1. LAS HEBRAS DEL DNA Y SUS INTERACCIONES La importancia de las interacciones débiles Jorge Arévalo

2. Fuerzas que participan en las interacciones del Duplex DNA DNA – Proteína La tecnología de la hibridación

3. Fuerzas que participan en las interacciones del Duplex DNA HERENCIA o el Flujo de Información vertical

6. 1 nm 10 nm 100 nm 1  DNA Virus Bacteria Proteins 0.1 nm Nanotube Nanowire Next Generation CMOS Current CMOS Technology evolutionary technology revolutionary technology Nano-bio interface Nanofiber

7. 10.2 Most bacterial repressors are dimers containing  helices that insert into adjacent major grooves of operator DNA Figure 10-13

8. 10.2 Ligand-induced conformational changes alter affinity of many repressors for DNA Figure 10-14 Tryptophan binding induces a conformational change in the trp aporepressor

9. The backbone of the DNA strand consists of alternating sugar and phosphate groups

14. ¿ Qué mantiene unidas a las hebras del DNA? Una re-visita con mayor detalle

15. Chemical bonds are the basis of both form and function Covalent bonds are the strongest But non-covalent interactions have dramatic influence on biochemical reactions –Electrostatic interactions –Hydrogen bonding –van der Waals interactions

16. Covalent Bonds Covalent bonds are the strongest and are formed when electrons are shared between two adjacent atoms. C-C bonds are about 1.54 Angstroms and have a bond energy of 85 kcal/mol while C-O double bonds have an energy of 175 kcal/mol.

17. Electrostatic interactions E=kq 1 q 2 /Dr Monovalent ions in solution produce electrostatic bonds of about 1.4 kcal/mole

18. The base pairs are stacked on top of each other along the length of the DNA double helix Specific base pairing (hydrogen bonding) between the bases of the two strands stabilizes the double helix

20. Water Non-covalent bonds are altered by the medium in which atoms are surrounded. For most biochemical reactions, the medium is water. Water is a asymmetric molecule. Due to it’s particular asymmetry, water is highly cohesive molecule and forms extensive hydrogen bonding networks with itself.

21. Water significantly weakens electrostatic and hydrogen bonds in molecules

22. Hydrogen bonds are dependent on geometry

23. Hydrogen bonds Hydrogen bonds are formed between hydrogen donors (that have hydrogen) and hydrogen acceptors (that lack hydrogen) and are essential partial electrostatic interactions. Typical hydrogen bonds have energies of 1-3 kcal/mole

24. Physical and chemical properties of DNA H bonding contributes little to the overall stability of ds DNA Stacking interactions result from hydrophobic forces –Van der Waals –dipole interactions Stability is sequence specific! StackedStacking energy dimer kJmol -1 CG -61 GC AT-16 TA Fig. 23-.20

25.  -  stacking : aromatic moieties possess electronic quadrupole moments, with  - delocalized  -electron cloud and  + for atoms outside the ring. Aromatic residues could produce an edge-to-face interaction, as opposed to the face-to-face  -  stacking of the rings. In DNA, approximately half of the bases are hetero-double cyclic. The enthalpic contribution of a single edge-to-face weakly polar interaction is independent of the number of rings in each member of the pair, but the energy of  -  (parallel ring) stacking increases markedly as the surface areas of the ring systems increase. For heterocycles, parallel stacking is competitive with -and may be more favorable than- perpendicular interaction.

26. van der Waals Interactions These interactions result from asymmetry of electrons within the atom. At an optimal distance between atoms (the van der Waals contact distance). The asymmetry between electrons can be complementary between two atoms. Typical van der Waals interactions have energies of 0.5 to 1.0 kcal/mole.

