Nucleic acid structure

Nucleic Acid Chemistry

Third term 2022-2023

Randy Bryant

Department of Biochemistry and Molecular Biology

Johns Hopkins Bloomberg School of Public Health

 Deoxyribonucleic acid (DNA)

DNA is a linear polymer containing linked

monomeric units

Monomeric units are arranged in a precise

linear sequence

The linear sequence encodes biological

information
 Deoxyribonucleotides

The monomeric units that form the DNA polymer are called
deoxyribonucleotides

Deoxyribonucleotides are made

up of three components:

1. sugar
2. base
3. phosphate
 Deoxyribose

The sugar portion of a deoxyribonucleotide is derived from

the 5-carbon pentose: 2´-deoxyribose (or deoxyribose)

5’
5´

4´
4’ 1’
1´

no –OH
3‛
3´ 2‛
2´
here

Deoxy- indicates that there is no hydroxyl group at the 2´-position

 Aqueous solution equilibria for deoxyribose

anomeric carbon
1´ anomeric carbon
2´
5´ 3´
1´
4´
1´
4´

5´

DNA

The cyclic deoxyribofuranose form is found in DNA nucleotides

 Base

The bases that are most commonly found in

DNA are:

adenine, guanine, cytosine, and thymine

All are structurally related to the heterocyclic

compounds: purine or pyrimidine
 DNA bases

purine bases

adenine guanine purine

(A) (G)

pyrimidine bases

pyrimidine
cytosine thymine
(C) (T)
Purine and pyrimidine bases: numbering system

7 6 4
5
1 5 3
8
2 6 2
9 4
3 1

purines pyrimidines

Exocyclic substituents are identified according to the number

of the atom within the ring to which they are attached
 Nucleoside α and β anomers

A base can be attached to a deoxyribose by a glycosidic bond

5´

1´

5´

1´

α-anomer β-anomer
base below deoxyribose ring base above deoxyribose ring
DNA
(relative to 5´-carbon) (relative to 5´-carbon)

Are two possible isomers: α-anomer and β-anomer

 Glycosidic bond

The bases in DNA are attached to the deoxyribose by a β-glycosidic

bond

base

C5´
N9
β-glycosidic
C1´
deoxyribose bond

Deoxyribose and base = deoxyribonucleoside

 Deoxyribonucleosides

purines
N9 N9

C1´ C1´

deoxyadenosine (dA) deoxyguanosine (dG)

pyrimidines
N1 N1

C1´ C1´

deoxycytidine (dC) deoxythymidine (dT)

 Phosphate group

Deoxyribonucleosides can be phosphorylated on the 5´-OH group

base

5´

phosphate deoxyribose

Deoxyribose, base, and phosphate group = deoxyribonucleotide

 Deoxyribonucleotides

deoxyadenosine 5´-monophosphate deoxyguanosine 5´-monophosphate

(dAMP) (dGMP)

deoxycytidine 5´-monophosphate deoxythymidine 5´-monophosphate

(dCMP) (dTMP)
 Phosphodiester bonds

Deoxyribonucleotides can be linked by 3´-5´phosphodiester bonds

3´
5´

3´
H+ + 5´
phosphodiester
pKa ̴ 0 3´
5´

Chain of deoxyribonucleotides = deoxyribonucleic acid (DNA)

 DNA polarity

Linear DNA molecules have chemically distinct 5´ and 3´-ends

5´-end 5´

3´
5´

3´
5´

3´
5´

3´
3´-end

The terminal 5´ and 3´-OH groups may be unphosphorylated or

phosphorylated
 DNA sequence

The sequence of a DNA molecule is specified in the 5´→ 3´ direction

5´-end
T

3´
A
5´

C
3´
5´

G
3´
5´

3´-end

The sequence of this DNA molecule would be: dTACG

 DNA chain length

Oligonucleotide: Refers to shorter DNA molecules

Generally less than ̴50 nucleotides

in length

Polynucleotide: Refers to longer DNA molecules

Generally more than ̴50 nucleotides

in length
 DNA strands

Single-stranded: DNA molecules consisting of a single

chain

Referred to as single-stranded DNA

(ssDNA)

Double-stranded: DNA molecules consisting of two

chains

Referred to as double-stranded DNA

(dsDNA) or duplex DNA

Most commonly occurring form of DNA

in biological systems
Watson-Crick paper

DNA structure

James Watson and Francis Crick

Nature 17, 737 (1953)
 B-form DNA

The DNA structure proposed by Watson and Crick in 1953

Was based on x-ray diffraction data obtained by Rosalind

Franklin with DNA fibers that were formed at high humidity
(95% RH)

