Modeling Nucleic Acid Structures

Prediction of Non-Canonical Base

Pairs in RNA

Dhananjay Bhattacharyya

Computational Science Division

Saha Institute of Nuclear Physics

Kolkata

E-mail: dhananjay.bhattacharyya@saha.ac.in

A Biological Cell

Major Biological Processes

• Cell Division (Mitosis or Meioses)

• Conversion of Chemical Energy to

Mechanical Energy (action of muscle)

• Reasoning or Thinking in Brain (through

exchange of Electrical Signal)

• Protein synthesis

• Others

Molecules present in a Cell

• Proteins, (such as Hemoglobin)

• Nucleic Acids (Deoxyribonuceic Acid or Ribonucleic Acid)

• Carbohydrate (Mono-Saccharide, Di-Saccharide, Poly-Saccharide)

• Lipid (in Cell Membrane)

• Water (maximum amount in a cell)

• Salt (Cations: Na+, K+, Mg2+ and Anions: Cl-)

• Others small molecules: Heme, Cholesterol, ATP, etc

OO

O

U U U A G C

G A A A U C G

Na4a3a2a1

mRNA

Gene

RNA polymerase Promoter

sequence

mRNA

Central Dogma of Molecular Biology: DNA RNA Protein

A

T

G

C

DNA as observed

R. Benfante, N. Landsberger, G. Tubiello and G. Badaracco

Nucl. Acids Res. 17, 8273 (1989)

C. Bustamante, J. Vesenka, C.L.Tang, W.

Rees, M. Guthold, and R. Kellers

Biochemistry 31, 22 (1992)

2d sin =n

L. Bragg

J. Kendrew

M. Perutz

L. Pauling

F. Crick

…And many

DNA double

helices without

any ligand

IUPAC-IUB suggested NUPARM

1. Dickerson, Bansal, Calladine, et al. (1989) EMBO J. 8: 1

2. Olson, Bansal, Burley, et al. J. Mol. Biol. (2001) 313:

229-237

Weisstein, Eric W. "Euler Angles." From MathWorld--A Wolfram Web

Resource. http://mathworld.wolfram.com/EulerAngles.html

E( )= 100

0)cos()sin(

0)sin()cos(

zR

)cos()sin(0

)sin()cos(0

001

xR

Quaternion Transformation

Q=a + ib + jc + kd (representing rotation)

with i2= -1, etc

=cos(a/2) + u sin(a/2), u = unit vector

Q-1 = a – ib – jc – kd

Position vector v=0 + ivx+ jvy+kvz

Rotated coordinates v’ = qvq-1

Base Pair Step Parameter (NUPARM v.1)

Y1

Y2

X2

X1

Xm = (X1 + X2) / | (X1 + X2) |

Ym = (Y1 + Y2) / | (Y1 + Y2) |

Zm = Xm x Ym

Tilt = 2 sin-1( Zm . Y1)

Roll = 2 sin-1( Zm . X1)

Twist = cos-1 (( X1 × Zm) . ( X2 × Zm))

Bansal, Bhattachary

ya & Ravi (1995)

CABIOS 11, 281

Shift, Slide and Rise, in

similar way

Definition and Nomenclature of Intra

Base Pair Parameters (IUPAC-IUB)

and NUPARM version 2.0

Mukherjee, Bansal & Bhattacharyya (2006) J Comput. Aided Mol. Des., 20, 629-45.

Base pair parameters Determination

Buckle = 2 sin-1( Zm . Y1)

Opening = 2 sin-1( Zm . X1)

Propeller = cos-1 (( X1 × Zm) . ( X2 × Zm))

Shear = -Xm . M

Stagger = Ym . M

Stretch = Zm . M

Xm = (X1 + X2) / | (X1 + X2) |

Ym = (Y1 + Y2) / | (Y1 + Y2) |

Zm = {(X1 + X2) x (Y1 + Y2)}/ {| (X1 + X2) | | (Y1 + Y2) |}

Mukherjee, Bansal & Bhattacharyya (2006), J Comput Aided Mol

Des, DOI 10.1007/s10822-006-9083-x

Partial list of DNA crystal structures

available at http://ndbserver.rutgers.edu

bd0001 12: A C C G A C G T C G G T

bd0003 12: A C C G G T A C C G G T

bd0004 12: C G C G A A T T C G C G

bd0006 10: G G C C A A T T G G

bd0011 12: C G C A A A T A T G C G

bd0014 12: C G C G A A T T C G C G

bd0015 10: C C G C C G G C G G

bd0017 9: C G C G C G G A G

bd0018 11: G C G A A T T C G C G

bd0019 12: G G C G A A T T C G C G

bd0022 12: A C C G G C G C C A C A

bd0023 10: C C A G T A C T G G

Bd0024 10: C C G A A T G A G G

Standard Reference frame of a Watson-

Crick base pair

Propeller

5’

5’

Calladine & Drew 1982 J. Mol. Biol.)

