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• Newer method to sequence whole genomes– Uses allyl protecting group:
Sequencing by Synthesis
O
O
PPPOB
O
O
OB
P
O
O
OH
O
OH
OB
P
O
O
OH
color
allyl
+DNA 3'-OH
Mixture of dNTPS
polymerase
colorDNA
RXN STOPS!Pd0
(30 s, deprotectsallyl moieties)
DNA
free to repeat
Copyright ©2006 by the National Academy of SciencesJu, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640
Sequencing by Synthesis
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
ON
O
O
P
O
O O
ON
O
O
ON
P
O
O O
N
NN
NH2
NH
O
O
N
NNH
O
NH2
OH(H)
OH(H)
OH(H)
• 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:
• If we change the number of or location of donor/acceptor groups, what interactions can occur?
N N
NN
O
N
H
H
H
sugar
NN
O
N
H
H
sugar
G C
D
D
D
A
A
A
For example: Expand the “genetic alphabet”
• Tm measurements vary dramatically due to changes in H-bonding properties & hydrophobicity
• DNA polymerase found to work with some modified bases (oligos)
• Such structures would be unlikely to form under prebiotic conditions
N N
NN
O
NH2sugar
N
N
NN
NH2
Osugar
A
D
A
D A
A
guanine
N
N
O
NHH
sugar
N
N
NH2
NHH
sugar
A
D
A
D
A
D
Cytosine
Employing Novel SugarsSome examples:
• 5- vs 6- membered ring
• Location of phosphodiester bond
• Hydroxyl groups (# & stereochemistry)
• Position of base
OBase
O
O
P
OBase
O
O
P
OH
OH
OBase
O
P
OH
O
BaseO
O
O
P
1'
2'3'
4'
5'
1'
2'3'
4'
5'
3'4'
5'
6'
1'
2'
6'
3'
4'1'
2'
6'
5'
Hexitol NA(HNA)
Homo-NA-D-Xylopyranosyl NA Altropyranosyl-NA
BaseO
O
O
P
1'
2'3'
4'
5'
6'
Hexitol NA(HNA)
• 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
NH2
NH
NH
NH
NH
OH
OR
R
O
R O
R
O
R O
NH2
N
NH
N
NH
N
O
Base
O
O
Base
O
CONH2
O
Base
O
O
OH
PO
-OOH
Base
O
O
O
O
O
PO
-O
OHOH
OH
Base
Base
ProteinPNA 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
• Glycol nucleic acid
• Can nucleic acid be made with such a simple sugar?– Is a ring structure important?– Minimum # of carbons?
OH
OH
glycol
Synthesis of GNA
O
OH
O
DMTO
NH
OH
DMTO N (Base)
POR2N
Cl
CNO
DMTO N (Base)
PR2N
OCN
DMT-Cl
each base(protected)
SN2(inversion @ leasthindered centre)
DNA synthesizer
R & S GNA made
• 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 single-
stranded oligonucleotide structures