Computational design of a circular RNA with prion-like behavior - - PowerPoint PPT Presentation

Computational design of a circular RNA with prion-like behavior

Stefan Badelt1, Christoph Flamm1 and Ivo L. Hofacker1,2 {stef,xtof,ivo}@tbi.univie.ac.at

1Institute for Theoretical Chemistry, University of Vienna,

W¨ ahringerstraße 17/3, A-1090 Vienna, Austria

2Research Group Bioinformatics and Computational Biology,

University of Vienna, W¨ ahringerstraße 29, A-1090 Vienna, Austria

July 31, 2014

1

RNA and artificial life

RNA synthetic biology

around 1980 discovery of catalytic RNA around 2000 Ribosome is an RNA Enzyme

Complexity of Organisms

e.g. ratio RNA/Protein scales with complexity engineering devices for altered gene expression 2006: ENCODE, practically whole genome is transcribed miRNA as regulatory elements to inhibit translation aptazymes (switches + ribozymes) to control gene expression

RNA functional diversity RNA world theory

genetic material + enzymatic function self-replicating RNA self-polymerizing RNA self-switching RNA

bottom up Artificial life inspired by nature

2

Prions and conformational self-replication

Prions are proteins known to be the infectious agents for several neurological diseases (e.g. Altzheimer, Creuzfeld-Jakob, ...) The “protein only hypothesis” states that a single mis-folded infectious prion can convert the other correctly folded proteins to the infectious agent.

N C

PrPC PrPSc oligomer PrPSc oligomer

N C N

+

PrP+

N C

PrPC PrPSc

N

PrPSc

N

PrPSc

N N C N

PrPC-PrPSc heterodimer

N N

PrPSc-PrPSc dimer PrPC PrPSc PrPSc protofibril

• Annu. Rev. Pathol. Mech. Dis. 2008.3:11-40. Downloaded from www.annualreviews.org

by University of Vienna - Main Library and Archive Services on 07/31/14. For personal use only.

Can we design a minimal RNA with prion-like behavior?

3

Prions and conformational self-replication

S1 S2 normal infectious

free energy [kcal/mol]

MFE E(B) maximize refolding barrier

Requirements for an RNA prion S1 S2 Energy Landscape

4

Prions and conformational self-replication

S1 S2 normal infectious

free energy [kcal/mol]

MFE E(B) maximize refolding barrier

Requirements for an RNA prion S1 S2 Energy Landscape

HIV Dis type kissing loop komplex

free energy [kcal/mol]

MFE E(B) minimize refolding barrier

S1 S2 S2 S2 S1 S2 S2 S2

+

S1 S2 S2 S2

4

Computational RNA folding

Sequence ⇒ Structure

free energy [kcal/mol]

• ✁ ✂✄☎

folding pathways

UGCGACGUCCGACCUCGUUUACGCCAGUACCCCACUUCUCUUUG

minimum free energy structure prediction suboptimal structure prediction MFE

G = −kTln(Z) Z =

S∈Ω e

−E(S) kT

P(S) = e

−E(S) kT

Z

5

Computational RNA design

Structure ⇒ Sequence (inverse of RNA folding problem) Simplest case: Find a sequence that forms a predefined structure ⇒ structure is the MFE of the designed sequence ⇒ maximize probability of the desired structure ⇒ sequence must be biologically reasonable (GC content) Even harder: Find a sequence that forms two predefined structures ⇒ sequence must be bi-stable (like a Prion)

(((...)))((...))((...)). .(((.((.(((...))).)).)))

6

Computational Prion design

• switch.pl with two conformations and HIV-Dis loop

....(((((((..((((...(((((...)))))...))))..))))))) (((((((.........)))))))....((((((.........)))))). NNNNNNNAACCGACGANNNNNNNNNNNNNNNNNAACGUCGGANNNNNNN

• Generate lots of sequences (128 different results)
• Select candidate with required prion features

7

Evaluation of prion-like behavior

M

dup

• ZM ... Partition function of the Monomer
• Zc1 ... Partition function constrained that c1 (cyan) is unpaired
• Zc2 ... Partition function constrained that c2 (green) is unpaired
• ZS1 ... Partition function constrained that S1 can form
• ZS2 ... Partition function constrained that S2 can form
• Zdup ... Partition function constrained that duplex can form

8

Evaluation of prion-like behavior

M

dup

• ZM ... Partition function of the Monomer
• Zc1 ... Partition function constrained that c1 (cyan) is unpaired
• Zc2 ... Partition function constrained that c2 (green) is unpaired
• ZS1 ... Partition function constrained that S1 can form
• ZS2 ... Partition function constrained that S2 can form
• Zdup ... Partition function constrained that duplex can form

