1. RNA & NKS Erik A. Schultes Hedgehog Research hedgehogresearch.info June 16, 2006

2. 5 3 Ribonucleic Acid: Universal biopolymer Linear, polarized

3. 5 3 C ytosine U racil A denine G uanine Ribonucleic Acid: Universal biopolymer Linear, polarized 4 distinct nitrogenous bases (nt) RNA can store genetic info (like DNA)

4. 5 3 C ytosine U racil A denine G uanine Ribonucleic Acid: Universal biopolymer Linear, polarized 4 distinct nitrogenous bases RNA can store genetic info (like DNA) Base-pairing rules: A with U C with G

5. 5 3 C ytosine U racil A denine G uanine 5 3 C ytosine U racil A denine G uanine

6. Double-stranded RNA Helix

7. 5 3 C ytosine U racil A denine G uanine Ribonucleic Acid: Universal biopolymer Linear, polarized 4 distinct nitrogenous bases RNA can store genetic info (like DNA) Base-pairing rules: A with U C with G RNA can act as an enzyme (like proteins)

8. Primary Structure Single-stranded RNA:

9. Primary Structure Secondary Structure Single-stranded RNA:

10. Primary Structure Secondary Structure Tertiary Structure Single-stranded RNA:

11. Hammer head VS HDV Group I Group II RNAse P Hairpin Single-stranded RNA:

12. 16S rRNA Single-stranded RNA:

13. 16S rRNA Universal Tree of Life

14. A New Kind of Science by Stephen Wolfram In our everyday experience with computers, the programs that we encounter are normally set up to perform very definite tasks.

15. In our everyday experience with computers, the programs that we encounter are normally set up to perform very definite tasks. Key idea: What happens if one instead just looks at simple arbitrarily chosen programs, created without any specific task in mind. A New Kind of Science by Stephen Wolfram

16. In our everyday experience with computers, the programs that we encounter are normally set up to perform very definite tasks. Key idea: What happens if one instead just looks at simple arbitrarily chosen programs, created without any specific task in mind. How do such programs typically behave? A New Kind of Science by Stephen Wolfram

17. computer (hardware) RNA molecule program (software) nucleotide sequence In our everyday experience with computers, the programs that we encounter are normally set up to perform very definite tasks. Key idea: What happens if one instead just looks at simple arbitrarily chosen programs, created without any specific task in mind. How do such programs typically behave? A New Kind of Science by Stephen Wolfram

18. In our everyday experience with RNAs, the sequences that we encounter are normally set up to perform very definite tasks. Key idea: What happens if one instead just looks at simple arbitrarily chosen sequences, created without any specific task in mind. How do such sequences typically behave? /. computer (hardware) RNA molecule /. program (software) nucleotide sequence A New Kind of Science by Stephen Wolfram

19. What do we mean by: RNA & NKS Arbitrary sequence? RNA behavior?

20. What do we mean by: RNA & NKS generated by random processArbitrary sequence? RNA behavior?

21. What do we mean by: RNA & NKS generated by random processArbitrary sequence? RNA behavior? folding dynamics

22. Never converges on a unique, specific fold Rapidly converges on a unique, specific fold What do we mean by: RNA & NKS generated by random processArbitrary sequence? RNA behavior? folding dynamics

23. Never converges on a unique, specific fold Rapidly converges on a unique, specific fold What do we mean by: RNA & NKS generated by random processArbitrary sequence? RNA behavior? folding dynamics Helix Poly(U) OrderedDisordered

24. What do we mean by: RNA & NKS generated by random processArbitrary sequence? RNA behavior? folding dynamics Evolved ComplexNever converges on a unique, specific fold Rapidly converges on a unique, specific fold Helix Poly(U) OrderedDisordered

25. What do we mean by: RNA & NKS generated by random processArbitrary sequence? RNA behavior? folding dynamics Evolved Complex Rapidly converges on a unique, specific fold Helix Ordered Never converges on a unique, specific fold Poly(U) Disordered Classes I & IIClass IV Class III