Is stabilized by extensive interactions with water molecules

Is the most common form of DNA under physiologicial

conditions
 Watson-Crick model

http://www.sciencemuseum.org.uk/images/I045/10313925.aspx
Rosalind Franklin and Photograph 51
 B-form DNA: The two ssDNA chains are coiled around a common axis

Form a right-handed double helix with

34 Å per helical turn

The two chains are in an antiparallel

orientation (opposite polarities)

The sugar-phosphate backbones of the

34 Å two chains are on the outside

The purine and pyrimidine bases are on

the inside

The bases are perpendicular to the central

axis

There are 1 ̴ 0 nucleotides per strand in each

helical repeat
Watson and Crick, Nature 17, 737 (1953)
 Base pairing

N6 O4

An adenine (A) in one strand is paired

N1 N3 with a thymine (T) in the other strand
(A:T base pair)
A:T pair

O6 N4

A guanine (G) in one strand is paired

N1 N3 with a cytosine (C) in the other strand
N2
(G:C base pair)
O2

G:C pair

The specificity of the base pairings is determined by hydrogen bonding

interactions between the bases
 Keto-enol tautomerization

keto

enol

Watson and Crick learned from chemist Jerry Donohue that the
keto forms of the bases would be the more stable tautomers

This insight allowed the base pairing interactions to be deduced

Original Watson-Crick base pair models
 Complementarity

Base pairing interactions are required in order for two DNA

strands to form a double helix

The sequence of one strand is

therefore necessarily related
to the sequence of the second
strand

The two strands are complementary in sequence

 Complementary base-paired DNA strands

5´ 3´

3´ 5´

The two strands in B-form DNA are oriented in an antiparallel manner

B-form DNA

Helix parameters B-DNA

Helix sense right-handed

Helical pitch 34 Å
Residues per turn 10
Twist per base pair 36o
Rise per base pair 3.4 Å
Base tilt -6o
Sugar pucker C2´-endo
Glycosyl angle anti
Groove width
major 11.7 Å
minor 5.7 Å
 Deoxyribose sugar pucker

The deoxyribose sugar is conformationally flexible

H H
HO H
3´ B 2´ B endo face
HO O

H 2´ H
H
⇌ HO

3´
O
H
H OH exo face
3'-endo 2'-endo
(north) (south)

B-DNA

The C2´-endo conformation is favored in B-form DNA

 Syn and anti conformations: purines

Have free rotation of the bases around the β-glycosyl bond

syn anti
N3 above sugar ring H8 above sugar ring

The anti conformation of purine nucleotides is found

in B-form DNA
 Syn and anti conformations: pyrimidines

Have free rotation of the bases around the β-glycosyl bond

6
2

syn anti
O2 above sugar ring H6 above sugar ring

The anti conformation of pyrimidine nucleotides is found

in B-form DNA
 Major and minor grooves

minor groove outer edges

5.7 Å

major groove
11.7 Å

inner edges

B-form DNA has a major groove and a minor groove

 Base stacking interactions

B-form DNA is stabilized by stacking interactions between

the planar bases
B-form DNA

Wikipedia commons
 A-form DNA

First described by Rosalind Franklin in 1953

Is the conformation found in DNA fibers that

were formed at low humidity (5% RH)

DNA oligomers in many crystal structures are

found in the A form
Watson-Crick paper

Alluded to existence of A-form DNA

A-form DNA

Helix parameters A-DNA

Helix sense right-handed

Helical pitch 29 Å
Residues per turn 11
Twist per base pair 32.7o
Rise per base pair 2.6 Å
Base tilt 20o
Sugar pucker C3´-endo
Glycosyl angle anti
Groove width
major 2.7 Å
minor 11.0 Å
 Deoxyribose sugar pucker: B and A-form DNA

C2´-endo C3´-endo
B-form A-form

The C3´-endo pucker produces a shorter phosphate-phosphate

distance in the DNA backbone

Results in a more compact helical conformation in A-form DNA

 Comparison of B and A-form DNA

B-DNA A-DNA

Bases are directly over the Bases are displaced 4.5 Å away
helix axis from helix axis
Creates a hollow core running
down the axis
 DNA helix parameters

trans-locational movements
of base pairs

rotational movements
of bases

There are many local variations in A and B-form DNA structures

 Z-form DNA

First described by Alexander Rich and colleagues in 1979

Observed in the crystal structure of the self-complementary

hexanucleotide:

