We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies’ SurePrint® DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).

We have previously described decreased immunostaining of nidogen-1/entactin; laminin chains alpha1, alpha5, beta1,gamma1; and epithelial integrin alpha3beta1 in human diabetic retinopathy (DR) corneas. Here, using 142 human corneas, we tested whether these alterations might be caused by decreased gene expression levels or increased degradation. By semiquantitative reverse transcription-polymerase chain reaction, gene expression levels of the alpha1, alpha5, and beta1 laminin chains; nidogen-1/entactin; integrin alpha3 and beta1 chains in diabetic and DR corneal epithelium were similar to normal. Thus, the observed basement membrane and integrin changes were unlikely to occur because of a decreased synthesis. mRNA levels of matrix metalloproteinase-10 (MMP-10/stromelysin-2) were significantly elevated in DR corneal epithelium and stroma, and of MMP-3/stromelysin-1, in DR corneal stroma. No such elevation was seen in keratoconus corneas. These data were confirmed by immunostaining, zymography, and Western blotting. mRNA levels of five other proteinases and of three tissue inhibitors of MMPs were similar to normal in diabetic and DR corneal epithelium and stroma. The data suggest that alterations of laminins, nidogen-1/entactin, and epithelial integrin in DR corneas may occur because of an increased proteolytic degradation. MMP-10 overexpressed in the diabetic corneal epithelium seems to be the major contributor to the observed changes in DR corneas. Such alterations may bring about epithelial adhesive abnormalities clinically seen in diabetic corneas.

WAF1 binds to cyclin-Cdk complexes and inhibits their activity, causing cell cycle arrest. Previous studies have shown that expression of WAF1 is induced through the p53-dependent pathway; WAF1 is induced in cells with functional p53 but not in cells with either mutant p53 or no 53. Human myeloblastic leukemia cells KG-1 had no constitutive expression of p53, and irradiation did not induce p53. However, irradiation increased WAF1 expression in KG-1 cells and other cell lines containing mutant p53. The KG-1 cells constitutively produced low levels of tumor necrosis factor (TNF); irradiation markedly increased the production of TNF. Notably, induction of WAF1 mRNA by irradiation was blocked by anti-TNF antibody. Furthermore, exogenously added TNF increased levels of WAF1 mRNA in these cells. Irradiation increased the rate of WAF1 transcription 3-fold, and the half-life (t1/2) of WAF1 mRNA in these cells increased from < 1 h in unirradiated cells to > 4 h in irradiated cells. These findings indicate that increased levels of WAF1 transcripts occur, at least in part, through a pathway of TNF production and that the increase in WAF1 mRNA observed after irradiation is regulated by both transcriptional and posttranscriptional mechanisms. Our present study strongly suggests that an alternative pathway of induction of WAF1 occurs independent of activation by p53.