RNA & NKS

Erik A. SchultesHedgehog Research

hedgehogresearch.info

June 16, 2006

5’

3’

Ribonucleic Acid:• Universal biopolymer• Linear, polarized

5’

3’

Cytosine

Uracil

Adenine

Guanine

Ribonucleic Acid:• Universal biopolymer• Linear, polarized • 4 distinct nitrogenous bases (nt)• RNA can store genetic info (like DNA)

5’

3’

Cytosine

Uracil

Adenine

Guanine

Ribonucleic Acid:• Universal biopolymer• Linear, polarized • 4 distinct nitrogenous bases• RNA can store genetic info (like DNA)• Base-pairing rules:

A with UC with G

5’

3’

Cytosine

Uracil

Adenine

Guanine

5’

3’

Cytosine

Uracil

Adenine

Guanine

Double-stranded RNA Helix

5’

3’

Cytosine

Uracil

Adenine

Guanine

Ribonucleic Acid:• Universal biopolymer• Linear, polarized • 4 distinct nitrogenous bases• RNA can store genetic info (like DNA)• Base-pairing rules:

A with UC with G

• RNA can act as an enzyme (like proteins)

PrimaryStructure

5 ’ G G A A U U G C G G G AA A G G G G U C A A C A GC C G U U C A G U A C C AA G U C U C A G G G G A AA C U U U G A G A U G G CC U U G C A A A G G G U AU G G U A A U A A G C U GA C G G A C A U G G U C CU A A C C A C G C A G C CA A G U C C U A A G U C AA C A G A U C U U C U G UU G A U A U G G A U G C AGUUCA3’

Single-stranded RNA:

ACGUGGAAUUGCGGGAAGAGGGCAACAGCCGUUCAGUACCAAGUCUCAGGGGAAACUUUGAGAUGGCCUGCAAAGGGUAUUGGUAAUAAGCUGACGUGGUCCUAACCCGCAGUGCCAAGUACGUAGCGCUAUAAGUUAGCUAUAUAGCGCAUUCU5’3’ACA

5 ’ G G A A U U G C G G G AA A G G G G U C A A C A GC C G U U C A G U A C C AA G U C U C A G G G G A AA C U U U G A G A U G G CC U U G C A A A G G G U AU G G U A A U A A G C U GA C G G A C A U G G U C CU A A C C A C G C A G C CA A G U C C U A A G U C AA C A G A U C U U C U G UU G A U A U G G A U G C AGUUCA3’

PrimaryStructure

SecondaryStructure

Single-stranded RNA:

ACGUGGAAUUGCGGGAAGAGGGCAACAGCCGUUCAGUACCAAGUCUCAGGGGAAACUUUGAGAUGGCCUGCAAAGGGUAUUGGUAAUAAGCUGACGUGGUCCUAACCCGCAGUGCCAAGUACGUAGCGCUAUAAGUUAGCUAUAUAGCGCAUUCU5’3’ACA

5 ’ G G A A U U G C G G G AA A G G G G U C A A C A GC C G U U C A G U A C C AA G U C U C A G G G G A AA C U U U G A G A U G G CC U U G C A A A G G G U AU G G U A A U A A G C U GA C G G A C A U G G U C CU A A C C A C G C A G C CA A G U C C U A A G U C AA C A G A U C U U C U G UU G A U A U G G A U G C AGUUCA3’

PrimaryStructure

SecondaryStructure

TertiaryStructure

Single-stranded RNA:

Hammer head

VS

HDV

Group I

Group IIRNAse P

Hairpin

Single-stranded RNA:

16S rRNA

Single-stranded RNA:

16S rRNA Universal Tree of Life

A New Kind of Scienceby Stephen Wolfram

In our everyday experience with computers, the programs that we encounter are normally set up to perform very definite tasks.

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 Scienceby Stephen Wolfram

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 Scienceby Stephen Wolfram

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 Scienceby Stephen Wolfram

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 Scienceby Stephen Wolfram

What do we mean by:

RNA & NKS

Arbitrary sequence? RNA behavior?

What do we mean by:

RNA & NKS

generated by random processArbitrary sequence? RNA behavior?

