TY - JOUR
T1 - Evidence for higher-order structure formation by the c-myb 18-mer phosphorothioate antisense (Codons 2-7) oligodeoxynucleotide
T2 - Potential relationship to antisense c-myb inhibition
AU - Vilenchik, M.
AU - Benimetsky, L.
AU - Kolbanovsky, A.
AU - Miller, P.
AU - Stein, C. A.
PY - 2001
Y1 - 2001
N2 - We have demonstrated the formation of higher-order structures (presumably tetraplexes) by an 18-mer phosphorothioate antisense c-myb oligodeoxyribonucleotide that has been shown to have activity in the treatment of leukemia xenograft models. Although not observable by conventionally employed techniques, such as PAGE and dimethyl sulfate (DMS) protection, the formation of such higher-order structures by this oligonucleotide was revealed by several techniques. These included capillary gel electrophoresis (CGE), which demonstrated the presence of molecules with greatly increased retention time compared with the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements. For the last, the 18-mer phosphorothioate oligonucleotide was synthesized with a 5′-fluorescein group. Similar to the molecular beacon model, its fluorescence was quenched when combined in solution with tetraplex-forming oligomers that contained a 3′-Dabcyl moiety. 7-Deazaguanosine inhibits the formation of tetraplexes by eliminated Hoogsteen base pair interactions. The wild-type and 7-deazaguanosine-substituted antisense c-myb oligomers differentially downregulated the expression of the c-myb proto-oncogene in K562 and HL60 cells, with the wild-type oligomer being the least active. The 18-mer c-myb molecule can, therefore, form highly complex structures, whose analysis in solution cannot be limited to examination of slab gel electrophoresis results alone.
AB - We have demonstrated the formation of higher-order structures (presumably tetraplexes) by an 18-mer phosphorothioate antisense c-myb oligodeoxyribonucleotide that has been shown to have activity in the treatment of leukemia xenograft models. Although not observable by conventionally employed techniques, such as PAGE and dimethyl sulfate (DMS) protection, the formation of such higher-order structures by this oligonucleotide was revealed by several techniques. These included capillary gel electrophoresis (CGE), which demonstrated the presence of molecules with greatly increased retention time compared with the monomer; magnetic circular dichroism spectroscopy, which demonstrated a band at 290 nm, a characteristic of antiparallel tetraplexes; and fluorescence energy transfer measurements. For the last, the 18-mer phosphorothioate oligonucleotide was synthesized with a 5′-fluorescein group. Similar to the molecular beacon model, its fluorescence was quenched when combined in solution with tetraplex-forming oligomers that contained a 3′-Dabcyl moiety. 7-Deazaguanosine inhibits the formation of tetraplexes by eliminated Hoogsteen base pair interactions. The wild-type and 7-deazaguanosine-substituted antisense c-myb oligomers differentially downregulated the expression of the c-myb proto-oncogene in K562 and HL60 cells, with the wild-type oligomer being the least active. The 18-mer c-myb molecule can, therefore, form highly complex structures, whose analysis in solution cannot be limited to examination of slab gel electrophoresis results alone.
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U2 - 10.1089/108729001750171317
DO - 10.1089/108729001750171317
M3 - Article
C2 - 11334144
AN - SCOPUS:0035029569
SN - 1087-2906
VL - 11
SP - 87
EP - 97
JO - Antisense and Nucleic Acid Drug Development
JF - Antisense and Nucleic Acid Drug Development
IS - 2
ER -