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Nucleic acid engineering
Nucleic acids “old basic” functions
1) Contain information about protein structure (DNA)
2) Participate in selection and ligation of amino acids needed for proteins (RNA)
Central dogma of molecular biology
The new paradigm
general
special
epigenetics
Epigenetics can be defined as ‘the study of heritable changes in genome function that occur without a change in DNA sequence
The primary structure of nucleic acids
Linear biopolymers of monomers
Proteins – 20 amino acids
Nucleic acids – 5 nucleotides
RNA ~ 10-106 nucleotides (mRNA; tRNA, rRNA, snRNAs)
DNA ~ up to 108 nucleotides
Nucleotide
Glycosidic bond
phosphate
base
Phosphodiester bond
sugar (pentose)
Nucleoside
Base
Adenine
Guanine
Nucleoside
Adenosine
A
Guanosine
G
deoxyadenosine
dA
deoxyguanosine
dG
Deoxynucleoside
Thymine
deoxythymidine dT
Cytosine
Uracil
Cytidine
C
Uridine
U
deoxycytidine
dC
Deoxyribonucleotides
Ribonucleotides
Guanosine 5’-monophosphate
The nucleic acids pentoses
5’
4’
1’
3’
2’
ribose
2-deoxyribose
The nucleic acids bases: purines
The nucleic acids bases: pyrimidines
Phosphodiester bond
3’
5’
C3’
C5’
After bond formation a pyrophosphate is released
3’
5’
The secondary structure of nucleic acids
Duplex DNA is a helix formed by two DNA strands aligned in a antiparallel fashion
H-bonding is possible only when the two chains are antiparallel
RNAs form intra-strand base-pairs from self-complementary regions along the chain.
Therefore, also present secondary structure (double helix)
Watson-Crick base pairing
Chargaff rules:
Adenine = Thymine (Uracil in RNA)
Guanine = Cytosine
Conformations of sugar puckers
C2’ endo sugar puckering for the guanosine
C3’ endo sugar puckering for the adenosine
Pentose flexibility
7.0 Å
5.9 Å
C3’ endo
 Allows different nucleic acid conformations
C2’ endo
Conformations of the glycosidic bond
anti glycosidic angle conformation for adenosine
syn glycosidic angle conformation for guanosine
Glycosidic bond flexibility
syn-Adenosine
anti-Adenosine
Polynucleotides
5’
A
Glycosidic bond
5’AGC3’
G
5’pApGpC
Phosphodiester bond
C
OH
3’
Sequences are always referred as 5’ to 3’ unless stated and the notation in groups of 3
nucleotides should be avoid unless codons highlighting is needed
CATGCGGTTACCGATACCTAGAACCTGGACTACG
CAT GCG GTT ACC GAT ACC TAG AAC CTG GAC TACG
Hydrogen bonding and base stacking hold the DNA double helix together
Hydrogen bonding
Dδ-
Hδ+ + :Aδ-  Dδ-
Covalent bond
Hδ+
:Aδ-
Hydrogen bond
-O
H + :O=C- 
-O H
:O=C-
N
H + :O=C- 
N H
:O=C-
N
H + :N

N H
:N
Bond
Length (Å)
Energy (kcal/mol)
Covalent
~ 1.5
80-100
Hydrogen
~ 3.0
2-3 (GC=7.5; AT=5.3)
Since the bases are planar, they can stack nicely on one another by hydrophobic and Van
der Waals forces (about 4-15 kcal/mol per dinucleotide)
base stacking (kcal/mol )
H bonding (kcal/mol )
- Energy of hydrogen bonding depends mainly on base composition.
- However, base stacking depends on the sequence of the DNA.
