3DNA web site – building visualization NA structures Nucleic Acid Database UNAFold Web Server – thermodynamic-based predictions Nucleic Acid Structure Generator

Nucleic Acid Folds

Yeast phenylalanine tRNA (4tna.pdb)

acceptor end

anticodon

trna1.pdb

triplex region

Figure 4-18

http://jmol.x3dna.org/

contiguous helix

Yeast phenylalanine tRNA (4tna.pdb)

acceptor end

anticodon

trna2.pdb

Η

Ο

Ο

Ν

Ν

Ν

Ν

Ν

Ν

Ν

Η

Η

CH3

CH3

Ν

ΝΝ

Ν

Ο

Η

Ν Η

Η NΝ

O

Ο

HHG18

ψ55

m1A58T54

Ν

Ν

Ο

Ο

Η

ΗΗΝ

Η

ΟΝ

Ν

ΝΝ

G4

U69

Ν

ΝΝ

Ν

Ο

Η

Ν Η

Η

Ν

Ν

Ν

Ο

Η ΗG15C48

ΝΝ

Ν

Ν

Ο

Η

Ν ΗΗ

ΗΗ

Ο

Ν

ΝΝ

Ν

Ν

Ν

Ν

ΟΗ

Ν

Η

ΗCH3

ΝΝ

Ν

Ν

Ν ΗΗ

Η

Ο

Ο

ΝΝ

ΝΝ

Ν

Ν

ΝΗΗ

File: trnabp.cw2

A9

A23 U12

m7G46

G22C13

Base Pairing Found in tRNA

Figure 4-20

reverse Hoogsteen wobble

reverse Hoogsteen

A B C

A. - binderB. + binder C. pre conformational changeD. post conformational change

D

*P

*P

*P*P

Hyper reactive sites

Accessiblesites

induce cleavage

induce cleavage

*P

*P

*P

*P

Chemical Probe Assays of Nucleic Acid Structure and Interactions

Struct probes.ppt

O

Base

O OP

O

OO- O-

O

O

P

O

O OP

O

OO- O-

O

O

P

OH

O

O OPO

OO- O-

OOP

H

OH H

H

hemiacetalabasic site

O

O OPO

OO- O-

OOP

H

OH B

O

OP

O

OO-

H

OH

H -O

O-O

O

P

B

OH

OH

-O

O-O

O

PO-P

O

OO-

3'-phosphate 5'-phosphate

enolization

β-elimination(retro-Michael Rxn)

β-elimination(retro-Michael Rxn)

hydrolysis

Mechanism of strand cleavagefollowing glycosidic bond hydrolysis

B

B H

Figure: cleave1.cw2

In the presence of piperidine, the β-elimination reactions may take place through the enamine.

Dimethyl sulfate (DMS). Alkylates sterically accessible N7 of purines.(See the Maxam-Gilbert G reaction in chapter on sequencing.)

Reactivity:1. The N7 of G in single strand and duplex DNA.2. The N7 of purines in the anti conformation.

N

N

G > A

CH3 O SO

OO CH3

DMS

N

N

CH3

Dimethyl Sulfate Probe for the Accessibility of N7. A Major Groove Accessibility Probe.

dms_rxn.cwg

File: DEPC.cwg

Diethylpyrodicarbonate (DEPC) probe ofsingle stranded purines

Approach is from the major groove side. DEPC is bulky, and reaction with N7 is inhibited in duplex DNA.

Reactive N7:1. Single strand DNA.2. Loops of cruciforms.3. Purines in the syn conformation in Z DNA.

DEPC

+

N

N

OO

CH3 O O

O O

O CH3

N

N

A or G

N

NO

O

H O

O

Os

N

N

O

O

CH3

H

N

NO

O

HCH3

H

O

O

Os

N

N

O

O

file: oso4.cwg

Osmium tetroxide and KMnO4 oxidation of5,6-double bond of pyrimidines

Reagent must approach from above or below the plane of the pyrimidinetherefore it will not work well on stacked DNA

Reactivity:1. Reacts with T's at junctions between B & Z DNA.2. Reacts with T's in cruciform loops.

