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A previously described self-complementary oligodeoxynucleotide termed triplex-forming oligodeoxynucleotide (TFO A), 54 bases in length, designed against the polypurine tract of HIV-1 RNA, inhibited viral replication at a 1 to 3 microM concentration in acutely infected cells, whereas antisense and scrambled sequence oligodeoxynucleotides were ineffective. Three HIV-1 viral isolates from patients of clinical categories A1, B, and C3 were transmitted to peripheral blood mononuclear cells and tested for production of p24 antigen and syncytium formation in the absence and in the presence of either TFO A or a control oligodeoxynucleotide of randomized sequence. No p24 antigen or syncytia were detected for up to 30 days when TFO A was added to the cells. Viability of the cells was found not to be affected by the drugs compared to controls within 2 weeks. Analysis of viral DNA synthesis by PCR for the LTR and gag gene indicated no DNA signal, suggesting that TFO A affects viral replication before formation of a DNA provirus. Measurements of the stability of TFO A indicate a half-life of about 2 hr. A two-dimensional computer fold analysis of TFO A suggested a self-complementary hairpin-loop configuration with GC-rich stems and single-stranded 5' and 3' ends. Since intracellular triplex formation may not be an efficient process, the observed inhibitory effect may be due to a direct inhibition of the RT and RNase H enzyme activities by the oligodeoxynucleotide. However, a triple-helix effect on the incoming RNA may play a role as well.

Phytoplasma-associated diseases are reported for more than 1,000 plant species worldwide. Only a few genome sequences are available in contrast to the economical importance of these bacterial pathogens. A new strategy was used to retrieve phytoplasma strain-specific genome data. Multiple displacement amplification was performed on DNA obtained from <3 g of plant tissue from tobacco and parsley samples infected with ‘stolbur' strains. Random hexamers and Phi29 polymerase were evaluated with and without supplementation by group-assigned oligonucleotides providing templates for Illumina's sequencing approach. Metagenomic drafts derived from individual and pooled strain-specific de novo assemblies were analyzed. Supplementation of the Phi29 reaction with the group-assigned oligonucleotides resulted in an about 2-fold enrichment of the percentage of phytoplasma-assigned reads and thereby improved assembly results. The obtained genomic drafts represent the largest datasets available from ‘stolbur' phytoplasmas. Sequences of the two strains (558 kb, 448 proteins and 516 kb, 346 proteins, respectively) were annotated allowing the identification of prominent membrane proteins and reconstruction of core pathways. Analysis of a putative truncated sucrose phosphorylase provides hints on sugar degradation. Furthermore, it is shown that drafts obtained from repetitive-rich genomes allow only limited analysis on multicopy regions and genome completeness.