5’ 3’
5’ 3’
A-T
C-G
T-A
G-C
3’ 5’
3’ 5’
10.51
12.8
6.57
12.8
Double helix
Base pairs are co-planar
Base pairs are perpendicular to the helix axis
Helix is stabilized by base stacking and hydrogen bonding
Double helix is right-handed
If the helix spirals in the same direction that the four fingers
of the right hand are pointing then it is a right handed helix
(Lehninger et al., Principles of Biochemistry)
Typical dimensions (B-DNA)
314 Å2
3.4 Å
34 Å
20 Å
Helix pitch
~ 10 nucleotides
Major vs Minor Groove – distinctly different environments – important for recognition & binding
The bases in a base pair are not directly across the helix axis from one another along some diameter but
rather are slightly displaced. This displacement, and the relative orientation of the glycosidic bonds
linking the bases to the sugar–phosphate backbone, leads to differently sized grooves in the cylindrical
column created by the double helix, the major groove and the minor groove, each coursing along its
length
The ABZs of DNA secondary structure: several alloforms A, B, C, D, Z
Topological changes between B-and Z-DNA
Comparison of the structural properties of A-, B-, and Z-DNA
minor groove
pitch
minor groove
tilt
rotation
rise
Double Helix Type
Overall proportions
Rise per base pair
Helix packing diameter
Helix rotation sense
Base pairs per turn of helix
Rotation per base pair
Pitch per turn of helix
Base-pair tilt
A
B
Z
Short and broad
2.3 Å
25.5 Å
Right-handed
~11
33.6°
24.6 Å
Longer and thinner
3.32 Å ± 0.19 Å
23.7 Å
Right-handed
~10
35.9° ± 4.2°
33.2 Å
Elongated and slim
3.8 Å
18.4 Å
Left-handed
12
260°/2
45.6 Å
+19°
-1.2° ± 4.1°
-9°
Wide and with
intermediate depth
Narrow and with
intermediate depth
Flattened out on helix
surface
Extremely narrow but very
deep
Major groove proportions
Extremely narrow but very deep
Minor groove proportions
Very broad but shallow
Adapted from Dickerson, R. L., et al., 1982. Cold Spring Harbor Symposium on Quantitative Biology 47:14.
A-DNA
B-DNA
Z-DNA
A, T, C
C3’endo
C2’endo
C2’endo
G
C3’endo
C2’endo
C3’endo
A, T, C
anti
anti
anti
G
anti
anti
syn
A, T, C
5.9
7.0
7.0
G
5.9
7.0
5.9
Pentose
Glycosidic bond
Distance between phosphates (Å)
-A B-DNA molecule can have stretches of Z-DNA
-Double strand RNA form A-type helices
- Z-DNA can appear under some conditions: high [salt], supercoiling, transcription, gene
expression
Major and minor grooves
Secondary structure of nucleic acids
DNA secondary structures
complementary sequences
5’ A G C T T G G C A T G C A G G G T T 3’
3’ T C G A A C C G T A C G T C C C A A 5’
duplex
unpaired bases
T
5’ A G C T T G G C T G C A G G G T T 3’
intra-helix
3’ T C G A A C C G A C G T C C C A A 5’
extra-helix
unpaired bases: bubble
T
G C
A
T
5’ A G C T T G G C T G C A G G G T T 3’
3’ T C G A A C C G A C G T C C C A A 5’
Unpaired bases
unpaired A
“extra-helix”
unpaired bases - bubble
duplex
unpaired A
“intra-helix”
duplex
DNA secondary structures
Invertead repeat
5’ G G A A T C G C A T G C G A T T C C 3’
3’ C C T T A G C G T A C G C T A A G G 5’
hairpin
cruciform
Direct repeat
5’ G G A C T C G C A G G A C T C G C A 3’
3’ C C T G A G C G T C C T G A G C G T 5’
Slipping strands
Stem-loop
unpaired T
unpaired A
unpaired T
Loop
unpaired G
AT
AT
GC
Stem
GC
AT
AT
DNA secondary structures
Mismatch
5’ A G C T T G G C G T G C A G G G T T 3’
3’ T C G A A C C G A A C G T C C C A A 5’
Junctions
3-ways
4-ways: Holliday junction
3 way junction
G•A mismatch
A
Holliday junction
G
Non-canonical base pairing
A·T Watson-Crick
T·A Watson-Crick
-R
-R
+R
reverse A·T
+R
+R
T·A Hoogsteen
+R
+R
A·T Hoogsteen
+R
-R
+R
Non-canonical base pairing
G·C Watson-Crick
-R
+R
G(anti)·G(syn)
reverse G·C
+R
-R
+R
+R
G(anti)·A (syn)
G·C+ Hoogsteen
-R
-R
+R
+
Protonation (pH≈5)
+R
Triplets
Triple helixes
Hypothesis 1
polypurine
polypyrimidine
3rd strand– parallel to purine strand
duplex
T
A
T
C+
G
C
Pyrimidine·Purine-Pyrimidine
T ·A
T ·A
T ·A
C+ · G
T ·A
C+ · G
-T
-T
-T
-C
-T
-C
3’Pyrimidine
5’Pyrimidine
3’Pyrimidine
triplex
Hypothesis 2
5’Purine
5’Pyrimidine
3’Purine
poliypurine
polyirimidine
3rd strand– anti-parallel to purine strand
duplex
A
A
T
G
G
C
Purine·Purine-Pyrimidine
3’Pyrimidine
A
G
A
G
A
G
·A
·A
·A
·G
·A
·G
-T
-T
-T
-C
-T
-C
triplex
5’Purine
3’Purine
5’Pyrimidine
5’Purine
3’Purine
Intramolecular triplex DNA
From DNA structure and function, R. Sinden
Biological role of triplexes
