Assembly Of Oligonucleotides Research Articles

Evaluating the quality of oligonucleotides that are immobilized on glass supports for biosensor development

Three different analytical techniques were compared to assess methodologies that could evaluate the success of oligonucleotide assembly on glass substrates functionalized with hexaethylene glycol (HEG) linker molecules. Post-synthesis cleavage of the linker-oligonucleotide conjugates from the solid support was done to prepare samples for analysis. Samples were investigated by electrospray ionization mass spectrometry (ESI-MS), high performance ion-exchange liquid chromatography (HPIEC) and polyacrylamide gel electrophoresis after radiolabeling ( 32 P -PAGE). The data from ESI-MS served to identify the various species detected by HPIEC and 32 P -PAGE. All three techniques were shown to be very sensitive to the presence and location (terminal or internucleotide) of HEG conjugated to the oligonucleotide sequence. This allowed differentiation and quantification of linker-oligonucleotides from non-conjugate oligonucleotides that originated from undesired synthesis directly on the glass surface. Furthermore, shorter linker-oligonucleotide conjugates that were formed by incomplete nucleobase-coupling during DNA synthesis on the linker could be identified by HPIEC and 32 P -PAGE, allowing purity assessment of the assembled strands. Despite the inherent higher sensitivity of PAGE of radiolabeled samples, HPIEC was shown to be the method of choice due to high sample throughput and facile quantitative analysis of the products.

Surprising fidelity of template-directed chemical ligation of oligonucleotides.

Nucleic acid replication via oligonucleotide ligation has been shown to be extremely prone to errors. If this is the case, it is difficult to envision how the assembly and replication of short oligonucleotides could have contributed to the origin of life and to the evolution of a putative RNA world. In order to assess the fidelity of oligonucleotide replication more accurately, chemical ligation reactions were performed with constant-sequence DNA templates and random-sequence DNA pools as substrates. In keeping with earlier results, constant-sequence hairpin templates were not faithfully copied by random-sequence substrates. Linear templates, however, showed exceptional replication fidelity, particularly when random hexamers were ligated at 25 degrees C. Surprisingly, at low temperatures the formation of G.A base pairs was common and sometimes occurred even more readily than the formation of the corresponding Watson-Crick A-T and G-C base pairs. The fidelity of ligation reactions increases with temperature and decreases with the length of the random-sequence substrates. Oligonucleotides with a defined sequence can be copied faithfully in the absence of enzymes. Thus, to the extent that short oligonucleotides could readily have been generated by prebiotic mechanisms, it is possible that the earliest self-replicators arose via oligonucleotide ligation.

Synthesis of oligodeoxynucleotides containing 5-aminouracil and its N-acetyl derivative

E. Ferrer, G. Neubauer, M. Mann and R. Eritja, J. Chem. Soc., Perkin Trans. 1, 1997, 2051 DOI: 10.1039/A701371J

Novel non-nucleosidic phosphoramidite building blocks for versatile functionalization of oligonucleotides at primary hydroxy groups

Synthesis of two non-nucleosidic phosphoramidite building blocks, 1a, b, that enable attachment of various tether groups to oligonucleotides at their 5′-terminus (or 1′-OH of 3′-deoxypsiconucleoside units) is described. Introduction of these linkers during the oligonucleotide assembly on a solid support, and their subsequent derivatization upon deprotection, afforded amino-, carboxy-, and sulfanyl-alkyl-tethered oligonucleotides.

Membrane-linked probes: 5′-(polysulfonylmethyloxyhexaglycol) oligonucleotides

A novel approach for the synthesis of functional, film-forming polymers which consists of the assembly of oligonucleotides anchored on a solid support with a soluble polymeric reagent is described. Phosphoramidites of hydroxymethyl-polysulfone and hexaglycoloxymethyl-polysulfone were synthesized and were linked by phosphate ester bonds to fragments of DNA anchored on controlled pore glass supports in the last step of an automatic synthesis of oligonucleotides. Microtiter plates were coated with the oligonucleotide-hexaethyloxymethyl-polysulfone and hybridization with a complementary biotin-labelled DNA probe was applied followed by avidine-peroxidase and detection by the usual procedure. Control experiments using a non-relevant probe for hybridization or using hybridization solutions with no oligonucleotide were also performed.

