

8 (- H2O)(- H2O)

9 (- H2O)(- H2O)

10 (-H 2 O)

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15 Hydrogen Bonding!

16 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

17 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)

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19 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

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23 Other forms of DNA

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26 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

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28 Step 1: helicase Step 2: DNA polymerase Step 3: DNA ligase

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34 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)

35 Traditional methods for Transcription Factor detection Expression Microarrays Gel Shift Assays The challenge: Most of these methods are indirect, slow (hours), or cant differentiate active and inactive protein. Western Blots

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

37 Optical Conformation Switching TF Switch Sensors

38 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]

39 TF Beacon Actual Performance

40 Quantitative Detection in 4 easy steps

41 transcription

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44 initiation

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47 elongation

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54 A – U – G – U – U – U – G – U – C – A – G – U..... mRNA tRNA peptide Met –––– Phe –––– Val –––– Ser.... (5') U – A – C – A – A – A – C – A – G – U – C – A

55 AU CU

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

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67 DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

68 DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

69 DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

70 DNA T – A – C – A – A – A – C – A – G – T – C – A..... A – T – G – T – T – T – G – T – C – A – G – T..... (3') (5') peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5')

71 peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U..... mRNA (5') peptide Met –––– Phe –––– Asp –––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U..... mRNA (5') Mutations

72 point substitution mutation peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') peptide Met –––– Phe –––– Asp –––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U..... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

73 point substitution mutation peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') peptide Met –––– Phe –––– Asp –––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

74 point substitution mutation peptide Met –––– Phe –––– Val –––– Ser.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') peptide Met –––– Phe ––– Asp ––– Ser.... A – U – G – U – U – U – G – A – C – A – G – U – A.... mRNA (5')

75 peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') frameshift mutation peptide Met –––– Phe –––– Val –––– Val.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

76 peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') frameshift mutation peptide Met –––– Phe –––– Val –––– Val.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

77 peptide Met –––– Phe –––– Val –––– Ser.... mRNA (5') frameshift mutation peptide Met –––– Phe –––– Val –––– Val.... A – U – G – U – U – U – G – U – C – A – G – U – A.... mRNA (5') A – U – G – U – U – U – G – U – C – A – G – U – A....

78 PCR Polymerase Chain Reaction

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80 DNA Fingerprinting

81 DNA Sequencing

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83 Shotgun Sequencing

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

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