Abstract
Abstract 2461
Poster Board II-438
3′ trailers of pre-tRNA or micro-pre-tRNA are removed by tRNA 3′ processing endoribonuclease (tRNase ZL) for pre-tRNA or micro-pre-tRNA to transform into mature tRNA or micro-tRNA. It has been demonstrated that 7-base-pair acceptor stem, about 5-base pair T-loop structure and a 3′-trailer are requisites for being recognized by tRNase ZL. A synthesized small guide RNA (sgRNA), which is designed to possess the complementary sequences with target RNA and form sgRNA/target RNA complex resembling a pre-tRNA or a micro-pre-tRNA, could be used for target RNA to be recognized and cleaved. As to the structure of sgRNA, a 3′-truncated tRNA (a clover leaf-like structure resembling tRNA and complementary to target RNA in the 7-base-pair acceptor stem) and a 5′-half-tRNA (a half of clover leaf-like structure and complemantary to target RNA of a 3′-half-tRNA structure) can form a pre-tRNA-like complex with target RNA. Heptamer sgRNA (7-base RNA structure complementary to target RNA and making target RNA form T-loop structur downstream of the complementary region) as well as hook RNA (a single T-loop-like structure with upstream 7-base RNA structure complementary to target RNA) can form a micro-pre-tRNA-like complex, which could be recognized and cleaved by tRNase Z. Heptamer sgRNA is thought to be superior to siRNA in mucleic acid medicine because of less off-target effect, less IFN-α production, easier introduction into target cells and less expensive for synthesis. In order to develop a unique sgRNA-based nucleic acid medicine, we investigated whether apoptosis could sbe induced and proliferation could be inhibited in leukemia cells by using tumor antigen heptamer sgRNA. The 5′, 3′-phosphorylated 2′-O-methyl sgRNAs with or without 3′LNA (locked nucleic acid), which were designed to form micro-pre-tRNA like structure with target mRNA of leukemia-associated antigens (LAA) such as WT1, Bcl2, PRAME, survivin, proteinase 3, hTERT and RHAMM, were synthesized and purified by high-performance liquid chromatography. Several leukemic cell lines and fresh leukemia cells from AML/ALL patients expressing relevant LAA were cultured with each heptamer sgRNA at the concecntrations of 0.1 μM to 10 μM for 24-72 hours. Effecs of sgRNA on apoptosis of the leukemia cells were evaluated by flow cytometry after staining with Annexin-V/7AAD or DiOC6(3). Proliferaton inhibition of the cells by sgRNA was evaluated by an improved MTT assay (TetraColor One). In addition, introduction of heptamer sgRNA into target cells was investigated using FITC-labeled sgRNA. Although EGFP sgRNA (negative control) did not induce apoptosis in leukemia cells such as K562, heptamer sgRNA against WT1, Bcl2, PRAME, surviving, proteinase 3, hTERT and RHAMM could induce apoptosis in concentration dependent manner of sgRNA irrespective of sgRNA with or without LNA. Apoptosis was enhanced by the combination of two different sgRNAs such as WT1 sgRNA and PRAME sgRNA. Likewise, proliferation was inhibited in leukemia cells cultured with sgRNA against WT1, PRAME, survivin, proteinase 3, hTERT and RHAMM. Apoptosis due to anti-leukemic agent such as etoposide or doxorubicine was enhanced by the addition of sgRNA more than the additive theoretical level of anti-leukemic agent and sgRNA. Apoptosis of some leukemia cells such as HL60, which were resistant to LAA sgRNA in apoptosis or proliferation inhibition, could be hardly introduced into with FITC-labeled sgRNA by only culturing with. sgRNA-based apoptosis in these cell lines such as HL60 was enhanced by using lipofection for sgRNA intruduction. These findings suggested a possibility that tumor antigen sgRNA could be applied as a novel nucleic acid medicine against leukemia and other tumors.
Disclosures:
No relevant conflicts of interest to declare.
Symmetry in Nucleic-Acid Double Helices
