N2 - The cellular mechanism of anti-DNA antibody synthesis in patients with systemic lupus erythematosus (SLE) was studied by DNA-specific solid-phase radioimmunoassay. Anti-DNA antibody synthesis in response to DNA was T-dependent, and the experiments with reconstituted lymphocytes from identical twins discordant for SLE showed that B cells and T cells from SLE patients must cooperate to synthesize anti-DNA antibody. Anti-DNA antibody synthesis by lymphocytes from patients with inactive SLE was enhanced by T4 cells and suppressed by T8 cells in response to DNA. Although T4 cells from patients with active SLE could enhance anti-DNA antibody synthesis by autologous B cells, their T8 cells could not suppress anti-DNA antibody synthesis by autologous B cells. These results indicate that elevated anti-DNA antibody synthesis in response to DNA in patients with active SLE is due to abnormalities of both SLE B cells and SLE T cells. They further indicate that dysfunction of T8 cells from patients with active SLE may, in part, be responsible for deficient regulation of anti-DNA antibody synthesis.
Successful oligonucleotide cleavage and deprotection require consideration of the deprotection conditions for each product and some products may require pretreatment or special deprotection conditions. Each synthesis should be reviewed to ensure the products have compatible deprotection conditions. Special deprotection requirements can be found on our Analytical Reports, Certificates of Analysis, Technical Bulletins, and Website: .
The authors note that the detailed studies of the molecular mechanisms of DNA repair pathways were made possible by using site-specifically modified oligonucleotides and that the availability of phosphoramidites to synthesize oligonucleotides with DNA lesions has contributed to the field. They illustrate the article using primarily structural studies in the following examples:
The cleavage reaction with concentrated ammonium hydroxide (28 to 33% NH3 in water), if carried out separately, is normally considered to be 1 hour at room temperature. Deprotection using ammonium hydroxide is the most traditional method and dates back to the earliest days of oligonucleotide synthesis. One of the critical issues when using ammonium hydroxide, which is water saturated with ammonia gas, is to keep the solution fresh. We aliquot and store ammonium hydroxide in the refrigerator in portions appropriate for use in 1 week. Using an old bottle of ammonium hydroxide is false economy since the resulting oligos are not going to be completely deprotected.
Phosphoramidites that allow the generation of oligonucleotides containing site-specific lesions have been vital components for studying the mechanism of DNA repair. New DNA lesions are still being discovered and the study of their biological consequences will require their site-specific incorporation into oligonucleotides. The authors conclude that the increased availability of phosphoramidites for the synthesis of lesion-containing oligonucleotides should facilitate many future discoveries in the broad area of DNA damage and repair.
More recently, different approaches have been used to develop a method called chemical primer extension, which involves the reaction of activated nucleotides with the end of a slightly modified DNA primer. Clemens Richert, Andreas Kaiser, and Sebastian Spies of the University of Stuttgart (Germany) have now developed this method further. They found a protective group that can be removed under gentle conditions so that the DNA duplexes made from the primer and template do not fall apart. This allows the reactivity of the nucleotides and the terminus of the primer to be switched on and off as desired, and the sequence information in the template strand can be read out nucleotide by nucleotide. For this method to work, the template and primer are both attached to tiny spheres. As in an automated synthesizer, the reagents and building blocks can flow over the spheres. The primer is bound to the template through base pairing. A suitable nucleotide from the surrounding solution docks at the next vacant binding site of the template. The nucleotide then binds to the reactive end of the primer through activated phosphate units. The sites that are supposed to react are chemically altered to become more reactive than in natural DNA. The special thing about this method is that the chain extension can be controlled to occur in either the 3" or the 5" direction. This is not known to take place in nature.
DCC activation has been used from the first days of the solid phase technique  and is still popular today. DIC  is also frequently used and presents the advantage that the corresponding urea is more soluble than the one obtained from DCC.
The rate-determining step in oligonucleotide synthesis is more than likely the removal of the protecting group on the G base. Ignore this at your peril since, traditionally, one of the most common reasons for poor performance of oligonucleotides is the presence of a small percentage of the G protecting groups remaining in the final product oligonucleotide. Chromatographic methods may miss the presence of the G protecting groups but these are readily revealed by mass spectral analysis. What are the options with attendant pros and cons for oligonucleotide deprotection?
The cellular mechanism of anti-DNA antibody synthesis in patients with systemic lupus erythematosus (SLE) was studied by DNA-specific solid-phase radioimmunoassay. Anti-DNA antibody synthesis in response to DNA was T-dependent, and the experiments with reconstituted lymphocytes from identical twins discordant for SLE showed that B cells and T cells from SLE patients must cooperate to synthesize anti-DNA antibody. Anti-DNA antibody synthesis by lymphocytes from patients with inactive SLE was enhanced by T4 cells and suppressed by T8 cells in response to DNA. Although T4 cells from patients with active SLE could enhance anti-DNA antibody synthesis by autologous B cells, their T8 cells could not suppress anti-DNA antibody synthesis by autologous B cells. These results indicate that elevated anti-DNA antibody synthesis in response to DNA in patients with active SLE is due to abnormalities of both SLE B cells and SLE T cells. They further indicate that dysfunction of T8 cells from patients with active SLE may, in part, be responsible for deficient regulation of anti-DNA antibody synthesis.
One question relating to the origin of life is: How was nature able to copy DNA or RNA strands before polymerases existed? Since the 1980s, DNA synthesizers have allowed chemists to produce DNA strands, but without a template or primer; the sequence is determined by the order of addition of the reagents. Only the use of protective groups that inhibit uncontrolled reactions and the programmed addition of the reagents ensure that the sequence of bases is correct. This is clearly not how nature does it. But how could template-directed primer extension function purely chemically, with no enzymes?
Glen Research is delighted to introduce a GalNAc modification strategy using a monomeric GalNAc support and the equivalent GalNAc phosphoramidite. Our experimental work has shown that these products are fully compatible with regular oligonucleotide synthesis and deprotection. Oligonucleotides containing GalNAc can be deprotected using standard procedures during which the acetyl protecting groups on the GalNAc group are removed. Glen Research offers these GalNAc C3 products under an agreement with AM Chemicals LLC.
We often recommend using the UltraMILD monomers (Pac-dA, Ac-dC and iPr- Pac-dG) and deprotection with potassium carbonate in methanol. In this way, some of these very sensitive oligonucleotides can be conveniently isolated. If capping is carried out using Cap A containing phenoxyacetic anhydride, it is possible to deprotect UltraMILD oligonucleotides in 4 hours at RT with 0.05M potassium carbonate in methanol or 2 hours at RT with ammonium hydroxide. Alternatively, using the regular Cap A containing acetic anhydride, it is necessary to deprotect overnight at room temperature to remove any Ac-dG formed during the capping step. For TAMRA containing oligonucleotides, an alternative deprotection3 may be carried out using t-butylamine/methanol/water (1:1:2) overnight at 55°C. Another option that we have found to be excellent uses t-butylamine/water (1:3) for 6 hours at 60°C. In this case, the regular protecting groups on the monomers may be used. An even milder approach has been described as “Ultra-UltraMild".4 In this technique, Q-supports5 are combined with UltraMild monomers to allow extremely gentle deprotection. After completion of the synthesis, the solid support is dried and treated overnight at 55°C with a solution containing 10% (v/v) diisopropylamine (iPr2NH) in 0.25 M ß-mercaptoethanol in MeOH.