Basepair parameters of bdl001

C G C G A A T T C G C G

Roll Variation in Crystal Structures

Roll of d(AA).d(TT) doublets

0

10

20

30

-15 -10 -5.4 -0.9 3.72 8.29 12.9

Roll

Oc

cu

rre

nc

e

Roll of d(CG).d(CG) doublets

0

10

20

30

40

-17 -10 -4.3 1.78 7.87 14 20.1

Roll

Oc

cu

rre

nc

e

Roll of d(GC).d(GC) doublets

0

5

10

15

20

-27 -21 -12 -5.4 1.15 7.68 14.2

Roll

Oc

cu

rre

nc

e

Input geometry parameter (Roll, Tilt, Twist etc.)

and (Propeller, Buckle, etc.)

Input Ideal Base Pair Coordinates

Convert Geometry parameters to

Helical Sense (Analytical Relations)

Apply Rotations/Translations to two

Base Pairs in Helical Sense

Repeat the procedure for

polymer generation

100º

1.5Å

5.4Å

Helix:

(R, , z) (x, y, z)

Orientation of nth residue == Orientation

of 1st residue

2/1

221

2/1

221

2/1

2/11221

4)1()1(2

cot2

1sin

4)1()1(2

cot2

1sin

sincos2

sinsin

RTTRTRR

RTTRTRT

TRTR

hh

hh

h

hhh

hhh

hhhhz

hhhhy

hhhhx

B

B

BS

BBS

BBS

coscos]cos1[

sinsincos2

sinsincos2

sin2sincos2

sin2sincos2

3

2

1

32

31

2sin;

2sin 22 TR

Curved DNA models built

from Crystal parameters(A3G7)n

(A6G4)n

(A10)n

Analysis and Generation of Double Helical Structure of

DNA: Sequence Directed DNA Curvature – Prediction of

Promoter Regions in Genomic sequences

Natural sequences having different RL

Synthetic sequence

(CGCAAAAAAG)n

with large RLApplied to predict Promoter Sequences

Parameter Variability

Double Helical DNA Flexibility depends on Base

Sequence

Sequence Persistence Length, P

566 ÅMixed Sequence

Poly(dG).poly(dC) 816 Å (Rigid, expt.)

Poly(dA).poly(dT) 1174 Å (Rigid, expt)

Poly(dA-dT).poly(dA-dT) ~1100 Å

Poly(dA-dG).poly(dC-dT) ~ 800 Å

Poly(dG-dC).poly(dG-dC) 412 Å

Poly(dA-dC).poly(dG-dT) ~600 Å

Poly(dC-dT-dG).

poly(dC-dA-dC)

397 Å (Flexible, expt.)

Poly(dC-dG-dG).

poly(dG-dC-dC)

410 Å (Flexible, expt.)

D. Bhattacharyya, S.

Kundu, A.R. Thakur & R.

Majumdar (1999) J. Biomol.

Struct. Dynam.17, 289.

“A DNA Structural Atlas for E-coli”, Pedersen, A.G. et

al (2000) J. Molecular Biology, 299, 907-930

TATA Box binding to TBP

OO

O

U U U A G C

G A A A U C G

Naaaa

mRNA

RNA polymerase Promoter

sequence

mRNA

Cellular functions: DNA RNA Protein

Intron

Structural Motifs of RNA

Helix

Hairpin

loop

Bulge

loop

Continuous

stack

Kissing

loop

D

H

A ABHA

R1

R2

van der Waals

Approximate Hydrogen Bond

van der Waals + Coulomb

Approximate H-Bond + Coulomb

Ener

gy

(kca

l/m

ol)

Hydrogen Bond

Possibility of Unusual Base

Pairing in RNA

Base Pair Finder

Took a base edge

Identify the H-bonding centers (N3G & N2G)

Look for H-bond partner through distance

calculation (N6A & N7A)

Check linearity of pseudo-angles

C6G-N3G-N6A

N3G-N6A-N1A

N1G-N2G-N7A

N2G-N7A-N9A

Confirm orientation through angle calculationGives rise to:

1822 A:U W-W(C);

6056 G:C W-W(C) and

847 G:U W-W(C) base pairs

Das, Mukherjee, Mitra & Bhattacharyya (2006) J Biomol Struct Dynam, 24, 149-161

127

Variants

with

TWO

H-

bonds

between

the

Bases/

sugars

G:C W:W C

A:U W:W C

G:U W:W C

A:G H: S T

A:U H:W T

A:A H:H T

G:A W:W C

G:A S:W T

A:A W:W T

A:U W:W T

A:A H: W T

A:U H:W C

G:G S:S T

G:G H:W T

A:C W:W T

C:U W:W T

A:C H:W T

G:G H:WC

G:C W:W T

A:G s:s T

AA HHT

AG SST

AG HST

AU HWT

AU HWT

Double helical fragment from

ribosomal RNA (PDB ID: 1N32)

S. Halder and D. Bhattacharyya (2010) J. Phys. Chem. B 114: 14028

Different Non-canonical Basepairing Motifs In RNA

Double Helices

Motif RNA Type Organism

G:A S:HT

A:G H:ST

23S rRNA

Haloarcula marismortui

Thermus thermophilus

Escherichia coli

Deinococcus radiodurans

16S rRNAEscherichia coli

Thermus thermophilus

Riboswitch Synthetic

A:A s:hT

A:U H:WT

A:G H:ST

16S rRNAEscherichia coli

Thermus thermophilus

G:A S:HT

A:G H:ST

A:G H:ST

23S rRNA

Haloarcula marismortui

Escherichia coli

Thermus thermophilus

U:G S:WC

U:U W:WC23S rRNA Haloarcula marismortui

G:A S:HT

A:U H:WT

A:G H:ST

23S rRNA Thermus thermophilus

A:G W:WC

A:G W:WC23S rRNA

Thermus thermophilus

Escherichia coli

Motif RNA Type Organism

G:A S:HT

G:A S:HT

A:G H:ST

U:U W:WC

16S rRNAThermus thermophilus

Escherichia coli

G:A S:HT

G:A S:HT

A:G H:ST

23S rRNA Thermus thermophilus

U:U W:WC

U:U W:WC

23S rRNA Haloarcula marismortui

IRES RNA Cricket paralysis virus

G:A S:HT

A:G H:ST

G:G H:zT

23S rRNA Escherichia coli

G:G z:HT

U:A W:HT

A:G H:ST

16S rRNAThermus thermophilus

(in very few structures)

U:C W:WC

U:U W:WC23S rRNA

Deinococcus radiodurans

(in very few structures)

B-DNA:

1BNA.pdb

A-DNA:

1ZEX.pdb

Regular RNA

oligonucleotide:

1QCU.pdb

Backbone generated by CHARMM through

Restrained Energy Minimization

RNA fragments with Non

Watson-Crick Base Pairs:

fragments from 2AW4.pdb and

1N32.pdb

Helix

RMSD with regenerated

structure

Average RMSD with

similar crystal structures

All-atom Base-atom All-atom Base-atom

B-DNA (1BNA) 0.535 0.148 0.438 0.318

A-DNA (1ZEX) 0.399 0.175 x x

A-RNA (1QCU) 0.332 0.110 x x

U:U W:WC

(1J5A)0.475 0.357 1.082 1.035

A:G W:WC

(1FJG)0.497 0.396 1.035 0.904

G:U W:WC

(1N33)0.529 0.251 1.007 0.988

G:A S:HT

A:G H:ST

(2AW4)

0.560 0.169 0.391 0.377

A:A s:hT

A:U H:WT

A:G H:ST

(1N32)

0.602 0.249 0.225 0.218

1N32-Helix

Buckle Open Propeller Stagger Shear Stretch

1G:18U W:WC-8.47 3.77 -12.25 0.07 -2.23 2.85

-8.19 3.80 -12.53 0.08 -2.20 2.91

2C:17G W:WC-4.28 2.66 -19.84 0.00 0.56 2.80

-4.16 2.62 -19.93 -0.01 0.57 2.80

3A:16A s:hT-19.05 0.97 3.15 -0.16 2.75 2.83

-19.04 0.63 3.40 -0.15 2.70 2.84

4A:15U H:WT-4.18 3.54 -7.30 -0.25 -0.41 2.87

-3.73 3.47 -7.45 -0.24 -0.36 2.93

5A:14G H:ST-15.18 14.72 -10.81 0.13 2.77 3.10

-11.74 15.56 -14.48 0.13 3.43 3.05

6C:13G W:WC15.09 1.93 -17.29 -0.35 -0.13 2.72

15.04 1.90 -16.94 -0.36 -0.11 2.73

7C:12G W:WC8.40 3.17 -1.20 0.01 -0.05 2.88

7.84 3.24 -2.92 0.01 -0.13 3.04

8G:11C W:WC-3.30 0.87 -7.51 -0.47 -0.06 2.68

-3.33 0.77 -7.35 -0.47 -0.07 2.69

9G:10C W:WC-22.23 0.40 -9.15 -0.14 0.19 2.82

-22.07 0.34 -9.34 -0.14 0.18 2.82

Electrostatic Potential

RNA Secondary

Structure Prediction –

Modern Biology is

based on it !....