What do we mean by:

RNA & NKS

generated by random processArbitrary sequence? RNA behavior? folding dynamics

Never converges on aunique, 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

Never converges on aunique, 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

HelixPoly(U)

OrderedDisordered

What do we mean by:

RNA & NKS

generated by random processArbitrary sequence? RNA behavior? folding dynamics

Evolved

ComplexNever converges on aunique, specific fold

Rapidly converges on a unique, specific fold

HelixPoly(U)

OrderedDisordered

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 aunique, specific fold

Poly(U)

Disordered

Classes I & IIClass IVClass III

1. Lead II chemical probingsecondary structureuniqueness of folding

2. Native gel electrophoresisuniqueness of foldingsize of fold

3. Analytical centrifugationsize & shape of fold

Analyzing RNA Behavior

RNA & NKS

Control SequencestRNAPHE (76nt)

HDV (85nt)Ligase (87nt)

Reference SequencePoly(U)

Choosing Arbitrary Sequences

RNA & NKS

Control SequencestRNAPHE (76nt)

HDV (85nt)Ligase (87nt)

Reference SequencePoly(U)85

Arbitrary Sequences10 Permuted HDV (85nt)10 Isoheteropolymer (85nt)

Choosing Arbitrary Sequences

RNA & NKS

Automated DNA Synthesis

OHOOBasePOOOOHOOBaseCH2CH2

Structure Probing with Pb++

Pb++

OHOOBasePOOOOHOOBaseCH2CH2

Pb++

Structure Probing with Pb++

OHOOBasePOOOHOOBaseCH2CH2OH

Pb++

Structure Probing with Pb++

CGGGCGGGCGGCGUCGCGUCCGACGUCCGUACGUCCGCGUACGUCCGACGUACGUCACGACGUACGUACGACGACGUUAGCGCGC5’3’

OH ladderT1 ladderTimeOHOOBasePOOOHOOBaseCH2CH2OH

HDV

Structure Probing with Pb++

CGGGCGGGCGGCGUCGCGUCCGACGUCCGUACGUCCGCGUACGUCCGACGUACGUCACGACGUACGUACGACGACGUUAGCGCGC5’3’

OH ladderT1 ladderTimeOHOOBasePOOOHOOBaseCH2CH2OH

HDV

Structure Probing with Pb++

Arbitrary sequences acquire sequence-specific folds

8% (29:1)100mM THE, pH7.530mM KCl 0, 1, 10mM MgCl2

3W, 2000VhrXC = 100mmT = 23-24 ºC

Native Gel Electrophoresis

-

+

Arbitrary sequences acquire compact folds

Inferring molecular size and shape from the concentration distribution of puresample under a centrifugal filed.

Sedimentation Velocity Experiments

Reference

Sample

Sedimentation Velocity Experiments

Meniscus

Sedimentation Velocity Experiments

RNA

A260

r (cm)

Fs

Sedimentation Velocity Experiments40,000 RPM100,000 Xg

tRNA

Sedimentation Velocity Experiments

A260

r (cm)

S=4.072 SD=8.45 FicksM=25.2 kDaRs=26.2 Å

Arbitrary sequences acquire compact folds

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)

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)

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 Equivalenceweak RNA PCE: complex, biologically relevant folds are abundant in seq space

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 Equivalenceweak RNA PCE: complex, biologically relevant folds are abundant in seq spacestrong RNA PCE: specific folds may beabundant in seq space

Distribution of Folds inRNA Sequence Space

3'AGGGGUCGGUCCACCUCCGCGGUCCGACUCGGGCAUCGAUGG CUAAGGACGAAUGC GGCGAUUAGGGGUUUUCCUAGGUACCUCUCGGC3'GGCAACUCGUCUAAACGCCGGGUGAAGAUGCCCpppCAGUCGGUGGAUGUUAGGGGCGGACCACGUUUUUUUUC U ACCA 2' HOGU CAAACGClass IIIligaseHDV

Prototype Ribozymes

AUUAAAAGCCGCUGGGCCGCCUCCUCGCGGCCGGCCCCGAUAAGGGAGGAAUUUUUCCACGGGACUCCGGAGUGUGGGCUGACHDV fold A GUA C3’U UGAACCCCUCUGGGGGCCCGCCUAACACGACGUCGUGCCGGAAACUppp2' HOCAGUCGGUGGGAGCUGCCGGAGGGAAGGAUG3’UCUUU AACCLigase foldCC

Intersection Sequence

Testing for Ligation & Cleavage

Connecting the Prototypesby Neutral Paths

Connecting the Prototypesby Neutral Paths

Prototype Ligase

42 mutations

Connecting the Prototypesby Neutral Paths

Prototype HDV

Prototype Ligase

42 mutations

44 mutations

Intersection of Fitness Landscapes

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

NKS and Neutral Networks

1. Are INT sequences typical or rare?

2. Are sequences on NN typical or evolved?

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?

Acknowledgments

David P. BartelWhitehead Institute

NSF/Alfred P. Sloan FellowshipTMF/Charles A. King Trust Fellowship