Hidefumi Yamakawa

Sequence-specific inhibition of a transcription factor by circular dumbbell DNA oligonucleotides.

The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA‐binding proteins can be inhibited by the use of double‐stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN‐κB) and circular (CDODN‐κB) dumbbell DNA oligonucleotides containing a NF‐κB binding site to analyze the inhibition of the NF‐κB‐dependent activation of the human immunodeficiency virus type‐1 (HIV‐1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double‐stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF‐κB sequence were found to compete with the native strand for NF‐κB binding. The circular dumbbell and double‐stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF‐κB binding proteins, while the nicked NF‐κB dumbbell was a less effective competitor. In Jurkat T‐cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV‐NL4‐3 Luc. The CDODN‐κB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN‐κB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non‐specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti‐HIV‐1 or other therapeutic effects.

Properties and Anti-HIV Activity of Nicked Dumbbell Oligonucleotides

Abstract We have designed a new type of oligodeoxyribonucleotide. These oligodeoxyribonucleotides form two hairpin loop structures with base pairs (sense and antisense) in the double helical stem at the 3′ and 5′-ends (nicked dumbbell oligonucleotides). The nicked dumbbell oligonucleotides are molecules with free ends that are more resistant to exonuclease attack. Furthermore, the nicked dumbbell oligonucleotide containing phosphorothioate (P=S) bonds in the hairpin loops has increased nuclease resistance, as compared to the unmodified nicked oligonucleotide. The binding of the nicked dumbbell oligonucleotide to RNA is lower than that of a single-stranded DNA. We also describe the anti-HIV activity of nicked dumbbell oligonucleotides. #This article is dedicated to Professor Yoshihisa Mimno on the occasion of his 75 th birthday.

PH-INDEPENDENT INHIBITION OF RESTRICTION ENDONUCLEASE CLEAVAGE VIA TRIPLE HELIX FORMATION BY OLIGONUCLEOTIDES CONTAINING 8-OXO-2'-DEOXYADENOSINE

The ability of homopyrimidine oligonucleotides containing 8‐oxo‐2′‐deoxyadenosine to form stable, triple helical structures with the sequence containing the recognition site for the class II‐S restriction enzyme, Ksp632‐I, was studied as a function of pH. The 8‐oxo‐2′‐deoxyadenosine‐substituted oligomers were shown to inhibit enzymatic cleavage and to bind within the physiological pH range in a pH‐independent fashion without compromising specificity.

Properties and Anti-HIV Activity of Circular Sense and Antisense Oligonucleotides

Abstract The circularization of a 48 mer oligonucleotide was investigated by using enzymatic ligation with T4 DNA ligase. This method yielded 30% circular molecules. The circular oligonucleotides are molecules with no free ends and are therefore more resistant to exonuclease attack. The circular oligonucleotide has remarkably increased Tm values as compared to the nicked and double stranded DNAs. However, the RNase H activity is lower than that with a single stranded DNA. We also describe the anti-HIV activity of a circular oligonucleotide.

Inhibition of Influenza Virus Replication in MDCK Cells by Circular Dumbbell RNA/DNA Chimeras with Closed Alkyl Loop Structures

We have demonstrated that a new type of circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON) with two closed nucleotide or alkyl loop structures (hexa-ethylene glycol) inhibits influenza virus A replication in MDCK cells. The enzymatic synthesis of circular dumbbell RNA/DNA chimeric oligonucleotides was achieved by enzymatically ligating a self-complementary phosphorylated oligonucleotide with T4-RNA ligase. The CDRDON-Al, with two closed alkyl loop structures, showed higher nuclease resistance, hybridization, and cellular uptake than the anti-S-ODN and the CDRDON, with two closed nucleotide hairpin-loop structures. The circular dumbbell RNA/DNA chimeric oligonucleotide (CDRDON-Al-PB2-as), containing an AUG initiation-codon sequence as the target of PB2, showed highly inhibitory effects on influenza A virus RNA expression. The limited toxicity of unmodified phosphodiester oligonucleotides and the sequence-specific binding to target mRNA indicate that circular dumbbell RNA/DNA chimeric phosphodiester oligonucleotides can be used with intact cells, and may prevent viral replication in culture.

Inhibition of Restriction Enzyme Ksp 632-I Via Triple Helix Formation by Phosphorothioate Oligonucleotides

Abstract The ability of pyrimidine-rich oligonucleotide phosphorothioate to form stable triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme Ksp 632-I was examined. First, we prepared double strand oligonucleotides corresponding to the major groove of SV40 DNA at 17 base pair homopurine-homopyrimidine sequences, and studied their interaction with homopyrimidine oligodeoxyribonucleotides including replacement of the PS group in the second nucleotide position from the 5′-terminus (SO-ODNs) and of the PS group at both the 3′- and 5′-ends (S2O-ODNs). The resulting perfect DNA triplexes were detected by the gel-mobility shift. The phosphorothioate oligonucleotide analogues (SO-ODNs) and (S2O-ODNs) were shown to inhibit enzymatic cleavage under conditions that allow for triple helix formation. Inhibition is sequence-specific and occurs in the micromolar concentration range. Of particular interest is the Sp-phosphorothioate analogue (Sp-SO-ODNs) which ...

ANTI-INFLUENZA VIRUS ACTIVITIES OF NICKED AND CIRCULAR DUMBBELL RNA/DNA CHIMERIC OLIGONUCLEOTIDES

Abstract We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.