27. Propiedades físicas del DNA que derivan de las interacciones arriba mencionadas Re-visita rápida

28. Denaturation changes physical properties of DNA Chemical Stability of nucleic acids –DNA is stable to mild H +, OH - –RNA is rapidly degraded by [OH-] Isolated Chromosomal DNA is viscous but m echanical forces readily break DNA ( viscosity decreases) Pipeting Sonication Shearing dsDNA is denatured by –alkaline pH –heat –solvent Fig. 23-15

29. Heat denaturation of DNA hyperchromic shift : –40% increased absorbance over a narrow temperature range –cooperativity melting curve –midpt is its melting temperature Tm ss polyA fig. 23-21

30. Overall GC content is reflected in buoyant density CsCl forms a density gradient in a centrifugal field The buoyant density of DNA differs between species –Satellite DNAs (repetitive DNAs) may have distinct GC content

31. T m is a function of GC content and ionic strength T m = 81.5-16.6(log 10 [Na + ]) + 0.41(%G+C)- 600/N –N= chain length –Accurate ~ 10 -3 M< [Na+] < 1 M Decreases in ionic strength decrease T m Primer (olignonucleotide) annealing: T m  2 o C X (A+T) + 4 o (G+C)

32. Denatured DNA can be renatured Melt and quick cooled ~25 o below T m Short complementary regions anneal Annealing is a cooperative process By manipulating temperature and salt concentration, reannealing is precise and sequence dependent Sequence specific reannealing underlies many experimental methods in molecular biology –Southern and northern blotting –Amplification of individual genes by PCR reactions

34. Reference N.M. Luscombe et al, Nucl. Acid Res 29, 2860-2874 (2001). Luger et al, Nature 389, 251-260 (1997)

35. La tecnología de la hibridación

36. Southern and Northern blotting and hybridization detect complementary sequences Detect mutant alleles (RFLPs) [Southern] Homologous genes between organisms mRNAs corresponding to a particular gene (Northern)

39. What is hybridization? Complementary base pairing of two single strands of nucleic acid  double strand product –DNA/DNA –RNA/RNA –DNA/RNA

40. What holds the two strands together? Hydrogen bonds between the base pairs

41. What holds the two strands together? Hydrophobic interactions of stacked bases van der Waals forces between stacked bases

42. Factors affecting the strength of strand pairing Number of GC pairs vs. AT pairs Mismatch Length of hybridizing strands [Salt] of hybridization solution Temperature Concentrations of denaturants

43. Factors affecting the strength of strand pairing Number of GC pairs vs. number of AT pairs –The more H-bonds between strands, the more strongly they are held together 3 H-bonds between G and C 2 H-bonds between A and T –So…the more GC pairs, the more H-bonds between strands

44. Factors affecting the strength of strand pairing % Mismatch –the greater the lack of complementarity, the fewer hydrogen bonds –the lower the strength of the hybrid

45. Factors affecting the strength of strand pairing Length of hybridizing strands –the longer the strands, the more hydrogen bonds and the more hydrophobic interactions, so –the greater the strength of the hybrid

46. Factors affecting the strength of strand pairing [salt] of solution  [salt]   strength of the hybrid –negative charges of the phosphate moieties of the sugar-phosphate backbones repel each other –+ ions from salts in solution act as counterions to reduce repulsion Monovalent cations (Na + ) Divalent cations (1 mM Mg ++ = 100 mM Na+) »Why does [Mg ++ ] affect specificity of PCR priming?

48. Factors affecting the strength of strand pairing Temperature –heat increases the kinetic energy of each of the two strands –sufficient heat makes kinetic energy > H-bond energy –strands separate

49. Factors affecting the strength of strand pairing pH –[OH - ], ~pH 12 enolic hydroxyl groups on bases ionize keto-amino H-bonds disrupted Concentration of denaturants –formamide, urea

50. Combined effects of these factors can be expressed as equations for the Tm What is Tm? Equation to estimate Tm for DNA oligonucleotides Equation to estimate Tm for polynucleotides

51. What is Tm? Tm = temperature of melting or separation of strands –Tm is a function of the DNA fragment or RNA strand under consideration and the solution in which the hybridization is occuring. Changing the temperature does not change the Tm!