Selection of a ribozyme that functions as a superior template in a self-copying reaction.

The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.

Regioselective 2′/3′-O-Allylation of Pyrimidine Ribonucleosides Using Phase Transfer Catalysis

Abstract A short and convenient procedure for regiospecific O-allylation of uridine is reported by employing dibutyltin oxide as a mild base in conjunction with a phase transfer catalyst tetrabutylammonium bromide. The resulting isomeric 2′/3′-O-allyl uridines were separated after conversion into their corresponding 5′-O-DMT derivatives. The 2′-O-allyluridine 3 was then transformed into 2′-O-allylcytidine 7 and both were individually converted into the corresponding β-cyanoethyl phosphoramidite monomers (9 and 10) and a phosphodiester monomer 11, required for oligonucleotide assembly. The utility of 11 is demonstrated by synthesis and characterization of a 2′-O-allyl ribodinucleotide UpU.

Expression of a modified Dutch elm disease toxin in Escherichia coli.

The fungal toxin associated with Dutch elm disease, cerato-ulmin, has been produced in the bacterium Escherichia coli by the assembly of oligonucleotides according to the unpublished amino acid sequence of the toxin. This toxin was produced at approximately 80 micrograms/L of cell culture as a fusion to glutathione S-transferase. We synthesized the toxin as a fusion protein to improve purification and stability. Recombinant cerato-ulmin was analyzed by immunoblot analysis and then separated from its fusion partner by thrombin. We incorporated this molecule into an appropriate medium to test the activity of the toxin on the growth of American elm callus cultures.

Antibody engineering using very long template-assembled oligonucleotides

We have previously constructed single-stranded DNA fragments up to 361 nucleotides long by the ligation of shorter synthetic fragments assembled on a closely homologous template and used them for site-directed mutagenesis. Here we have extended the utility of such long oligonucleotides for protein engineering in two ways. First oligonucleotides have been assembled on a template to which they show only relatively weak sequence homology (<70% identity), allowing conversion of genes encoding rather distantly related proteins. We have converted a gene encoding human light chain constant domain from the kappa to the lambda isotype, which share <40% amino acid identity. Second, libraries of random mutants were constructed within antibody variable domains with up to 20 targeted codons separated by up to 186 nucleotides. These libraries were created by template-directed assembly of degenerate oligonucleotides followed by PCR amplification and subcloning. Nucleotide sequence analysis of clones demonstrated that this is an effective method for the simultaneous random mutagenesis of several codons distant within the target gene. This should provide a useful tool for modifying the ligand binding properties of proteins, e.g., the antigen binding affinity and specificity of antibodies.

A Multisubunit Ribozyme That Is a Catalyst of and Template for Complementary Strand RNA synthesis

Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

Oligonucleotides, part 5+: synthesis and fluorescence studies of DNA oligomers d(AT)5containing adenines covalently linked at C-8 with dansyl fluorophore

The synthesis of oligodeoxynucleotides d(AT)5 in which specific adenines are linked at C-8 position with dansyl fluorophores via a variable polymethylene spacer chain are reported. This was achieved by a strategy involving prelabelling at the monomeric stage followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled oligonucleotides. Several mono and polydansyl d(AT)5 derivatives in which the fluorophore is linked via ethylene, tetramethylene and hexamethylene spacer arms were synthesised for a systematic study of their fluorescence characteristics. It was observed that (i) enhancements in fluorescence intensity and emission quantum yields are seen due to multiple labelling, (ii) the magnitude of enhancements are related to labelling configuration and (iii) quenching efficiency is minimal with shorter and rigid spacer arms. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids.

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.