DNA and RNA

1 Nucleotide Properties
1a Nucleotide Nomenclature
1b DNA Sequencing
 The Molecular Dogma of Life

DNA carries genetic information in the form of its sequence of nucleotides that is ultimately expressed
in the form of proteins — it accomplishes such a remarkable feat via three major mechanisms:
(1) DNA Replication—a process by which DNA copies or “replicates” itself, or simply produces two identical
strand replicas from a parent DNA double helix
(2) RNA Transcription—a process of converting or “transcribing” the deoxyribonucleotide sequence
within one strand of DNA into single-stranded RNAs such as the messenger RNA (or mRNA)
(3) RNA Translation—a process whereby the genetic information carried by mRNA is “translated” into
the synthesis of a polypeptide chain in conjunction with the ribosomal machinery
But, before we can shed light on these remarkable phenomena, we need to understand the structure
and thermodynamics of DNA and its RNA counterpart @ the very minimum:
- Structure (or shape) — what do they look like? It is all in the looks!
- Thermodynamics (or stability) — why do they look the way they do?! What are the “invisible”
forces responsible for the stability of their shapes?
SECTION 1A: Nucleotide Nomenclature
Synopsis 1A
- Nucleotides are the building blocks of nucleic acids such as DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid)

- A nucleotide consists of:

- a nitrogenous base (a heterocyclic ring)
- a ribose (RNA) or deoxyribose (DNA) sugar
- one or more phosphate groups

- The nitrogenous bases of nucleotides can be classified into two

heterocyclic ring systems:
(1) Purines—adenine (A), guanine (G), and hypoxanthine (I)
(2) Pyrimidines—cytosine (C), thymine (T), and uracil (U)

- While DNA is exclusively comprised of AGCT, T is replaced with U in

RNA — tRNA also contains hypoxanthine (I)
 Nucleoside versus Nucleotide

Nucleoside Nucleotide

Nucleoside:
- Nitrogenous base (a heterocyclic aromatic ring)
- (Deoxy)ribose sugar —a pentose with a furanose ring

Nucleotide:
- Nitrogenous base (a heterocyclic aromatic ring)
- (Deoxy)ribose sugar — a pentose with a furanose ring
- One or more phosphate groups (attached to the sugar component)
 Nucleotide Bases: Heterocyclic Rings

Imidazole
- Nucleotide bases are derivatives of either purine or pyrimidine — the two most
widely occurring heterocyclic aromatic ring compounds in nature

- A purine is essentially a pyrimidine ring fused to imidazole — the sidechain

functional group of the AA histidine --->

- In the context of nucleic acids:

- Purines include adenine (A), guanine (G), and hypoxanthine (I)
- Pyrimidines include cytosine (C), thymine (T) and uracil (U)
- One-letter codes are also used to generalize bases/nucleotides:
N  Nucleotide (any base or nucleotide)
R  Purine (any one of the three purines)
Y  Pyrimidine (any one of the three pyrimidines)
 Nucleotide Bases: Purines and Pyrimidines

6
6 6

Hypoxanthine (I)
Purines  Adenine (A) Guanine (G)

6 6
H3C 6
5
2 2 2

Cytosine (C) Uracil (U)

Pyrimidines Thymine (T)
- While DNA is solely comprised of AGCT, RNA contains AGCU — uracil (U) replaces thymine (T).
- Hypoxanthine (I) is commonly found in transfer RNA (tRNA) as an essential modification but in DNA as
a mismatch due to its preferential base-pairing with cytosine (C)
- In both DNA and tRNA, hypoxanthine (I) results from the deamination of adenine (A)
Nucleotide Sugars: Ribose and Deoxyribose

- In the context of nucleic acids, ribose and deoxyribose constitute the sole sugar
components of nucleotides

- The atoms in sugar components of nucleic acids are suffixed (or appended) with
a prime (’) so as to distinguish them from the atoms of nitrogenous bases!