N

N

O

NH2

N

N

O

NHO

H

N

N

O

N

NHOH

HO

H

labile to piperidine

file: HAma.cwg

Hydroxylamine reactions with C

Reagent must approach from above or below the plane of the pyrimidinetherefore it will not work well on stacked DNAReactivity:1. Reacts with C's at junctions betwee B & Z DNA.2. Reacts with C's at junctions between out of phase Z DNA blocks such as the sequence shown below:

5'-GCGCGC-CGCGCG-3' 3'-CGCGCG-GCGCGC-5'

3. Reacts with C's in cruciform loops.

SHAPE analysis of RNA Selective 2’-hydroxyl acylation analyzed by primer extension

Shaperxn.ppt

1. Mortimer, S. A. and K. M. Weeks (2007) A Fast-Acting Reagent for Accurate Analysis of RNA Secondary and Tertiary Structure by SHAPE Chemistry. J Am Chem Soc 129, 4144-4145.

Overview of SHAPE-Seq. (A) Experimental pipeline. A DNA bar code is added to the 3′ end of template molecules, enabling SHAPE chemistry and sequencing library generation to be done on a mixture of bar-coded RNAs. PNAS 2011, 11063

Multishape.ppt

Figure 4-15

5'- AGGAAG GAAGGA 3'3'- TCCTTC CTTCCT 5'

5'- AGGAAG3'- TCCTTC

TCCTTC

3'5'

AGGAAG single strand

triplex

Figure: Hdna1.cw2

GAAGGA

5' 3'

CTTCCT

CTTCCT 3'GAAGGA 5'

single strand

triplex

H DNA, Hoogsteen DNA or Hinged DNA forms in reverse repeat purine DNA under high negative supercoiling

reverse repeat not inverted repeat

Chemical Probing of H-DNA Johnston, B.H. (1988) The S1-sensitive form of d(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science, 241, 1800-4.

HDNAgel.ppt

a) normal superhelical density b) Higher superhelical density (higher torsional stress)

HDNAanal.ppt

The End Replication Problem

Succesive rounds of replication lead to progressive shortening of the ends of DNA

RNA RNA RNA

missing DNA

replication of this strand results in a shorter DNA

Telomerase solves the End Replication Problem, RNA templated DNA synthesis

elongation elongation

translocation

ribonucleoprotein

Annu. Rev. Pharmacol. Toxicol. 2003. 43:359–79

Figure 4-21b

Schematic structure of a telomere

single strand end protected by DNA displacement loop formation POT

protection of telomere binds TTAG3

The G’s in the telomere sequence can form Quartets via Hoogsteen Base Pairing, • Hoogsteen base pairing leads to circluar tetrad. • Center of quartet has large negative electrostatic

potential that can bind cations • all anti glycosyl conformation leads to parallel

stranded quadruplex

Figure 4-22

N

NN

N

O

N

H

H

H

HN

N

N N

O

NHH

H

H

N

NN

N

O

N

H

H

H

H

H

HH

HN

O

N N

NN

Figure: G_tetra1.cw2

+

Four possible orientations of Gn strands

Figure 4-26

parallel antiparallel

mixed (3+1) antiparallel

Ways of forming intramolecular quadruplex formation with [GxNy]z with 3 types of loops: propeller, lateral, diagonal

Figure 4-27

3'

5'

3'

5'

5'3'

diagonal loop

lateral loop

externalloop

externalorpropellerloop

lateral loop

lateral loop

lateral loop

5'

3'

lateral loop

lateral loop

lateral loop

propeller

basket chair

hybrid

a

aa

a

aa

ss

ss

s s

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

s

s

ss

s

s

aa

a

a

aa

s

ss

s

s s

Flip central Base quad

Glycosyl conformation depends on strand orientation. Bases in one base quad can all flip from anti to syn, and syn to anti

Figure 4-28

Front. Chem. 4:38. doi: 10.3389/fchem.2016.00038

J. Phys. Chem. B, Vol. 110, No. 32, 2006 16077

Li+ is strongly hydrated and cannot bind

Na+ fits in the plane K+ fits between the planes

Cramer and Truhlar

prop

ella

r

lateral

lateral2

3

syn

anti

A21

T20

T19

G18

G17

G16

A15T14

T13

G12

G11

G10A9

T8

T7

G6

G5

G4

A3

1

1

2

3

G11

G12

T13

T14

A15

G23

G24

T20

A21

T7

G10

A9T8

A3

G4

G5

G6

syn

anti

G22

T19

K+

T8

T7

G4

G5

G10

G11

X

T13A15

G17

G18

T19A21

G12

G16

syn

anti

T20

T14

G6

A9

Y

5'3'

diagonal

lateral lateralT8

T7G10

G11

T13

T14

A15

G17

T19

A21

G12

syn

anti

A9T18

Na+

Na+

Na+

G5

G6G18

G16 G4

laterallateral

diagonal

hybrid-1 hybrid-2

basket form 3

Characterized human telomeric DNA G-quadruplex structures in solution by NMR

Folding and Unfolding Pathways for the Human Telomeric G-Quadruplex

J. Mol. Biol. (2014) 426, 1629–1650

Chemical Probes of G-quartet structures. Hoogsteen base pairing interferes with G reaction (reaction at N7).

