1
3. Chemical oligonucleotide and gene synthesis
Basic strategy: consecutive coupling of mononucleotide building blocks
Chemical requirements:
- protection of reactive groups (a.o. on the nucleotide bases)
- protection of 5’-end at one side and 3’-end at other side
- protection stable under conditions of coupling reaction
- protection sufficiently labile to allow deblocking without strand cleavage
- extremely high yield: ==> theoretical yield versus number of cycles
(nb. several steps per cycle)
Chemistry
- phosphodiester: G. Khorana in the 70’s
- phosphotriester: protection of the 2nd acidic phosphate group
- phosphite and phosphoramidite: phosphotriester methods with initial 3-valent phosphorus
reaction with nucleoside to phosphotriester intermediate
- phosphite : e.g. X=Cl
- phosphoramidite : e.g. X=N(iPr)2
- phosphonate: OH of phosphate replaced by H
=> phosphite, phosphoramidite and H-phosphonate methods require oxidation step
to phosphate. Oxidation of phosphonate only once at the global end of synthesis.
In practice
- immobilisation of 3’-end on solid support, e.g. silica glass
- addition/removal of reagent readily carried out
- simple washing procedure between the steps
- automatable
- direction of synthesis: towards 5’-end
- blocking of termini
- blocking of 5’-end with dimethoxytrityl (DMT)
- 3’-end bound to solid support via a “linker”
G. Volckaert
Chemical DNA synthesis
2/17/2016
2
Example : the phosphoramidite synthesis cycle
- 4 cycling steps: (many substeps)
1. detritylation of 5’-end
2. coupling reaction
3. capping reaction to block irreversibly any unreacted 5’-OH
4. oxidation of 3-valent to 5-valent phosphorus (I2 as weak oxidant)
- at end of synthesis
- break the attachment to the carrier
- hydrolysis of the protecting groups of heterocyclic bases
- cleave the phosphate protection (cyano-ethyl)
- remove the final 5’-trityl group together with or after other deprotections
Result of oligonucleotide synthesis
- single-stranded fragments : but double-stranded with perfectly palindromic oligonucleotides
- fragments have 5’- and 3’-OH : ligation requires 5’-phosphate and 3’-OH
- sizes from n=2 to n=200 are feasible; but standard sizes rather from 10 to 60
Oligonucleotides with variations in the phosphate group
- phosphorothioates (1 S instead of O in phosphate)
- phosphorodithioates (both acidic O’s replaced by S)
- methylphosphonates (CH3 instead of OH)
- phosphate-pentose backbone completely replaceable by e.g. a peptide backbone
=> PNA
- LNA : “locked” nucleic acid : bonds (via O) between C2’ and C4’
=> more rigid structure with particular (and better) hybridisation properties
G. Volckaert
Chemical DNA synthesis
2/17/2016
3
Other possibilities
- incorporation of : - modified bases, e.g. hypoxanthine (nucleoside = inosine)
- modified phosphates
- synthesis of -anomeres
- oligonucleotides with reactive NH2 group at 5’-end, enabling covalent coupling of
other components such as biotin, digoxygenin
- direct incorporation of biotin at internal position or at 5' or 3' end.
similarly for incorporation of fluorescent (and other) groups
Definitions:
- linker, adaptor, connector
- primer, degenerated primer
- probe, mixed probe, degenerated probe
- relation amino-acid sequence <> nucleotide sequence
e.g.
M - A - W - R - Y - T 5’ ATG-GCN-TGG-AGR-TAY-GCNCGN
4
2 4
2 (4)
= 64 oligonucleotides
the 17-mer mixture represents all potential coding sequences (and more) for
that particular peptide sequence
remark: not all 64 oligomers encode the exact sequence (see arginine triplet....)
Applications
- sequencing primers
- PCR amplifications (two primer)
- RAPD and variants (one primer)
- manipulation of DNA fragments : with linkers, adaptors, connectors
G. Volckaert
Chemical DNA synthesis
2/17/2016
4
- hybridisation: “fishing” in libraries with probes, mixed probes, ...
- site-specific mutagenesis: mismatch primers, DNA cassettes
- gene fragment and total gene synthesis
- Khorana : synthesis of a tRNA gene
- different approaches combining chemical and enzymatical synthesis
- synthesis of large mixtures (degeneracies)
- anti-sense DNA and RNA, also sense oligonucleotides, RNAi
- structural analysis: ==> discovery of Z-DNA
- primers for radioactive labeling by polymerase reactions (“repair-synthesis”)
Fluorescent analogs
"Molecular beaken"
- for detection in hybridisation experiments: fluorophor + quencher at termini.
- stem-loop ==> quenching
- hybridisation disrupts stem ==> activation
In the Taqman system (for qPCR)
- quenching upon hybridisation
- exonucleolytic removal of fluorescent group leads to fluorescence.
Use of FRET : (fluorescence resonance energy transfer).
- distance between the donor-acceptor pair of primary importance :
==> increased fluorescence
ET-primers (Engery Transfer) (for DNA sequencing)
G. Volckaert
Chemical DNA synthesis
(cfr. bigdyes)
2/17/2016
5
Photolithographic DNA synthesis
- large scale synthesis of oligonucleotides in micro-arrays ('DNA-chips'):
- light-directed synthesis (adapted from semiconductor industry): selective
removal of 5'- (or 3'-) protecting moieties on glass support by controlled
exposure to UV light (365 nm) through a photolithographic mask
=> parallel synthesis of thousands of (immobilized) oligonucleotides.
- terminology
- WAFER : disc containing the array
- FEATURE : each single element in the array
- at present features of 24x24 m feasible (=> 1,6 cm2 chip)
- features of 10x10 m being worked on => more than 106 sequences/chip
- Photodeprotection at 365 nm (I-line of Hg lamp) (wavelengths below 360 nm
may lead to modification at bases). Reaction time less than 1 minute.
- Efficiencies of about 90-95% per step => 20-mer synthesis => about 10% of
final product is the cognate oligomer sequence.
- Selection at will of oligomer sequences to be synthesized.
but: the maximum number of different oligomers is 4n (n = size of the sequence)
e.g. 46 equals 4096 hexamer, 48 = 65,536 octamers,
410 = more than1 million decamers (1,048,576)
==> with current technology, the synthesis of all 16-mers (4,294,967,296) on
a single disk is virtually impossible (with features of 10x10 m, this
would require a surface of 65.5 x 65.5 cm).
G. Volckaert
Chemical DNA synthesis
2/17/2016