http://www.scq.ubc.ca/

• 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