Structural Motifs of RNA

Helix

Hairpin

loop

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

-12 -8 -4 0 4 8 12 16 20 24 28 32 36

Roll

Fre

qu

en

cy

0

0.05

0.1

0.15

0.2

0.25

0.3

-4 0 4 8 12 16 20 24 28 32 36 40 44

Twist

Fre

qu

en

cy

Non-canonical

base step

Canonical

base step

A:A s:hT

G:C W:WC

Tk

FF

P

P

B

nwcwc

iwc

inwc exp

U:A W:HT

G:A H:ST

RNA 2d-structure

prediction – Free-

energy of stacking

MD Simulations from Crystal Structure

S. Halder and D. Bhattacharyya (2010) J. Phys. Chem. B 114: 14028

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11

Fre

qu

en

cy

C1'-C1' distance in Å

30S subunit of ribosome

Requirements:

Free-energy of Stacking

Modeling base pair steps

1FJG 1J5A 1N33 1N32 2AW4

6.08 5.15 5.53 5.27 5.35

5.75 5.67 5.62 5.05 5.80

5.38 5.09 5.52 7.31 5.50

5.27 6.39 5.33 5.34 5.21

5.33 5.16 5.30 5.00 6.10

5.89 5.39 5.65 5.90 4.99

5.54 5.72 5.47 5.19 5.49

5.30 4.99 5.13 5.86 5.50

6.25 6.29 5.57 5.92 5.45

4.79 5.26 5.60 5.78 5.76

5.54 5.29 5.70 5.40 6.40

5.62 5.36 5.62 5.32 5.67

5.28 5.85 6.37 5.57

5.56 5.35 5.36 5.14

5.34 4.98 4.68

5.45 5.11 7.17

5.51 5.27

5.45 5.64

5.72 6.39

5.60 4.90

5.86

5.19

5.90

5.00

5.34

7.31 5.055.27

5.92

5.78

5.40

5.32

6.37

5.36

4.98

5.11

Overlap between Successive Base Pairs

N1 surface dots

N2 surface dots

N surface dots

Overlap area = {(N1+N2)-N}/5

Implemented in NUPARM v2

3

5

7

9

11

18

20

22

24

26

28

30

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

C1

'-C

1' d

ista

nce

in Å

Ove

rlap

in Å

2OverlapC1'-C1' 1st strandC1'-C1' 2nd strand

A:A H:HTC:G W:WC

Twist

5

7

9

11

13

12

14

16

18

20

22

24

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90

C1

'-C

1' d

ista

nce

in Å

Ove

rlap

in Å

2

OverlapC1'-C1' 1st strandC1'-C1' 2nd strand

A:G W:ST

Twist

-20,-2.5 +20,-2.5

+20,+2.5-20,+2.5

Generated Structures for different

combinations of Roll and Slide

Calculated Interaction Energy by

quantum Chemical Method (DFT, etc)

ΔE=Ec-∑Em

Stacking Energy using Dispersion Corrected DFT

Mukherjee, SenthilKumar, Bansal & Bhattacharyya (2013) Bioopolymers (Epublished)

Summary

• Analyze Structures of DNA double helix, get their

avg. structural parameters Predict Structure and

Flexibility of chromosomal sequence

• Obtain structures of different non Watson-Crick base

pairs stacked on canonical ones

• Obtain Feasible Structures with good stacking

• Obtain Stacking Interaction energy using DFT

• Predict Relative Free Energy of different sequences

• Predict Secondary Structure of RNA ????

Funded and Supported by

CSIR, DBT and CDAC-Pune & DAE

Dr. Shayantani Mukherjee

Sukanya Halder

Prof. Manju Bansal (IISc)

Jhuma Das

Sankar Basu

Sanchita Mukherjee

SenthilKumar, D.K. (IISc)

Prof. Rahul Banerjee

Prof. Devapriya Choudhuri (JNU)

Prof. Abhijit Mitra (IIIT-H)

Purshottam Sharma (IIIT-H)

Arvind Marathe (IISc)

Sudhanshu Sankar (JNU)

Dr. Sudipta Samanta

Rahul Pal

Thank You