Structural Studies of Non-

canonical Base pairs in RNA

Dhananjay Bhattacharyya

Biophysics Division

Saha Institute of Nuclear Physics

Kolkata

dhananjay.bhattacharyya@saha.ac.in

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

Viral RNA

Signaling RNA

miRNA

siRNA

tRNAIle Crystal Structure

(PDB ID: 1QU2)

AG

T

C

Basepair Parameters

(IUPAC/IUB recommendation

Base pair parameter definition in

NUPARM

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) |}

M

S. Mukherjee, M. Bansal; D. Bhattacharyya (2006) J. Comp. Aided Mol. Des. 20; 629

A:U H:WT

Non-canonical Basepairing

W: Watson-Crick edgeH: Hoogsteen edgeS: Sugar edgew/h/s: Involves weak C-HO/N interactionC/T: Cis- or Trans-orientation of the two glycosidic bonds

DH

A ABHA

R1

R2

),,(fr

R

r

Rd

r

qq)r(V

bondedHij

ijmin,

ij

ijmin,

ij

ij

ji

1012

Van der Waals

Approximate Hydrogen Bond

Van der Waals & Coulomb

Approximate H-Bond & Coulomb

Base Pair Finder

Took a base edge

Identify the H-bonding centers (N3G & N2G)

Look for H-bond partner through distance calculation (N6A & N7A)

Calculate pseudo-angles (such as C6G-N3G-N6A, N3G-N6A-N1A, N1G-N2G-N7A, N2G-

N7A-N9A in figure) for

planarity

Confirm orientation through angle calculation

Calculate E= i(di-3.0)2 + k( k- i are for

two H-bond distances and k are for four pseudo

angles

Gives rise to:

6959 A:U W-W(C);

21965 G:C W-W(C) and

2786 G:U W-W(C) base pairs

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

161

G:U W:W Cis (846)

U:U W:W Cis (84)

A:G W:W Cis (150)

A:G H:S Trans (558)

A:U H:W Trans (410)

A:A H:H Trans (109)

Property of good and stable

Base PairDISTRIBUTION OF PROPELLER VALUE

0

0.05

0.1

0.15

0.2

0.25

0.3

-50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45

PROPELLER VALUE

RE

LA

TIV

E F

RE

QU

EN

CY

GC_WWC

AG_HST

AU_HWT

DISTRIBUTION OF STAGGER VALUE

0

0.1

0.2

0.3

0.4

0.5

-3

-2.7

-2.4

-2.1

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3 0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

STAGGER VALUE

RE

LA

TIV

E F

RE

QU

EN

CY

GC_WWC

AG_HST

AU_HWT

DISTRIBUTION OF SHEAR VALUE

0

0.1

0.2

0.3

0.4

0.5

-2

-1.7

-1.4

-1.1

-0.8

-0.5

-0.2

0.1

0.4

0.7 1

1.3

1.6

1.9

2.2

2.5

2.8

3.1

3.4

3.7

SHEAR VALUE

RE

LA

TIV

E F

RE

QU

EN

CY

GC_WWC

AG_HST

AU_HWT

DISTRIBUTION OF STRETCH VALUE

0

0.1

0.2

0.3

0.4

2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9

STRETCH VALUE

RE

LA

TIV

E F

RE

QU

EN

CY

GC_WWC

AG_HST

AU_HWT

DISTRIBUTION OF OPEN ANGLE

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

-25

-17 -9 -1 7 15 23 31 39 47

OPEN ANGLE VALUE

RELA

TIV

E F

REQ

UEN

CY

GC_WWC

AG_HST

AU_HWT

Geometry Optimization by different

Methods

Selected structures of BPs from PDB

Optimized Structures by B3LYP/6-31G**

Optimized by MP2/6-31G**

Optimized by HF/CC-pVDZ

Optimized by GGA-BW91/DZP

Optimized by semi-empirical methods (AM1, PM3)

Optimized by AMBER force-field

Compared structure and dynamics with Molecular Dynamics Simulations

Failure of AM1 (most popular semi-

empirical method)

G:C W:W C

E = -26

kcal/mol

A:U W:W C

E = -14

G:U W:W C

E = -15

A:G H:S T

E = -10

A:G s:s T

E = -6

A:U H:W T

E = -13

A:A H:H T

E = -10

G:A W:W C

E = -15

G:A S:W T

E = -11

A:A W:W T

E = -12

A:U W:W T

E = -13

A:A H:W T

E = -11

2=>NHO

1=>NH...N

1=>NHO

1=>NHN 2=>NH...O

2=>NH...N

1=>NHN

1=>CH...O

1=>NH...O

1=>NH...N2=>NH...N

1=>NH...O

1=>NHN

2=>NH...N

2=>NH...N

1=>NH..O

1=>NH...N

1=>NH...O

1=>NHN

Strengths of different H-bonds from 33 non-canonical Base Pairs

Ray, Panigrahi, Bhattacharyya & Bhattacharyya (2008) J. Phys. Chem. B112, 3786.

Considered Energy components, ENHO, ENHN, etc are additive.

Additional stabilities, i may come from van der Waals, dipole-

dipole etc interactions.

