Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have re-examined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3′ sequences of the ODNs determine their effect. At concentrations of 10 μmol/L (for phosphorothioate ODNs) or 25 μmol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3′ positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5′ monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2′deoxycytidine 5′-monophosphate] is not cytotoxic), on their concentration (d-GMP [2′-deoxyguanosine 5′-monophosphate], TMP [thymidine 5′-monophosphate], and d-AMP [2′-deoxyadenosine 5′-monophosphate] are cytotoxic at concentrations between 5 and 10 μmol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3′ hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.
We have examined the effect of BCR/ABL junctional antisense phosphodiester oligodeoxyribonucleotides (ODNs) on BV173 and other chronic myeloid leukemia (CML) cell lines. Various control ODNs were used to understand the mechanism of the observed antiproliferative effect. Not only the antisense ODNs but also several control ODNs inhibit the proliferation of the leukemic cell lines. All the ODNs that inhibit the cell proliferation share a TAT consensus sequence at their 3′ end. A 1-base mismatch within this consensus sequence abolishes the antiproliferative effect. Mismatches of several bases at any other position within the sequence of the active ODNs do not suppress the observed effect. Similar experiments on normal or CML CD34+ cell fraction led to the same observations. We conclude that the antiproliferative effect of the phosphodiester BCR/ABL antisense ODNs cannot be attributed to an antisense mechanism but rather to a nonelucidated effect of a 3′ terminal TAT sequence. This effect is not CML specific.
Objectives The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primers/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection Methods We compared the clinical performances of the recommended primers/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID‐19. An additional 30 “pre-intervention screening” samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets. Results The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives. Conclusion An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.
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