52. What is Tm? For complementary oligonucleotides (10 - 23 nt) –Temp at which 50% of complementary molecules exist as single strands 50% 5’ - - - - - - - - - - - - - 3’ 3’ - - - - - - - - - - - - - 5’ 50% 5’ - - - - - - - - - - - - - 3’ 3’ - - - - - - - - - - - - - 5’

53. What is Tm? For complementary polynucleotides (>~25nt) –Tm is the temp at which 50% of hydrogen bonds within any one hybrid are broken

54. Combined effects of factors contributing to strength of a hybrid can be expressed as equations for Tm for DNA oligonucleotides in 1.0M Na + Tm ( o C) = 4 (G+C) + 2 (A+T) Note: how does this equation account for –length? –% GC? –The conditions of the solution

55. Combined effects can be expressed as equations for Tm for DNA polynucleotides and oligos as short as 14 nt Tm = 81.4 + 16.6 log [(M + )/1+0.7(M + )] + 0.41 (%G+C) - 600/L - %mismatch - 0.65 (% formamide) M + = monovalent cation concentration L = length of probe sequence

56. Tm for polynucleotides (cont’d) How does the equation on the previous slide account for –length? –% GC? –The conditions of the solution

57. Membrane hybridization One nucleic acid component is affixed to membrane; the other is in solution –probe(s) affixed; sample in solution HLA-DQalpha –samples affixed; probe(s) in solution 14;18 translocation Membrane material binds DNA or RNA –nylon –charged nylon –nitrocellulose

58. Steps in membrane hybridization blocking or prehybridization hybridization wash or rinse visualization

59. Blocking/prehybridization Why? –Remember, membrane binds nucleic acid, so labeled nucleic acid in hybridization solution can bind everywhere on membrane   background

60. Blocking/prehybridization How? –Membrane with affixed nucleic acid is bathed in blocking solution at hybridization temperature –Components of blocking solution bind non- specifically to membrane to prevent labeled nucleic acid from binding except to complementary strands

61. Blocking/prehybridization common blocking agents –sodium dodecyl sulfate (SDS) –nonfat dry milk –bovine serum albumin –Ficoll (carbohydrate polymer) –polyvinylpyrollidone (PVP)

62. Hybridization What? –Labeled nucleic acid in solution is allowed to anneal to affixed complementary strands Conditions –Must be determined empirically –Hybridization solution includes [Salt] determined from Tm formulas Membrane blocking agents Denatured labeled nucleic acid; denatured by –High temperature (95 o C) or –Alkaline (high pH) conditions

63. Hybridization Conditions (cont’d) –Temp set below Tm to optimize rate of hybridization oligonucleotides: 15 o below Tm polynucleotides: 15-35 o below Tm

64. Wash/rinse Why? –To remove labeled probe/sample that is in excess non-specifically bound bound with loose complementarity

65. Wash/rinse How? Bathe membrane in solution lacking labeled probe/sample Use stringency conditions that minimize non-specific hybridization –stringency = likelihood that two strands will separate Be aware that wash conditions for oligonucleotide and polynucleotide hybridizations differ because: –oligonucleotide hybrids are not in equilibrium –polynucleotide hybrids are in equilibrium

66. Choosing wash conditions –To wash polynucleotide hybridizations (equilibrium) raise stringency conditions to make it harder for imperfect hybrids to remain annealed perform washes just below the Tm –  stringency   likelihood that two strands will separate Lower the salt concentration Raise the temperature Include denaturants

67. Choosing wash conditions (cont’d) To wash oligonucleotide hybridizations –Use stringency similar to or lower than hybridization condtions Same or lower temperature Same or higher salt concentrations –Short time periods

68. Visualization requires a visible signal –radioactive –non-radioactive, enzyme linked –non-radioactive, non-enzymatic e.g., use of fluorescent label for enzyme-linked signal generation –additional block and rinse steps required avoid conditions which will disrupt hybrids

69. Websites to use molecular models Molecules in Motion http://www.umass.edu/molvis/freichsman/index.html http://www.umass.edu/molvis/freichsman/index.html Molecules from Chemistry http://www.ouc.bc.ca/chem/molecule/molecule.html http://www.ouc.bc.ca/chem/molecule/molecule.html Protein Data Back http://www.ncbi.nlm.nih.gov/Structure/ Klotho Biochemical Structure Database http://www.ibc.wustl.edu/moirai/klotho/