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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

Formation of the complex between the ADP-1 peptide and TAR RNA and inhibition of the formation of the complex by helicates.(a) Binding of the ADP-1 peptide to the TAR RNA (2 nM). Lane C; TAR RNA in the absence of the ADP-1. Lanes 1–6; TAR RNA in the presence of 0.5, 1, 2, 4, 8, and 16 nM ADP-1, respectively. (b) Inhibition of the complex between the ADP-1 peptide (4 nM) and TAR RNA (2 nM) by M- and P-[Fe2L3]Cl4. Lane C; TAR RNA in the absence of the ADP-1 and helicates. Lanes 1–5; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of M-[Fe2L3]Cl4. Lanes 6–10; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of P-[Fe2L3]Cl4.
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f4: Formation of the complex between the ADP-1 peptide and TAR RNA and inhibition of the formation of the complex by helicates.(a) Binding of the ADP-1 peptide to the TAR RNA (2 nM). Lane C; TAR RNA in the absence of the ADP-1. Lanes 1–6; TAR RNA in the presence of 0.5, 1, 2, 4, 8, and 16 nM ADP-1, respectively. (b) Inhibition of the complex between the ADP-1 peptide (4 nM) and TAR RNA (2 nM) by M- and P-[Fe2L3]Cl4. Lane C; TAR RNA in the absence of the ADP-1 and helicates. Lanes 1–5; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of M-[Fe2L3]Cl4. Lanes 6–10; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of P-[Fe2L3]Cl4.

Mentions: The EMSA was also employed to determine if the binding of the helicates to the TAR RNA can inhibit the Tat-TAR interaction. In these experiments, we used the ADP-1 polypeptide (see Fig. 1c for its sequence) that has been previously demonstrated4 to carry the minimal RNA recognition region of the HIV-1 Tat protein and closely mimic Tat binding specificity. The autoradiogram in Fig. 4a presents binding of the ADP-1 to the TAR RNA in the absence of the helicates. The autoradiogram in Fig. 4b shows how the formation of the ADP-1-TAR RNA complex is inhibited in the presence of M- and P-[Fe2L3]Cl4. It can be seen that the ADP-1-TAR RNA interaction is almost completely inhibited in the presence of both enantiomers at the concentration of 16 nM. It might be possible that the helicates disrupt formation of the ADP-1-TAR complex by binding to the peptide rather than to the TAR-RNA. In order to probe this eventuality, we recorded CD spectra of M-[Fe2L3]Cl4 at constant helicate concentration (14 μM) and increasing ADP-1 concentrations (see Supplementary Fig. S5). No changes in the CD spectra were noticed with increasing concentrations of ADP-1, which is consistent with the view that the helicates did not bind significantly to ADP-1 peptide under conditions of our experiments.


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)

Formation of the complex between the ADP-1 peptide and TAR RNA and inhibition of the formation of the complex by helicates.(a) Binding of the ADP-1 peptide to the TAR RNA (2 nM). Lane C; TAR RNA in the absence of the ADP-1. Lanes 1–6; TAR RNA in the presence of 0.5, 1, 2, 4, 8, and 16 nM ADP-1, respectively. (b) Inhibition of the complex between the ADP-1 peptide (4 nM) and TAR RNA (2 nM) by M- and P-[Fe2L3]Cl4. Lane C; TAR RNA in the absence of the ADP-1 and helicates. Lanes 1–5; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of M-[Fe2L3]Cl4. Lanes 6–10; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of P-[Fe2L3]Cl4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940744&req=5

f4: Formation of the complex between the ADP-1 peptide and TAR RNA and inhibition of the formation of the complex by helicates.(a) Binding of the ADP-1 peptide to the TAR RNA (2 nM). Lane C; TAR RNA in the absence of the ADP-1. Lanes 1–6; TAR RNA in the presence of 0.5, 1, 2, 4, 8, and 16 nM ADP-1, respectively. (b) Inhibition of the complex between the ADP-1 peptide (4 nM) and TAR RNA (2 nM) by M- and P-[Fe2L3]Cl4. Lane C; TAR RNA in the absence of the ADP-1 and helicates. Lanes 1–5; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of M-[Fe2L3]Cl4. Lanes 6–10; TAR RNA mixed with the ADP-1 and increasing concentrations (1, 2, 4, 8, and 16 nM, respectively) of P-[Fe2L3]Cl4.
Mentions: The EMSA was also employed to determine if the binding of the helicates to the TAR RNA can inhibit the Tat-TAR interaction. In these experiments, we used the ADP-1 polypeptide (see Fig. 1c for its sequence) that has been previously demonstrated4 to carry the minimal RNA recognition region of the HIV-1 Tat protein and closely mimic Tat binding specificity. The autoradiogram in Fig. 4a presents binding of the ADP-1 to the TAR RNA in the absence of the helicates. The autoradiogram in Fig. 4b shows how the formation of the ADP-1-TAR RNA complex is inhibited in the presence of M- and P-[Fe2L3]Cl4. It can be seen that the ADP-1-TAR RNA interaction is almost completely inhibited in the presence of both enantiomers at the concentration of 16 nM. It might be possible that the helicates disrupt formation of the ADP-1-TAR complex by binding to the peptide rather than to the TAR-RNA. In order to probe this eventuality, we recorded CD spectra of M-[Fe2L3]Cl4 at constant helicate concentration (14 μM) and increasing ADP-1 concentrations (see Supplementary Fig. S5). No changes in the CD spectra were noticed with increasing concentrations of ADP-1, which is consistent with the view that the helicates did not bind significantly to ADP-1 peptide under conditions of our experiments.

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