Eukaryotic DNA contains many polypurine and polypyrimidine stretches:
→potential role of triplexes in biological functions
•Specific sites for regulatory proteins
•Gene transcription inhibitors
•Replication terminators
•Recombination sites
•Telomer terminals
Gene regulation by triplexes
Quadruplexes
Consist of four guanines stabilized by Hoogsteen hydrogen bonding. These tetrads can
stack on each other, forming a G-quadruplex structure (these may be inter and/or
intramolecular). Distributed widely in the human genome as targets for regulating gene
expression and chromosomal maintenance (telomeres are single stranded DNA 3’ ends of
eukaryotic chromosomes)
The arrangement of guanine bases in the G-quartet, shown
together with a centrally placed metal ion. Hydrogen bonds are
shown as dotted lines, and the positions of the grooves are
indicated. From NAR 34, 5402 (2006)
Biological role of DNA supercoiling
- Packing of DNA inside cells
- Supercoiled DNA has stored energy needed for replication and transcription(energy is
needed to open the double helix)
-Supercoiling make possible contact between distant DNA regions
-Two types of coiling:
protein
Tertiary structure of nucleic acids: Chromosome organization
Tertiary Structure in DNA: Supercoils
Double-stranded circular DNA (or linear DNA duplexes whose ends are not free to rotate),
form supercoils if the strands are underwound (negatively supercoiled) or overwound
(positively supercoiled)
Plasmid supercoiling
Type I Topoisomerase (cuts 1 strand)
e.g.: E. coli Topoisomerase I relaxes DNA (no ATP need)
Type II Topoisomerase (cuts 2 strands)
eg: E. coli gyrase is able to introduce negative coiling (need ATP)
DNA denaturation
The midpoint of the melting curve is defined as the melting temperature, Tm
When duplex DNA molecules are subjected to conditions of pH, temperature, or ionic
strength that disrupt hydrogen bonds, the strands are no longer held together. That is,
the double helix is denatured and the strands separate as individual random coils
Why DNAs differ in their Tm values?
Depends on GC content
Why DNA in different ionic strengths melts with different Tm values?
At 0.2 M Na+, Tm = 69.3 + 0.41(% G + C). Ions suppress the electrostatic repulsion between the
negatively charged phosphate groups in the complementary strands of the helix, thereby stabilizing it.
DNA denaturants
- At pH>10, extensive deprotonation of the bases occurs, destroying their hydrogen bonding
potential and denaturing the DNA duplex.
- At pH<2.3, extensive protonation of the bases disrupts base pairing.
- Alkali is the preferred denaturant because, unlike acid, it does not hydrolyze the glycosidic
linkages in the sugar–phosphate backbone.
- Small solutes, formamide and urea, that readily form H bonds are also DNA denaturants.
Nucleic acid renaturation
Steps in the thermal denaturation and renaturation of DNA. The nucleation phase of the
reaction is a second-order process depending on sequence alignment of the two strands. This
process takes place slowly because it takes time for complementary sequences to encounter
one another in solution and then align themselves in register. Once the sequences are
aligned, the strands zipper up quickly.
DNA-Protein interactions
 Polymerases
 Cutting (nucleases, restriction enzymes)
PROTEINS
 Repairing
 Topological changes (helicases)
 Regulatory
 Structural (histones)
+
DNA
PROTEIN
Recognition and binding
Specific
recognizes a specific sequence
(e.g. restriction enzymes)
Non specific
independent of nucleotide sequence
(e.g. polymerases)
DNA-Protein interactions
Phosphates: electrostatic interactions with basic amino acids
Non specific
DNA bending: fitting
Bend
Grooves
Grooves: fitting and hydrogen bond and electrostatic interactions
phosphates
between bases and amino acids,
- direct sequence reading
Specific
Phosphates: base sequences define a spatial arrangement for sugarphosphates,
- indirect sequence reading
Hydrogen bonding sites in major and minor grooves
Triangles: hydrogen bonding; Rectangles: electrostatic interactions
Electrostatic interactions with phosphate group
Protein-DNA hydrogen bonding
Arginine-GC
Glutamine -AT
Potential hydrogen bonds and acceptors on the amino acid side chains
Structures of the amino acids containing different side chains. The triangle designates a hydrogen bond donor or acceptor.
