RNA folding, anti-HIV aptamer design, and human telomerase RNA activity

Shi-Jie Chen

Department of Physics & AstronomyDepartment of Biochemistry

University of Missouri-Columbia

RNA (ribonucleic acid) Primary Structure

P

O c

P

O

c

c

7 torsional angles per nt to specify the 3D structure of an RNA

Base pairing and stacking

U

2D (contact map) and 3D structures

Theimer and Feigon et al, Mol. Cell., 17, 671-682, 2005

Human telomerase RNA

kcal/molST

kcal/molH

kcal/molG

STHG stems

tens

tens

several

Telomerase controls the elongation of telomere

When the telomere become critically short, the cell is unable to replicate. Thus, telomerase is important in cell division and normal development.

Secondary structure of human telomerase RNA (hTR)

Chen, Blasco & Greider, Cell, 100, 503 (2000)

Need an entropy theory.Conf entropy is intrinsically a 3D problem.

Vfold model

S-J Chen “RNA Folding: Conformational Statistics, Folding Kinetics, and Ion Electrostatics” Annual Review of Biophysics 2008

RNA conformations described by torsions of the virtual bonds

W. Olson & Flory 1972S Cao & S-J Chen 2008

C O

N1 (primidine: U, C)N9 (purine: A, G)

C4

C5O5

P

Backbone virtual bond torsions are rotameric

(t, g-)

(t, g-)

(t, t) (g-, t)(t, t)

(g+,t)

C3’-endoC2’-endo

Wadley and Pyle et al. JMB, 2007

P

C4

PN1/9

diamond lattice

θη

C4 C4

P P

RNA conformational ensemble Random walk of the virtual bonds in diamond lattice

The Vfold model – a general tool

The model requires no any fitting parameters.The computations are from first principles.

Actual loop is quite rigid, how to account for this effect in the loop conformational enumeration in the Vfold model?

: The free energy of the coaxial stacking between two stems (S1 and S2)

Sequence structure

structure alexperiment in the pairs base ofNumber

pairs base predicted correctly ofNumber )( yselectivitSE

structure predicted in the pairs base ofNumber

pairs base predictedcorrectly ofNumber )( yspecificitSP

Vfold model gives better predictions than Pknots, which ignores the contribution of loop entropy. Cao & Chen, Nucleic Acids Res, 2006

Pseudoknot motifs(a) H-type pseudoknot (b) H-type pseudoknot

with structured loops

(c) Secondary structure + pseudoknot (d) Several H-type pseudoknots

TYMV TMV

BWYVSARS

2D structure prediction

SE=SP=1 for perfect accuracy

28-91 nt, 22 sequences

Ren, J., Rastegari, B., Condon, A., Hoos, H.H. (2005) RNA.Cao & Chen (2009) RNA

Free energy landscape

Shi-Jie Chen. Annual Review of Biophysics 2008

RNA folding energy landscape is bumpy

Sashital, Cornilescu & Butcher. NSMB 2004; Madhani & Guthrie. Cell 1992

Cao & Chen. JMB 2005

Secondary structure of human telomerase RNA (hTR)

Loop-stem (helix) tertiary interactions

: The free energy of the coaxial stacking between two stems (S1 and S2)

Loop-helix interactions are functionally important in RNA pseudoknot human

disease

Theimer and Feigon et al, Mol. Cell., 17, 671-682, 2005

Loop-stem base triple interaction

9

Predicting loop-stem base triple interactions

: triplebaseeach for ) ,(Fit (2) SH

Protonated C.(C-G) and C.(G-C): (-14 kcal/mol, -38 cal/mol.K)

unprotonated:

(-7 kcal/mol, -19 cal/mol.K)

(1) Vfold chain entropy

+

Disruption of the loop-stem base triple

The Vfold model gives good predictions on structures and folding stabilities

Nucleotide sequence 2D structure, stability, free energy landscape

RMSD = 2.2 A

Multiscale all-atom tertiary structure prediction

Sugarcane Yellow Leaf Virus (ScYLV)

Secondary structure can be slave to tertiary contacts.

Correct structure

Inhibition of the tertiary contact structural switch

loop-helix contacts

Wrong structure

Anti-HIV RNA aptamer design

• Aptamers that bind reverse transcriptase (RT) inhibit its activity in enzymatic assays and block viral replication whe expressed in cells.

• Many RNA aptamers to RT form pseudoknots

Donald Burke

AGA

ACUGAA

UUCCGU

AGGGC UGACUU

A AA

U

Jaeger, Restle, Steitz (1998) EMBO J

AGA

ACUGAA

UUCCGU

AGGGC UGACUU

A AA

U

Jaeger, Restle, Steitz (1998) EMBO J

Anti-HIV aptamer design

Can computational approach guide an experimental search for new aptamers? and can experimentation guide refinement of computational theory?

80.63 GCCACACUCCACUCUCGACCGUUUCUUGGGUUCUUCGGGAAAAAAAGCAACCUACUAUUGACUAUCGACGAAGAUCUGUU 134gauucggaugcuccgguagcucaaccug 3’

The location of a fluorescently labeled primer on a denaturing gel

Physics theory guides drug design

loop-helix contacts

?

80.63 GCCACACUCCACUCUCGACCGUUUCUUGGGUUCUUCGGGAAAAAAAGCAACCUACUAUUGACUAUCGACGAAGAUCUGUU 134gauucggaugcuccgguagcucaaccug 3’

The location of a fluorescently labeled primer on a denaturing gel

Physics theory guides drug design

full length

D. Burke

Pseudoknot folding kineticsand

human Telomerase RNA activity

Telomerase controls the elongation of telomere

When the telomeres become critically short, the cell is unable to replicate. Thus, telomerase is important in cell division and normal development.

Secondary structure of human telomerase RNA (hTR)

Chen, Blasco & Greider, Cell, 100, 503 (2000)

Conformational switch and hTR function

pseudoknot

hairpin

179

Comolli et al. 2002Theimer et al. 2003

Conformational switch and hTR function

179

Chen & Greider 2005 (179AG/110CU mutation to destabilize the hairpin)

AG

AG

UC

UC

X

pseudoknot

hairpinX

Rate model

Reduced conformational ensemble

40 nt: 10 confs 6000 6

Cao & Chen, Biophys J. 2009

Native-like & misfolded

Theory-experiment agreement

PK5Wyatt, Puglisi, and Tinoco 1990

Cao & Chen 2005 JMB

hTR: hairpin as a kinetic intermediateHairpin pseudoknot switch existsThe function may be kinetically controlled.The mutation expt alone cannot negate the role of conf switch.

Cao & Chen 2005 JMB

We proposed two structures that are correlated to the telomerase activity: A long-lived transient hairpin intermediate & the native pseudoknot.

Mutants such as 107AG and ∆U177 which forbid the formation of the native pseudoknot or hairpin intermediate result in the loss of telomerase activity.

AcknowledgmentSong CaoGengsheng ChenLiang Liu

Zoia Kopeikin (MU)Zhijie Tan (Wuhan U)Wenbing Zhang (Wuhan U)

Donald Burke (U Missouri) Juli Feigon (UCLA) David Giedroc (Indiania U)Samuel Butcher (U Wisconssin)

NSF MCB 0920067, NSF MCB 0920411NIH R01 GM 063732

Ion electrostaticsFolding kineticsTertiary structural folding