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Sequencing by Synthesis
• Newer method to sequence whole genomes
– Uses allyl protecting group:
color
PPPO
DNA
3'-OH
O
+
B
Mixture of dNTPS
O
allyl
polymerase
O
DNA
color
O P O
O
OH
B
O
RXN STOPS!
Pd0
(30 s, deprotects
allyl moieties)
O
DNA
O P O
O
OH
OH
B
free to repeat
Sequencing by Synthesis
Ju, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640
Copyright ©2006 by the National Academy of Sciences
DNA/RNA Analogues
• There have been several recent
reports of the modification of
oligonucleotides
• Modifications have included:
– Altering nitrogenous base
structure
– Employing different sugar
structures
– Modifying the backbone
– Or in combination
H2N
N
N
O
O
N
N
O
O
OH(H)
O P O
NH
O
O
N
O
O
O
H
N
OH(H)
N
O P O
O
O
O
N
OH(H)
N
NH2
• Why make oligo analogues?
– Structure/activity relationships (i.e., catalytic versatility)
– Antisense technology
– Insight into evolutionary process?
• Synthetic oligos can be designed to bind with itself, DNA,
RNA or all of the above
• Effects:
– H-bonding (base-pairing)
– Other types of interactions (i.e. Van de Waals)
– Overall shape (i.e., double helix? Hairpin loop?)
• How do we examine interactions?
–
–
–
–
Melting temp. Tm = 40.5 °C (DNA), 42.5 °C (RNA)
NMR (as well other spectroscopic methods)
Xray crystallography
Calculations
Antisense Technology
• Employs a synthetic oligo that is complementary (antisense)
to an mRNA sequence of interest
• One of Two main effects can occur:
– Translation arrest (no protein)
– Recruits RNAase that degrades mRNA
• Potential for therapeutic use
– Vitravene (acts on CMV virus)
Unnatural Base Pairs
• Recall the natural base pairing (Watson-Crick) in DNA:
D
A
H
H
N
O
H
N
N
N
sugar
N
sugar
A
N D
N
N
O
H
A
D
H
G
C
• If we change the number of or location of donor/acceptor
groups, what interactions can occur?
For example: Expand the “genetic alphabet”
N
•
Tm measurements vary
dramatically due to changes
in H-bonding properties &
hydrophobicity
D
D
NH2
N
N
N
N A
N
A
N
sugar
N
sugar
NH2
Adenine
D
D
H N
H
D
H N
H
A
H N
N
• DNA polymerase found to
work with some modified
bases (oligos)
H N
A
N O
N
O
N
H
D
H
D
N
N
A
N
A
sugar
A
A
NH2
NH2 D
N
D
sugar
sugar
• Such structures would be
unlikely to form under
prebiotic conditions
NH2
Cytosine
sugar
Cytosine
A
D
O
N
N
NH2
N A
N
N
N
N
sugar
NH2
D
guanine
N
sugar
A
O
A
Employing Novel Sugars
Some examples:
O
• 5- vs 6- membered ring
5'
Base
O
• Location of phosphodiester bond
1'
4'
2'
3'
• Hydroxyl groups (# & stereochemistry)
O
• Position of base
P
OH(H)
DNA/RNA
O
6'
5'
4'
O
Base
O
OH
3'
1'
5'
4'
O
Base
1'
5'
4'
3'
2'
O
P
-D-Xylopyranosyl NA
O
6'
O
3'
2'
P
Hexitol NA
(HNA)
Base
O
OH
1'
2'
O
P
6'
OH
Altropyranosyl-NA
5'
O
O
Base
1'
4'
O 3'
2'
P
Homo-NA
O
6'
5'
4'
O
O
Base
3'
2'
P
Hexitol NA
(HNA)
1'
• Adopts chair conformation in
oligo
• Forms helical duplex with RNA
(Tm = + 3°C) & with DNA (Tm =
+ 3°C)
• Adding more hydroxyl groups,
increases affinity for RNA
– Also increases thermal stability
Modification of the Backbone
• Peptide Nucleic Acid (PNA)
– Aminoethyl glycine units linked by a peptide bond
– achiral
OH
OH
NH2
NH2
R
O
HN
Base
Base
N
R
O
O
O
HO
O
NH
O
R
NH
Base
O
HN
P
-O
O
O
O
Base
R
O
O
N
NH
R
O
HO
O
Base
NH
-O
P
O
O
O
OH
O
Base
HO
N
O
CONH2
Protein
PNA
RNA
• Duplex formation
– PNA-DNA Tm = 68.8 °C (~20 °C higher than DNA-DNA!)
– PNA-RNA Tm = 72.2 °C
•  PNA has the recognition properties of DNA (can carry
genetic information) & has a higher stability than RNA
and DNA
• Stability?
– Lack of phosphate groups → neutral backbone  no
electrostatic repulsion!
– Not recognized by proteases
• Did a PNA molecule precede RNA?
– Has demonstrated numerous forms: hairpins, triplexes, etc
– Simple in structure (i.e., achiral) & stable
– Amino building blocks in primordial soup
Another Modified Nucleic Acid - GNA
OH
glycol
• Glycol nucleic acid
OH
• Can nucleic acid be made with
such a simple sugar?
– Is a ring structure important?
– Minimum # of carbons?
Synthesis of GNA
O
DMT-Cl
O
DMTO
HO
H N
each base
(protected)
O
CN
R2N
R2N P O
P
O
HO
CN
Cl
DMTO
SN2
(inversion @ least
hindered centre)
N (Base)
R & S GNA made
DNA synthesizer
DMTO
N (Base)
• Tested duplex formation:
– Duplex formation Tm = 63 °C (22 ° higher than same DNA
or RNA sequence!)
–  GNA more stable than DNA!
– Demonstrates that cyclic sugar not necessary!
• Was ribose in fact the sugar in the first pre-biotic nucleic
acids?
• Is GNA a pre-RNA candidate?
– A glycerol derivative, which is related to triose
– Recall, we looked at 3C sugars (i.e., glyceraldehyde) in relation
to the prebiotic formation of sugars
An example of chemical biology:
• Uses biological concepts, DNA structure
• Uses chemical ideas → conformation & functional
groups
• Uses chemical synthesis principles
• Makes a non-natural molecule with novel properties
• Relates those properties to the natural system
• Problem: Still difficult to predict and analyze singlestranded oligonucleotide structures