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Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication.

Malina J, Hannon MJ, Brabec V - Sci Rep (2016)

Bottom Line: The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription.The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA.These studies provide a new insight into the biological potential of metallosupramolecular helicates.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-61265 Brno, Czech Republic.

ABSTRACT
The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat-TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

No MeSH data available.


Related in: MedlinePlus

RNase A cleavage of TAR RNA in the presence of helicates.RNase A cleavage of 5′-32P end-labeled TAR RNA (2 μM) in the presence of increasing concentrations of M- and P-[Fe2L3]Cl4. Lane C; TAR RNA cleaved by RNase A in the absence of helicates. Lanes 1–4; TAR RNA mixed with M-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lanes 5–8; TAR RNA mixed with P-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lane C0; TAR RNA in the absence of helicates and RNase A. Phosphodiester bonds cleaved by RNase A are indicated on the left side of the gel.
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f3: RNase A cleavage of TAR RNA in the presence of helicates.RNase A cleavage of 5′-32P end-labeled TAR RNA (2 μM) in the presence of increasing concentrations of M- and P-[Fe2L3]Cl4. Lane C; TAR RNA cleaved by RNase A in the absence of helicates. Lanes 1–4; TAR RNA mixed with M-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lanes 5–8; TAR RNA mixed with P-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lane C0; TAR RNA in the absence of helicates and RNase A. Phosphodiester bonds cleaved by RNase A are indicated on the left side of the gel.

Mentions: In order to identify potential binding sites of M- and P-[Fe2L3]Cl4 on the TAR RNA, we probed the helicate:TAR RNA complexes with RNase A. Since this enzyme prefers cleavage of single-stranded to double-stranded regions of the RNA, it is particularly well suited for investigating drug binding to the single stranded parts, including the bulge and loop regions. The autoradiogram of the RNA cleavage-inhibition patterns for the TAR RNA is shown in Fig. 3. The strong cleavage at positions 23–26 corresponding to the 5′-UCU bulge is markedly reduced in the presence of both enantiomers of [Fe2L3]Cl4. In contrast, the cutting is increased between nucleotides C30 and U31 located in the loop. It is likely that the base protection effects induced by the helicates result from the direct interaction with the helicates, but it cannot be excluded that the protection arises from helicate-induced structural changes. In any case, the results of the RNase A footprinting suggest that the M- and P-[Fe2L3]Cl4 preferentially bind to the TAR RNA bulge or in its close proximity.


Iron(II) supramolecular helicates interfere with the HIV-1 Tat-TAR RNA interaction critical for viral replication.

Malina J, Hannon MJ, Brabec V - Sci Rep (2016)

RNase A cleavage of TAR RNA in the presence of helicates.RNase A cleavage of 5′-32P end-labeled TAR RNA (2 μM) in the presence of increasing concentrations of M- and P-[Fe2L3]Cl4. Lane C; TAR RNA cleaved by RNase A in the absence of helicates. Lanes 1–4; TAR RNA mixed with M-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lanes 5–8; TAR RNA mixed with P-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lane C0; TAR RNA in the absence of helicates and RNase A. Phosphodiester bonds cleaved by RNase A are indicated on the left side of the gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: RNase A cleavage of TAR RNA in the presence of helicates.RNase A cleavage of 5′-32P end-labeled TAR RNA (2 μM) in the presence of increasing concentrations of M- and P-[Fe2L3]Cl4. Lane C; TAR RNA cleaved by RNase A in the absence of helicates. Lanes 1–4; TAR RNA mixed with M-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lanes 5–8; TAR RNA mixed with P-[Fe2L3]Cl4 at 0.5:1, 1:1, 1.5:1, and 2:1 (helicate:TAR RNA) ratios, respectively, cleaved by RNase A. Lane C0; TAR RNA in the absence of helicates and RNase A. Phosphodiester bonds cleaved by RNase A are indicated on the left side of the gel.
Mentions: In order to identify potential binding sites of M- and P-[Fe2L3]Cl4 on the TAR RNA, we probed the helicate:TAR RNA complexes with RNase A. Since this enzyme prefers cleavage of single-stranded to double-stranded regions of the RNA, it is particularly well suited for investigating drug binding to the single stranded parts, including the bulge and loop regions. The autoradiogram of the RNA cleavage-inhibition patterns for the TAR RNA is shown in Fig. 3. The strong cleavage at positions 23–26 corresponding to the 5′-UCU bulge is markedly reduced in the presence of both enantiomers of [Fe2L3]Cl4. In contrast, the cutting is increased between nucleotides C30 and U31 located in the loop. It is likely that the base protection effects induced by the helicates result from the direct interaction with the helicates, but it cannot be excluded that the protection arises from helicate-induced structural changes. In any case, the results of the RNase A footprinting suggest that the M- and P-[Fe2L3]Cl4 preferentially bind to the TAR RNA bulge or in its close proximity.

Bottom Line: The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription.The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA.These studies provide a new insight into the biological potential of metallosupramolecular helicates.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-61265 Brno, Czech Republic.

ABSTRACT
The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat-TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

No MeSH data available.


Related in: MedlinePlus