Post on 27-Dec-2015
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