- Ribose is a 5-C sugar (pentose) exclusively found in ribonucleotides (the building

blocks of RNA)

- Reduction of 2’-OH group in ribose generates deoxyribose — the sole sugar

component of deoxyribonucleotides (the building blocks of DNA)
 Ribonucleotides and Deoxy(ribo)nucleotides
OH OH

Ribose 5’-phosphate Deoxyribose 3’-phosphate

N-glycosidic
bond

- Nitrogenous bases are covalently linked to the 1’-C atom of ribose (or deoxyribose)
via the so-called “N-glycosidic bond”
- One or more phosphate groups are covalently linked to 5’-C or 3’-C atoms of ribose
(or deoxyribose) via the so-called “phosphoester bonds”
Relationship Between Nucleosides and Nucleotides

- Nucleosides are solely comprised of a sugar (ribose or deoxyribose) bonded to a

nitrogenous base

- In nucleotides, one (or more) phosphate group(s) is/are attached to either the 5’-O
atom or the 3'-O atom of ribose or deoxyribose
Bases >> Ribonucleosides >> Ribonucleotides
if X = H if X = ribose if X = ribose phosphate

Adenine (A) Adenosine (A) Adenosine monophosphate

(AMP)

Guanine (G) Guanosine (G) Guanosine monophosphate

(GMP)

Hypoxanthine (I) Inosine (I) Inosine monophosphate

(IMP)

Cytosine (C) Cytidine (C) Cytidine monophosphate

(CMP)

Uracil (U) Uridine (U) Uridine monophosphate

(UMP)

Thymine (T) Thymidine (T) Thymidine monophosphate

(TMP)
Bases >> Deoxyribonucleosides >>Deoxyribonucleotides

if X = H if X = deoxyribose if X = deoxyribose phosphate

Adenine (A) Deoxyadenosine (dA) Deoxyadenosine

monophosphate (dAMP)

Guanine (G) Deoxyguanosine (dG) Deoxyguanosine

monophosphate (dGMP)

Hypoxanthine (I) Deoxyinosine (dI) Deoxyinosine monophosphate

(dIMP)

Cytosine (C) Deoxycytidine (dC) Deoxycytidine

monophosphate (dCMP)

Uracil (U) Deoxyuridine (dU) Deoxyuridine monophosphate

(dUMP)

Thymine (T) Deoxythymidine (dT) Deoxythymidine

monophosphate (dTMP)
Polynucleotide Structure
O
Ester || N-glycosidic
[acyl group bonded R2—C—O—R1 bond
to an R moiety via
an O atom]

O
Thioester ||
[acyl group bonded -O—C—S—R1
to an R via an S
atom]

O
Phosphoester ||
-O—P—O—R1
[phosphoryl group
bonded to an R moiety |
via an O atom]
O-

Phosphodiester
[phosphoryl group O Phosphodiester
bonded to two R ||
moieties via O bond
R2—O—P—O—R1
atoms] Nucleic acid is a biopolymer of nucleotides
|
O- held together by phosphodiester bonds!
 Schematic Representation of a Polynucleotide

- Vertical lines denote nucleosides

- Diagonal lines indicate phosphodiester linkages holding the nucleosides together
- Strand polarity is generally defined as running from the 5’-C atom of (deoxy)ribose
at one end (the 5’-end) to the 3’-C atom at the other end (the 3’-end)—or simply
indicated as 5’ 3’
 Exercise 1A

- Identify the purines and pyrimidines commonly found in nucleic

acids

- Draw the structures of adenine, adenosine, and adenosine

monophosphate (AMP)

- Describe the chemical differences between a ribonucleoside

triphosphate and a deoxyribonucleoside monophosphate
Section 1B: DNA Sequencing
Synopsis 1B

- In the laboratory, nucleic acids can be cleaved at specific

sequences by restriction enzymes

- Nucleic acid fragments can be separated on the basis of

their size using non-denaturing agarose gel electrophoresis (AGE).

