...ganisms, for purposes such as energy generation or bioremediation. The field is advancing in other aspects as well. Several sources are available for gene design software (Hoover and Lubkowski, 2002; =-=Rouillard et al., 2004-=-; Jayaraj et al., 2005), so the groundwork of disassembling a particular sequence to optimised short oligonucleotides is no bottleneck and should foster the spread of synthetic gene production. 3. New...
While the ability to make increasingly long stretches of DNAefficiently and at lower prices is a technological driver of thisfield, increasingly attention is being focused on improving thedesign of genes for specific purposes. Early in the genomesequencing era, gene synthesis was used as an (expensive) source of's thatwere predicted by genomic or partial cDNA information but weredifficult to clone. As higher quality sources of sequence verifiedcloned cDNA have become available, this practice has become lessurgent. However, producing large amounts of from gene sequences (or at least theprotein coding regions of genes, the ) found in naturecan sometimes prove difficult. Many of the most interestingproteins sought by molecular biologist are normally regulated to beexpressed in very low amounts in . Redesigning these genes offers ameans to improve gene expression in many cases. Rewriting the openreading frame is possible because of the redundancy of the geneticcode. Thus it is possible to change up to about a third of thenucleotides in an open reading frame and still produce the sameprotein. The available number of alternate designs possible for agiven protein is astronomical. For a typical of 300 there are over 10150 combinations that willencode an identical protein. Using optimization methods such asreplacing rarely used codons with more common codons sometimes havea dramatic effects. Further optimizations such as removing can also beincluded. At least in the case of E. coli, proteinexpression is maximized by predominantly using codons correspondingto tRNA's that retain amino acid charging during starvation (14).Computer programs are written to perform these and othersimultaneous optimizations are used to handle the enormouscomplexity of the task. A well optimized gene can improve proteinexpression 2 to 10 fold, and in some cases more than 100 foldimprovements have been reported. Because of the large numbers of changes madeto the original DNA sequence, the only practical way to create thenewly designed genes is to use gene synthesis.
Oligonucleotide Design for in vitro Gene Synthesis
In the 1950s, and co-workers developed a method where3’-O-acetylnucleoside-5’-O-phosphate2 was activated withN,N’-dicyclohehylcarbodiimide (DCC) or4-toluenesulfonylchloride (Ts-Cl) and a 5’-O-protectednucleoside 1 was reacted with the activatedspecies to give a protected dinucleoside monophosphate3. Uponthe removal of 3’-O-acetyl group using base-catalyzedhydrolysis, further chain elongation was carried out. Followingthis methodology, sets of tri- and tetradeoxyribonucleotides weresynthesized and enzymatically converted to longer oligonucleotides,which allowed elucidation of the . The major limitation of thephosphodiester method consisted in the formation of pyrophosphateoligomers and oligonucleotides branched at the internucleosidicphosphate. The method seems to be a step back from the moreselective chemistry described earlier; however, at that time, mostphosphate-protecting groups available now had not yet beenintroduced. The lack of the convenient protection strategynecessitated taking a retreat to a slower and less selectivechemistry to achieve the ultimate goal of the study.
oligonucleotide capture probes for gene expression profiling.
Oligonucleotides are chemically synthesized using nucleosidephosphoramidites. A nucleoside phosphoramidite is a derivative ofnatural or synthetic nucleosides with protection groups added toits reactive exocyclic amine and hydroxy groups. As mentionedearlier (see Phosphodiester synthesis above), the naturallyoccurring nucleotides (nucleoside-3'- or 5'-phosphates) areinsufficiently reactive to afford the synthetic preparation ofoligonucleotides. A dramatically more reactive N,N-diisopropylphosphoramidite group is therefore attached to the 3'-hydroxy groupof a nucleoside to form nucleoside phosphoramidite. To preventundesired side reactions, all other functional groups ofnucleosides have to be rendered unreactive (protected) by attaching. Upon the completion ofthe oligonucleotide chain assembly, all the protecting groups areremoved. Below, the protecting groups currently used incommercially available and most common nucleoside phosphoramiditebuilding blocks arebriefly reviewed:
RNA Oligos Oligonucleotides from Gene Link
In the 1970s, substantially more reactive P(III) derivatives ofnucleosides, 3'-O-chlorophosphites, were successfully usedfor the formation of internucleosidic linkages. Thisled to the discovery of the triester methodology. The group ledby M. Caruthers took the advantage of less aggressive and moreselective 1H-terazolidophosphites and implemented themethod on solid phase. Veryshortly after, the workers from the same group further improved themethod by using more stable nucleoside phosphoramidites as buildingblocks. Theuse of 2-cyanoethyl phosphite-protecting group in place of a lessuser-friendly group led to the nucleosidephosphoramidites currently used in oligonucleotide synthesis (seePhosphoramidite building blocks below).Many later improvements to the manufacturing of building blocks,instrumentation, and synthetic protocols made the phosphoramiditechemistry a very reliable and expedite method of choice for thepreparation of synthetic oligonucleotides.