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Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Background. Macrophages constitute 38% to 60% of infiltrating cells during acute renal allograft rejection. Their contribution to tissue damage during acute rejection was examined by depleting macrophages in a rat model. Methods. Lewis rats underwent bilateral nephrectomy and then received a Dark Agouti renal allograft and liposomal-clodronate, control phosphate-buffered saline liposomes, or saline intravenously (n=7 per group) on days 1 and 3 postsurgery. Grafts were harvested on day 5. Results. Liposomal-clodronate treatment resulted in a 70% reduction in blood ED1+ monocytes and 60% reduction in intragraft ED1+ macrophages (both P < 0.01). Half of all remaining interstitial ED1+ cells were undergoing apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling +/ED1+), and thus functional depletion of more than 75% of macrophages was achieved. Histologic and functional parameters of acute rejection were attenuated: interstitial infiltrate, tubulitis, and glomerulitis (P < 0.01); tubular cell apoptosis (P < 0.001); tubular cell proliferation (P < 0.001); and serum creatinine (P < 0.01). Production of inducible nitric oxide synthase by infiltrating cells and urinary nitric oxide excretion was reduced by 90% (P < 0.001). In contrast, no reduction in the number of other leukocytes was seen (CD3+, CD4+, CD8 +, and natural killer cells). Activation of lymphocytes (CD25 +) and production of lymphocyte effector molecules (granzyme B) were unaltered. Conclusion. This study demonstrates that macrophages contribute to tissue damage during acute rejection.

Background. Macrophages constitute 38% to 60% of infiltrating cells during acute renal allograft rejection. Their contribution to tissue damage during acute rejection was examined by depleting macrophages in a rat model. Methods. Lewis rats underwent bilateral nephrectomy and then received a Dark Agouti renal allograft and liposomal-clodronate, control phosphate-buffered saline liposomes, or saline intravenously (n=7 per group) on days 1 and 3 postsurgery. Grafts were harvested on day 5. Results. Liposomal-clodronate treatment resulted in a 70% reduction in blood ED1+ monocytes and 60% reduction in intragraft ED1+ macrophages (both P < 0.01). Half of all remaining interstitial ED1+ cells were undergoing apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling +/ED1+), and thus functional depletion of more than 75% of macrophages was achieved. Histologic and functional parameters of acute rejection were attenuated: interstitial infiltrate, tubulitis, and glomerulitis (P < 0.01); tubular cell apoptosis (P < 0.001); tubular cell proliferation (P < 0.001); and serum creatinine (P < 0.01). Production of inducible nitric oxide synthase by infiltrating cells and urinary nitric oxide excretion was reduced by 90% (P < 0.001). In contrast, no reduction in the number of other leukocytes was seen (CD3+, CD4+, CD8 +, and natural killer cells). Activation of lymphocytes (CD25 +) and production of lymphocyte effector molecules (granzyme B) were unaltered. Conclusion. This study demonstrates that macrophages contribute to tissue damage during acute rejection.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase which in vitro adds deoxynucleotides to the 3' OH end of a primer without a template. This function and its distribution in bone marrow pre-T cells and thymocytes suggest a role for this enzyme in the development of the immune system. We measured TdT activity in thymus biopsy specimens of patients with immunodeficiency disease to gain possible insight into the biology of this enzyme and/or the pathogenesis of immune deficient states. Thymuses from four patients with severe combined immunodeficiency disease (SCID) and one with DiGeorge syndrome were assayed for enzyme activity with and without ATP, a specific inhibitor of TdT. In two patients with SCID, specific enzyme activity (pmol/0.5 g tissue) was 50 and 157%, respectively, of age and sex-matched controls; one patient had no enzyme activity. TdT activity in the only adenosine deaminase deficient patient was 23% and was not inhibited by ATP. Crude extract from the DiGeorge thymus had 42% activity, but was not tested with ATP. Purification of crude extracts by DEAE, phosphocellulose and oligodT chromatography was performed to determine if specific enzyme activity had been masked. After phosphocellulose chromatography, ATP noninhibitable activity was removed from one fraction pool in one patient, but not from a second pool. Two other patients had TdT activity in low salt eluates (0.05 M KCl) rather than where expected. Purification of these samples by oligodT chromatography resulted in specific enzyme activity comparable to age-matched controls. The known inhibition of TdT activity by ATP suggests that the nucleotides and deoxynucleotides which accumulate in lymphocytes in SCID may account for the abnormal TdT activity seen in these patient specimens. Examination of TdT activity in immunodeficient patients may allow delineation of this enzyme's biological function.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase which in vitro adds deoxynucleotides to the 3' OH end of a primer without a template. This function and its distribution in bone marrow pre-T cells and thymocytes suggest a role for this enzyme in the development of the immune system. We measured TdT activity in thymus biopsy specimens of patients with immunodeficiency disease to gain possible insight into the biology of this enzyme and/or the pathogenesis of immune deficient states. Thymuses from four patients with severe combined immunodeficiency disease (SCID) and one with DiGeorge syndrome were assayed for enzyme activity with and without ATP, a specific inhibitor of TdT. In two patients with SCID, specific enzyme activity (pmol/0.5 g tissue) was 50 and 157%, respectively, of age and sex-matched controls; one patient had no enzyme activity. TdT activity in the only adenosine deaminase deficient patient was 23% and was not inhibited by ATP. Crude extract from the DiGeorge thymus had 42% activity, but was not tested with ATP. Purification of crude extracts by DEAE, phosphocellulose and oligodT chromatography was performed to determine if specific enzyme activity had been masked. After phosphocellulose chromatography, ATP noninhibitable activity was removed from one fraction pool in one patient, but not from a second pool. Two other patients had TdT activity in low salt eluates (0.05 M KCl) rather than where expected. Purification of these samples by oligodT chromatography resulted in specific enzyme activity comparable to age-matched controls. The known inhibition of TdT activity by ATP suggests that the nucleotides and deoxynucleotides which accumulate in lymphocytes in SCID may account for the abnormal TdT activity seen in these patient specimens. Examination of TdT activity in immunodeficient patients may allow delineation of this enzyme's biological function.