- After separation, DNA fragments can be sequenced via a

method referred to as the “chain-terminator method”

- The human genome contains ~23,000 genes, corresponding

to about 1.2% of its 3 billion nucleotides
 Restriction Endonucleases: Restriction Sites
Restriction Restriction
Enzyme Site Microorganism

Restriction endonucleases are protein enzymes that cleave double-

stranded DNA (dsDNA) at specific sites called the “restriction sites”
 Restriction Endonucleases: DNA Cleavage

“Sticky ends” “Blunt ends”

- Restriction endonucleases generally cleave DNA at palindromes—sequences that are identical

on both strands and related by a two-fold symmetry (they read the same when rotated by
180° about an axis horizontally perpendicular to the plane of the page)
- Words such as ROTOR and RADAR are palindromes—they read the same forward and backward!
- Restriction endonucleases can cleave DNA in a staggered manner to generate dsDNA fragments
with “overhangs” or “sticky ends”
- Restriction endonucleases can also cut DNA straight through or at the symmetry axis to produce
dsDNA fragments with “blunt ends”
 Agarose Gel Electrophoresis

Gel Apparatus Electrophoretogram

Separation of DNA fragments on a gel Analysis of separated DNA
after treatment with a restriction enzyme fragments on the gel
- After treatment with restriction endonucleases, DNA fragments can be separated on
agarose gel electrophoresis (AGE) — a non-denaturing method that separates
molecules on the basis of their size without the need for SDS!
- Following their separation, the gel can be stained with DNA-binding fluorescent dyes (eg
ethidium bromide) and the DNA fragments can be visualized and purified for sequencing
 Chain-Terminator Method: DNA Polymerase

- DNA polymerase converts single-stranded DNA (ssDNA) template into double-stranded (dsDNA)
- ssDNA template can be generated by heating dsDNA to break up the hydrogen bonds holding
individual strands together
- DNA polymerase adds nucleotides to the 3’-end of a nascent chain base-paired to the
complementary strand — i.e. it requires the “priming” of the template strand!
- Such priming is achieved using a short polynucleotide (typically 20-40bp long) complementary in
sequence to the 3’-end of the ssDNA template—this is called the “primer”
- DNA polymerase elongates the 3’-end of such a primer by stepwise addition of complementary
nucleotides
 Chain-Terminator Method: Sanger Sequencing
ddATP ddGTP

2’,3’-Dideoxynucleoside triphosphate (ddNTP)

ddCTP ddTTP

- In the classical Sanger method, DNA template is incubated with:

(a) DNA polymerase
(b) Complementary primer
(c) 4 x deoxynucleoside triphosphates (dNTPs)
(d) 4 x differentially-tagged fluorescent ddNTPs

- When the ddNTP is incorporated into the complementary strand in place of dNTP
analog, chain growth is terminated due to the lack of a free 3’-OH group!

- This generates a series of truncated DNA fragments (that differ by one

nucleotide) with differential fluorescent-tagged 3’-ddNTP

- Such differentially-tagged fluorescent DNA fragments are separated on the basis

of their size via non-denaturing agarose gel electrophoresis (AGE)
- The first complete map of human genome in 2001 was obtained using the Sanger Frederick Sanger
(1918-2013)
method
 Chain-Terminator Method: Automation
- In the modern automated chain-
terminator method, electrophoresis is
conducted in a capillary tube in order
to achieve higher sample throughput

- In this so-called “capillary electrophoresis (CE),“

negatively charged DNA fragments migrate
through an electrolyte gel (e.g. a solution
containing hydroxyethyl cellulose) in lieu of a
traditional agarose gel

- After fractionation via CE, DNA

fragments (differing by one nucleotide) are
detected with a LASER on-the-fly — i.e. as
they exit the capillary tube instead of being
analyzed later!
- This generates a series of colored peaks
that are automatically recorded and
converted to a chromatogram for
further analysis
 Chain-Terminator Method: Data Analysis

- Each colored profile—comprised of successive peaks—represents the sequence

pattern of a DNA fragment harboring one of the four ddNTPs

- Each peak—within a colored profile—denotes the 3’-end sequence of that pattern

- The sequence indicated is complimentary to the original template strand

 Exercise 1B

- Explain how restriction enzymes generate either sticky ends or blunt

ends

- Why do the smallest fragments of DNA move the farthest during

electrophoresis?

- List all the components and explain their purpose in the reaction
mixture used for the dideoxy DNA sequencing method