Recognition motifs
DNAseI
•DNA binds between domains from DNAseI
•The protein loop inserts in minor groove
•Non-specific electrostatic interactions between basic amino acids and phosphates
•There is no interaction with bases
Recognition motifs
Dimers (when target sequence is symmetric)
Binding DNA-Dimer
Restriction enzyme specific for an inverted repeat:
Monomer 1
GAATTC
GAA
TTC
+
CTTAAG
CTT
AAG
Monomer 2
Eco RI is a dimer: its symmetry fits to the target sequence
Non-specific and Specific interactions:
-The enzyme binds non specifically (to the phosphates) and then
slides till the specific sequence
-Four a-helices (2 per monomer) interact with the major groove
-Interaction is mediated by arginine and glutamine residues
Recognition motifs
HTH (a helix–turn–a helix)
Interaction with amino acids in the major groove
Recognition motifs
b sheet
Non specific Interaction between b sheet and phosphates in the minor groove
HU protein (bacterial histone-like protein)
Recognition motifs
Zinc fingers
Coordination between 2 Cys and 2 His and Zn are essential for the 3D structure finger
TFIIIA
Protein with 9 Zn fingers
Helix interacts with the major groove
Recognition motifs
Saddles
Protein essential for transcription start that binds to eukaryotic promoters TATA sequences
DNA bends and binds to the middle of the saddle by the minor groove to four Phe which bind
to bases
DNA-drug interactions
Netropsin
intercalators
Groove binders
- Intercalators: planar molecules (e.g. actinomycin D, ethidium bromide, TOTO)
- Groove binders (e.g. netropsin)
TOTO
Intercalating agents distort the double helix
Intercalating substances insert with ease into the double helix, indicating that the van der
Waals interactions they form with the bases sandwiching them are more favorable than
similar bonds between the bases themselves. Furthermore, the fact that these agents slip
in suggests that the double helix must temporarily unwind and present gaps for these
agents to occupy. That is, the DNA double helix in solution must be represented by a set of
metastable alternatives to the standard B-conformation. These alternatives constitute a
flickering repertoire of dynamic structures
RNA
Various Kinds of RNA Found in an E. coli Cell
Number of
Percentage of
Type
NucleotideResidues Total Cell RNA
mRNA
75–000
~2
tRNA
73–94
16
120
rRNA
82
1542
2904
Organization and composition of prokaryotic and eukaryotic ribosomes
The proposed secondary structure for E. coli 16S rRNA, based on comparative sequence analysis in
which the folding pattern is assumed to be conserved across different species. The molecule can be
subdivided into four domains—I, II, III, and IV—on the basis of contiguous stretches of the chain that
are closed by long-range base-pairing interactions. I, the 5'-domain, includes nucleotides 27 through
556. II, the central domain, runs from nucleotide 564 to 912. Two domains comprise the 3'-end of the
molecule. III, the major one, comprises nucleotides 923 to 1391. IV, the 3'-terminal domain, covers
residues 1392 to 1541.
tRNA
There are more than 80 modifications (post-transcription) in RNAs which increase its stability
pseudouridine (Y)
inosine (I)
dihydrouridine(D)
5-methyluridine (m5U)
5-methylcitidine (m5C)
PNAs (peptide nucleic acids) ARE SYNTHETIC POLYMERS in which the sugar–phosphate
backbone is replaced by a peptide backbone
PNAs are resistant to nucleases (and also to proteases) and thus show great promise as
specific diagnostic probes for unique DNA or RNA nucleotide sequences. PNAs also have
potential application as antisense drugs
Nucleic acid applications
Research (basic and applied)
- structure and function studies
- gene cloning
- sequencing
- etc
Diagnostics
- paternity tests
- diseases screening
- food contaminations
- etc
Forensics
- individual identification
- etc
Prophylaxis
- conventional vaccines
Gene therapy
- DNA vaccines
- antisense therapy
- etc
Nucleic acid synthesis
•Biological synthesis (in vivo)
Cell culture
Purification
Tissue
•Enzymatic synthesis (in vitro)
Purification
Fragment amplification
•Chemical synthesis
Purification
oligonucleotides