5´- GCGCGC -3´

3´- CGCGCG -5´

More generally found in oligomers containing alternating

purine and pyrimidines (especially G and C)

Consists of a left-handed double helix

Z-form DNA

Helix parameters Z-DNA

Helix sense left-handed

Helical pitch 45 Å
Residues per turn 12
Twist per base pair -30o
Rise per base pair 3.8 Å
Base tilt 7o
Sugar pucker
dG C3´-endo
dC C2´-endo
Glycosyl angle
dG syn
dC anti
Groove width
major convex
minor deep
Backbone conformations of B and Z-form DNA

B-form Z-form
(right-handed) (left-handed)
 Major conformations of DNA

B-form A-form Z-form

 Ribonucleic acid (RNA)

RNA is a linear polymer containing linked

monomeric units

Monomeric units are arranged in a precise

linear sequence

The linear sequence encodes biological

information
 Ribonucleotides

The monomeric units that form the RNA polymer are called
ribonucleotides

Ribonucleotides are made up

5´
of three components:
4´ 1´
1. sugar - ribose
3´ 2´
2. base
3. phosphate

Ribonucleotides have a hydroxyl group at the 2´-position

 Ribonucleotides

adenosine 5´-monophosphate guanosine 5´-monophosphate

(AMP) (GMP)

cytidine 5´-monophosphate uridine 5´-monophosphate

(CMP) (UMP)
 Uracil and thymine

4 4
3 3
5 5
2 2
6 6
1 1

thymine uracil
(DNA) (RNA)
 RNA structure

Ribonucleotides can be linked by 3´- 5´phosphodiester bonds

Linear chain of ribonucleotides = ribonucleic acid (RNA)

Watson-Crick paper

Predicted duplex RNA would

have a different structure
 Steric clash of the 2´-OH group in RNA

3´-endo 2´-endo

The 2´-endo conformation results in a steric clash between the

2´-OH group of the ribose and the adjacent phosphate group
The 3'-endo conformation relieves this steric clash
A-form RNA

Helix parameters A-RNA

Helix sense right-handed

Helical pitch 29 Å
Residues per turn 11
Twist per base pair 32.7o
Rise per base pair 2.6 Å
Base tilt 20o
Sugar pucker C3´-endo
Glycosyl angle anti
Groove width
major 2.7 Å
minor 11.0 Å
 RNA-DNA hybrids

Solutions studies indicate a mixed A/B conformation

Generally resemble the A-form, with the RNA sugars in

the 3´-endo conformation (A-form), but some or all of the
DNA sugars in the 2´-endo conformation (B-form)

Specific structure is dependent on experimental conditions

and the base composition of the RNA and DNA strands
 RNA-DNA hybrid duplex

RNA-DNA duplex consisting of d(GGCGCCCGAA) and r(UUCGGGCGCC)

 Spectroscopic properties of nucleic acids

The base components of nucleotides absorb with

λmax values in the range of 250 - 270 nm

The sugar and phosphate components, however, do

not exhibit significant UV absorption above 230 nm

So nucleotides have UV absorption profiles that are

similar to those of the constituent bases
 Absorption spectra for common nucleotides

Absorbance measurements are typically made at 260 nm for

mixtures of nucleotides (DNA or RNA)
 Hypochromicity

Bases in duplex DNA and RNA structures are stacked in a

face-to-face manner

Results in a reduction in the intensity of UV absorption

by the bases (up to 30%), relative to the free bases or
nucleotides.

This effect is referred to as hypochromicity

Is reversed when bases are unstacked (as in ssDNA or

ssRNA)
UV absorbance spectra of single and double-stranded DNA

ssDNA

dsDNA
 Thermal denaturation of DNA

increase temperature

decrease temperature

duplex single strands

(ordered) (randomly coiled)

At a sufficiently high temperature, duplex DNA can be converted

to the separated single-strands (thermal denaturation)
This process can be followed by monitoring the UV absorbance
of the DNA bases
 Thermal denaturation curve

ssDNA

Tm = midpoint of thermal
transition
Tm
= melting temperature

dsDNA

The melting temperature (Tm) provides an indication of the

stability of a particular nucleic acid structure
 Duplex stability

In general, duplex stability increases with:

Increasing duplex length

Increasing G:C base pair content

RNA-RNA > RNA-DNA > DNA-DNA