8

Evaluation of prion-like behavior

M

dup

Partition function of the Dimer: ZD = Zc1 ∗ Zc2 ∗ Zdup (1) Partition function of all Structures that are neither S1 nor S2: Z!S1 & !S2 = ZM − ZS1 − ZS2 Equilibrium Constant for Dimerization: [M]+[M] ⇔ [D] K = [D] [M]2 = ZD Z 2

M 9

Evaluation of prion-like behavior

1e-09 1e-08 1e-0

Monom ❡

Dim

e number of species concentra

K[D] = [M] ∗ [M]

10

Evaluation of prion-like behavior

M

1e-09 1e-08 1e-0✪

Monom

Dim

e number of species concentra

[S1] = ZS1 ZM · [M] + ZS1+c1 Zc1 + ZS1+c2 Zc2

• · [D]

(2) [S2] = ZS2 ZM · [M] + ZS2+c1 Zc1 + ZS2+c2 Zc2

• · [D]

11

Evaluation of prion-like behavior

S1 and S2 are separated by a high energy barrier:

length of refolding path [base-pair moves] free energy [kcal/mol]

20 40 60 80

5

• 5
• 10
• 10.70 kcal/mol

S2

• 12.70 kcal/mol

S1

6.00 kcal/mol

12

Evaluation of prion-like behavior

S2 catalyzes reaction from S1 to S2:

20 40 60 80

• 20
• 25
• 30
• 35

S1 + S2

• 23.40 kcal/mol
• 33.60 kcal/mol
• 17.00 kcal/mol
• 16.10 kcal/mol
• 29.70 kcal/mol

S2 + S2 + kiss

length of refolding path [base-pair moves] free energy [kcal/mol]

20 40 60 80

5

• 5
• 10
• 10.70 kcal/mol

S2

• 12.70 kcal/mol

S1

6.00 kcal/mol

• 31.80 kcal/mol
• 22.00 kcal/mol

Energy Model 2 Energy Model 1

• 39.00 kcal/mol

12

Evaluation of prion-like behavior

S2 catalyzes reaction from S1 to S2:

20 40 60 80

• 20
• 25
• 30
• 35

S1 + S2

• 23.40 kcal/mol
• 33.60 kcal/mol
• 17.00 kcal/mol
• 16.10 kcal/mol
• 29.70 kcal/mol

S2 + S2 + kiss

length of refolding path [base-pair moves] free energy [kcal/mol]

20 40 60 80

5

• 5
• 10
• 10.70 kcal/mol

S2

• 12.70 kcal/mol

S1

6.00 kcal/mol

• ❍
• ■❏

• ❱

• 31.80 kcal/mol
• 22.00 kcal/mol

Energy Model 2 Energy Model 1

• 39.00 kcal/mol

12

Summary

• RNAprions are a from of conformational self-replication
• Computatinal RNA folding and design
• HIV-Dis loops can be used to favor the infectious

conformation for dimers

• Different energy models for refolding pathways all show that

S2 can act as a catalyst

13

thanks to

This work: Ivo L. Hofacker Christoph Flamm General: Sabine M¨ uller Peter F. Stadler the TBI group

Flamm et al. (2001) Design of multi-stable RNA Molecules Weixlbaumer et al. (2004) Determination of Thermodynamic Parameters for HIV-1 DIS Type Loop-Loop Kissing Complexes Lorenz et al. (2011) ViennaRNA Package 2.0 The research was funded by the Austrian Science Fund (FWF): W1207-B09, I670-B11

14

Computational RNA folding

GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUGAAGAUCUGGAGGUCCUGUGUUCGAUCCACAGAAUUCGCACCA

D-Loop T-Loop Acceptor Stem

A secondary structure is a list of base pairs (i, j), where:

• A base may participate in at most one base pair.
• Base pairs must not cross,

i.e., no two pairs (i, j) and (k, l) may have i < k < j < l.

• Only isosteric base-pairs GC, CG, AU, UA, GU, UG are allowed.
• Hairpin loops have at least length 3 (|j − i| > 3)

15

Computational RNA folding

H H M I I I I I H loop p I M

E(S) =

• l∈S

E(l)

Nearest Neighbor Energy Model: The free energy E of a secondary structure S is the sum of the energies of its loops l

• Energies depend on loop type and size,

with some sequence dependence.

• Most relevant parameters are measured experimentally.

16

Computational RNA design

switch.pl in a nutshell:

• build a dependency graph
• mutate an initial sequence guided by dependency graph
• accept/reject mutations according to a cost function

(((...)))((...))((...)). .(((.((.(((...))).)).)))

Cost Function: ⇒ E(x, S1) + E(x, S2) − 2G(x) + ξ(E(x, S1) − (E(x, S2) + ǫ))2

17