PNAS 2002 99 11593-11598

iMotif, forms from the strand complementary to the d(TTTGGG)n at low pH (pH 6). Intercalated.

H. A. Day et al. / Bioorg. Med. Chem. 22 (2014) 4407–4418

d(TC5) 225D.pdb human telomeric

1A83.pdb

1EL2.pdb

Holliday Junction

1juc.pdb

Part of a cruciform, or an intermediate in recombination

5’-CCGGTA CCGG-3’ 3’-GGCCAT GGCC-5’ | | 5’-CCGG TACCGG-3’ 3’-GGCC ATGGCC-5’

5’-CCGGT ACCGG-3’ 3’-GGCCA TGGCC-5’

ACCG

G-3

’ TG

GCC

-5

ACCGG

-3’ TCCG

G-5

recombination representation

cruciform represen- tation

RNA tertiary structural motifs

1. Hendrix, D. K., S. E. Brenner and S. R. Holbrook (2005) RNA structural motifs: building blocks of a modular biomolecule. Quarterly reviews of biophysics 38, 221-243.

RNAs can adopt a variety of structures

GNRA tetraloops –tetraloop receptor

1. Fiore, J. L. and D. J. Nesbitt (2013) An RNA folding motif: GNRA tetraloop–receptor interactions. Quarterly Reviews of Biophysics 46, 223-264.

430.pdb

A14

G15

A16

G17

G17

A14

GNRA loop

1. Correll, C. C., A. Munishkin, Y.-L. Chan, Z. Ren, I. G. Wool and T. A. Steitz (1998) Crystal structure of the ribosomal RNA domain essential for binding elongation factors. Proceedings of the National Academy of Sciences 95, 13436-13441.

G10

U11 A20

GNRA Loop

bulge loop

Pseudoknots folding resulting from base pairing to loop of a hairpin

1. Giedroc, D. P., C. A. Theimer and P. L. Nixon (2000) Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting11Edited by D. E. Draper. Journal of Molecular Biology 298, 167-185.

viral RNA pseudoknot at atomic resolution 1L2X.pdb

red

blue

∑∑ ∆−∆+∆−∆=∆+∆=∆x

xxiix

xitotal sThsThggG

+∆G

coil - helix equilibrium

Figure: co_he1.cdr

Free energy of duplex formation is thesum of initiation and propagation terms

propagation

4 H-bonds

4 H-bonds

H

HO H O

H

HO H O

H

H

H

HO

NN

O

O

CH3

HH

HOH

H

H

HHN

N

N

NN

OH

H

H O

H

NN

O

O

CH3

HH

H

H

HHN

N

N

NN

Base Pairing in water conservesthe number of H-bonds

lect5_f1.cw2

Therefore Δhi ~ 0

eusKkcalsTsThg iiiii 18,298@65 −≈∆∴°−≈∆−≈∆−∆=∆

∆gi

Initiation

Free energy of initiation is purely entropic (no H-bonding in water)

Figure: hel_init.cdr

Therefore free energy of initiation can be considered to be purely entropic (Δhinitiation = 0) and corresponds to loss of 3 degrees of translational and 3 degrees of rotational freedom

Equipartition theory kT/2 per degree of freedom =3kT or 3*RT = 3*1.98*298 =

eucoiltheofnsorientatiohelixtheofnsorientatio

Rsx 2.26)233273/(1ln(98.12##

ln2 −=⋅⋅⋅⋅⋅⋅⋅=

⋅⋅=∆

Base Pair Stack Energy(kcal/mole)

GC/GC -14.6GT/AC -10.5GA/TC -9.8CG/CG -9.7CC/GG -8.3CT/AG -6.8AT/AT -6.6CA/TG -6.6TT/AA -5.4

6 rotatable bonds +1 pseudorotation angle

O BaseO

O-OP

O

O

Free energy of propagation has a favorableenthalpy term (pi stacking) but unfavorable entropy term (lower degrees of freedom)

Figure: hel_prop.cdr

∆gx

Δ Back of the envelop calculation of entropy term: Δsx = R * ln(# conformations helix/#conformation ss) Δsx = R*ln((1/(3^5*2*2)^2)=1.98*-14 = -28 eu therefore avg Δgx = -8.2 * 298*-28/1000 = -8.2+8.3 ~ 0

TA/TA -3.8

Experimentally derived thermodynamic parameters for helix formation from oligodeoxynucleotides from Breslauer et al. (1986) [Breslauer, 1986 #63]. Values in parentheses are from SantaLucia et al. (1996) [SantaLucia, 1996 #1971]. ∆gi for GC containing helices is set at 5 kcal and for helices containing only AT base pairs, 6 kcal, both I assume at 1 M NaCl, 25oC, and pH 7.