Least Squares Fit indicates i, errors should be smallest for best Fit

i

CHNCHN

i

CHOCHO

i

OHNOHN

i

NHNNHN

i

NHONHO

i

i EnEnEnEnEnEint

2

int

2

i

CHNCHN

i

CHOCHO

i

OHNOHN

i

NHNNHN

i

NHONHO

i

i

i

i EnEnEnEnEnE

Type of H-bond E (kcal/mol)

N-HO -7.82

N-HN -5.62

O-HN -6.89

C-HO -1.33

C-HN -0.67

Roy, Bhattacharyya, Panigrahi, Bhattacharyya, (2008) J. Phys. Chem. B B112, 3786

Comparison with X-ray

DISTRIBUTION OF BUCKLE VALUE OF GC_WWC

0

500

1000

1500

2000

2500

3000

-100 -9

0-8

0-7

0-6

0-5

0-4

0-3

0-2

0-1

0 0 10 20 30 40 50 60

BUCKLE VALUE

FR

EQ

UE

NC

Y

Series1

MP2:-4.92

CCD:-4.2

DFT:-0.3

ADF:-1.5

DISTRIBUTION OF PROPELLER VALUE OF AU_HWT

0

0.05

0.1

0.15

0.2

0.25

0.3

-50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45

PROPELLER VALUE

FR

EQ

UE

NC

Y

Series1

MP2:-1.93

CCD:-1.63

DFT:1.91

ADF:-0.91

DISTRIBUTION OF SHEAR VALUE OF AG_HST

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

-2

-1.7

-1.4

-1.1

-0.8

-0.5

-0.2

0.1

0.4

0.7 1

1.3

1.6

1.9

2.2

2.5

2.8

3.1

3.4

3.7

SHEAR VALUE

FR

EQ

UE

NC

Y

Series1

MP2:1.79

CCD:1.9

DFT:1.95

ADF:2.09

Hydrogen Bond Geometries

DISTRIBUTION OF N-H...O BOND

0

2

4

6

8

10

12

1.7 1.8 1.9 2 2.1 2.2 2.3

RANGE

FR

EQ

UE

NC

Y MP2

DFT

CCD

ADF

AMBER_WATER

AMBER

PM3

DISTRIBUTION OF N-HN BOND

0

5

10

15

20

25

1.7 1.8 1.9 2 2.1 2.2 2.3

RANGES

FR

EQ

UE

NC

Y

MP2

DFT

CCD

ADF

AMBER_WATER

AMBER

PM3

D

H

A ABHA

R2

Non-polar Base Pairs

Frequency: 289

Frequency: 48

Structures of others with weaker H-bonds

-6

-4

-2

0

2

4

6

8

1 3 5 7 9

11 13 15 17 19 21 23 25 27

BASEPAIR

SH

EA

R V

ALU

E

MP2/631G**

HF/ccPVDZ

B3LYP/631G**

B91/DZP

AMBER

AMBER+WATER

PM3

CRYSTAL_DATA

-120

-100

-80

-60

-40

-20

0

20

40

60

80

100

1 3 5 7 9

11 13 15 17 19 21 23 25 27

BASEPAIR

PR

OPELL

ER

VA

LUE

MP2/631G**

HF/ccPVDZ

B3LYP/631G**

B91/DZP

AMBER

AMBER+WATER

PM3

CRYSTAL_DATA

-120

-100

-80

-60

-40

-20

0

20

40

60

80

1 3 5 7 9

11 13 15 17 19 21 23 25 27

BASEPAIR

OPEN

AN

GLE

VA

LUE

MP2/631G**

HF/ccPVDZ

B3LYP/631G**

B91/DZP

AMBER

AMBER+WATER

PM3

CRYSTAL_DATA

nnnnnn

nn

nn

jijjij

TVTV

TVTV

TVTVTV

VT

kxdt

xdm

2

1

2

1

2

2

221

2

21

1

2

112

2

1211

2

11

2

2

....

........

....

..

0

0

= 0

ji

ijxx

VV

2

V is Total (QM) Potential Energy

Theory of Harmonic Vibration

k,where

,xdt

xd

2

2

2

2

0 Tk/)(k Boe)(

22

1

k/Tk.ln B22calc =

0.2

0.4

0.6

0.8

1

0.2 0.4 0.6 0.8 1

Crystallographic

Ca

lcu

late

d

Roy, Panigrahi, Bhattacharyya & Bhattacharyya, J. Phys. Chem. B (2008) B112, 3786

Sen, K.; Basu, S.; Bhattacharyya, D. Int. J. Quant. Chem. (2006) 106, 913

0

0.25

0.5

0.75

1

-3 -2 -1 0 1 2 3

Assignment of Vibration modes (frequency) to type of Motion:

Generated two sets of coordinates of all the atoms, Ximax & Ximin

Ran NUPARM on both to find major differences in parameters

Base pairs vibrate mostly along five (instead of six) directions

Vibrations by breaking H-bonds are often prohibited

Vibrations are in the time scale of pico second

Force constants can be used for CG simulations

Conclusions / AppealsNon canonical base pairs are important for RNA structure prediction

Many of these are sufficiently strong

Estimated Force-constants can be used for CG modeling

Their stacking interactions (combination of p-p interactions and hydrophobic effect)

needs to be estimated.

Major contributors:

Malyasri Bhattacharyya

Shayantani Mukherjee

Jhuma Das

Swati Panigrahi

Ashim Roy

Sukanya Halder

Collaborators:

Prof. Manju Bansal

Prof. Abhijit Mitra

Prof. Jaydeb Chakrabarti

Prof. Jiri Sponer

Supporters:

CSIR and DBT (Govt. of India)

CAMCS and CBAUNP (DAE, SINP)

CDAC (Pune)