26. 1. Lead II chemical probing secondary structure uniqueness of folding 2. Native gel electrophoresis uniqueness of folding size of fold 3. Analytical centrifugation size & shape of fold Analyzing RNA Behavior RNA & NKS

27. Control Sequences tRNA PHE (76nt) HDV (85nt) Ligase (87nt) Reference Sequence Poly(U) Choosing Arbitrary Sequences RNA & NKS

28. Control Sequences tRNA PHE (76nt) HDV (85nt) Ligase (87nt) Reference Sequence Poly(U) 85 Arbitrary Sequences 10 Permuted HDV (85nt) 10 Isoheteropolymer (85nt) Choosing Arbitrary Sequences RNA & NKS

29. Automated DNA Synthesis

30. Structure Probing with Pb ++ Pb ++

31. Structure Probing with Pb ++

32. Pb ++ Structure Probing with Pb ++

33. OH ladder T1 ladder Time HDV Structure Probing with Pb ++

34. OH ladder T1 ladder Time HDV Structure Probing with Pb ++

35. Arbitrary sequences acquire sequence-specific folds

36. 8% (29:1) 100mM THE, pH7.5 30mM KCl 0, 1, 10mM MgCl 2 3W, 2000Vhr XC = 100mm T = 23-24 ºC Native Gel Electrophoresis - +

37. Arbitrary sequences acquire compact folds

38. Inferring molecular size and shape from the concentration distribution of pure sample under a centrifugal filed. Sedimentation Velocity Experiments

39. Reference Sample Sedimentation Velocity Experiments

40. Meniscus Sedimentation Velocity Experiments RNA

41. A 260 r (cm) Sedimentation Velocity Experiments 40,000 RPM 100,000 Xg

42. tRNA Sedimentation Velocity Experiments A 260 r (cm) S=4.072 S D=8.45Ficks M=25.2kDa R s =26.2 Å

43. Arbitrary sequences acquire compact folds

44. The behavior of arbitrary RNA Arbitrary sequences frequently have compact, sequence specific folding - properties that have always been assumed to be evolutionarily derived. So far: 20 seq, 2 compositions = (1/2 postdoc)

45. The behavior of arbitrary RNA Arbitrary sequences frequently have compact, sequence specific folding - properties that have always been assumed to be evolutionarily derived. So far: 20 seq, 2 compositions = (1/2 postdoc) Next step: 35,000 seq, 1700 compositions = ($100M)

46. The behavior of arbitrary RNA Arbitrary sequences frequently have compact, sequence specific folding - properties that have always been assumed to be evolutionarily derived. Principle of Computational Equivalence weak RNA PCE: complex, biologically relevant folds are abundant in seq space

47. The behavior of arbitrary RNA Arbitrary sequences frequently have compact, sequence specific folding - properties that have always been assumed to be evolutionarily derived. Principle of Computational Equivalence weak RNA PCE: complex, biologically relevant folds are abundant in seq space strong RNA PCE: specific folds may be abundant in seq space

48. Distribution of Folds in RNA Sequence Space

49. Prototype Ribozymes

50. Intersection Sequence

51. Testing for Ligation & Cleavage

52. Connecting the Prototypes by Neutral Paths

53. Connecting the Prototypes by Neutral Paths Prototype Ligase 42 mutations

54. Connecting the Prototypes by Neutral Paths Prototype HDV Prototype Ligase 42 mutations 44 mutations

55. Intersection of Fitness Landscapes

56. 1. RNA NN exist - seq space is highly redundant in folds 1. New folds from existing folds 2. RNAs with different sequences and folds could still share ancestry 2. Different NN are proximal Conclusion Biological Implications

57. NKS and Neutral Networks 1. Are INT sequences typical or rare? 2. Are sequences on NN typical or evolved?

58. NKS and Neutral Networks 1. Are INT sequences typical or rare? 2. Are sequences on NN typical or evolved? 3. Does CA rule space have NN? 4. If so, are there INT rules?

59. Acknowledgments David P. Bartel Whitehead Institute NSF/Alfred P. Sloan Fellowship TMF/Charles A. King Trust Fellowship