Base Pair Stack ∆H ∆S ∆G (1 M NaCl, 25oC, pH 7)

CG•CG -11.9 (-10.1) -27.8 (-25.5) -3.6 (-2.09)

GC•GC -11.1 (-11.1) -26.7 (-28.4) -3.1 (-2.28)

GG•CC -11.0 (-6.7) -26.6 (-15.6) -3.1 (-1.77)

AA•TT -9.1 (-8.4) -24.0 (-23.6) -1.9 (-1.02)

CA•TG -5.8 (-7.4) -12.9 (-19.3) -1.9 (-1.38)

CT•AG -7.8 (-6.1) -20.8 (-16.1) -1.6 (-1.16)

GA•TC -5.6 (-7.7) -13.5 (-20.3) -1.6 (-1.46)

AT•AT -8.6 (-6.5) -23.9 (-18.8) -1.5 (-0.73)

GT•AC -6.5 (-8.6) -17.3 (-23.0) -1.3 (-1.43)

TA•TA -6.0 (-6.3) -16.9 (-18.5) -0.9 (-0.60)

Table thermo DNA.ppt

Thermodynamic parameters depend on sequence

Table. Calculation of ∆G for a series of oligonucleotides using the Breslauer thermodynamic parameters.

sequence ∆gi ∆gx ∆G G•C +5 0 5 GA•TC +5 0-1.6 3.4 GAC•GTC +5 0-1.6-1.3 2.1 GACG•CGTC +5 0-1.6-1.3-3.6 -0.5 GACGT•ACGTC +5 0-1.6-1.3-3.6-1.3 -1.8

Short DNA duplexes are unstable because the unfavorable ∆G for initiation dominates, but as it gets longer, the favorable ∆G propagation term dominates

Table ODN thermo calc.ppt

-3-2-10123456

0 2 4 6

ΔG

ODN length (bp)

Fig RNA example.ppt

RNA produced in vivo by transcription is single stranded and can thus fold intramolecularly by base pairing

One has to add, however, the entropic penalty for the single stranded sections, whose conformations are reduced relative to the unfolded single strand RNA

Table. Free energies at 25oC for base stacking in RNA duplexes.

Base-paired region ∆G (25oC)

AA•UU -1.2 AU•AU, UA•UA -1.8 AC•GU, CA•UG, AG•CU, GA•UC

-2.2

CG•CG -3.2 GC•GC, GG•CC -5.0 GU•GU -0.3 GX•YU, XG•CY (XY=Watson Crick bp)

0

Simple RNA folding parameters

Table RNA thermo.ppt

Approximate loop free energies at 25º C for RNA

Structure Number of bases unbonded ∆G (25oC)

Interior 2-6 +2

Loop 7-20 +3

m >20 1+2*log(m)

Bulge 1 +3

Loop 2-3 +4

4-7 +5

8-20 +6

m > 20 4+2*log(m)

Hairpin Loop

Closed by GC Closed by AU

3 +8 >8

4-5 +5 +7

6-7 +4 +6

8-9 +5 +7

10-30 +6 +8

m > 30 3.5+2*log(m) 5.5+2*log(m)

Table loop E.ppt

Secondary folding predictions at the Mfold web server

Lets fold yeast tRNA Phe GCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUGAAGAUCUGGAGGUCCUGUGUUCGAUCCACAGAAUUCGCACCA

UNAFold Web Server

Folding prediction for yeast tRNA

Observed secondary structure -21.90 kcal/mol

-22.9 kcal/mol

actual

5S RNA from yeast 70S ribosome (3o58.pdb1)

Mfold-predicted structure Mfold_5S.ppt

Mfold calculations for 5S and 5.8S RNAs

-42.9 -38.0 Mfold predicted structures

5.8S RNA from yeast 70S ribosome (3o58.pdb1)

Mfold_5.8Sa.ppt

-45 -39

-40 -45.5

Mfold_5.8Sb.ppt

-37 -41.6

-43.3 -40.7

Mfold_5.8Sc.ppt