The polypurine tract, PPT, of HIV as target for antisense and triple-helix-forming oligonucleotides

Volkmann, Silke; Dannull, Jens; Moelling, Karin

doi:10.1016/0300-9084(93)90027-p

Cited by 29 publications

(22 citation statements)

References 22 publications

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“…NC proteins may improve RNA -RNA interaction by their ability to unwind or melt RNA as described previously (Sykora and Moelling, 1981; Khan and Giedroc, 1992;Dib-Hajj et al, 1993). This melting effect is, however, only of minor importance for the reverse transcriptase in vitro (Volkmann et al, 1993). Whether it is more important in vivo has not yet been shown.…”

Section: Discussionmentioning

confidence: 96%

“…Retroviruses code for the structural protein Gag which harbours the nucleocapsid (NC) protein as well as the matrix and capsid proteins. In HIV-1 it is located at the C-terminus of the Pr55Gag polyprotein precursor and is processed through the action of the retroviral protease to the NCp15 protein which is further cleaved off the NCp7 (the actual NC protein), to a proline-rich p6 and a pl byproduct Wondrak et al, 1993). NC proteins play an essential role during retroviral replication.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, the NC proteins contribute to the binding of the tRNA primer to the primer binding site (PBS) on the viral RNA (Barat et al, 1989;Prats et al, 1991). It also improves reverse transcription of viral RNA to DNA in vitro although only to a low extent (Volkmann et al, 1993). The specificity of interaction between NC protein and PSI RNA has been exploited for the design of packaging cell lines which express viral structural proteins from a retroviral mRNA from which the PSI has been deleted.…”

Section: Introductionmentioning

confidence: 99%

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Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

et al. 1994

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The nucleocapsid (NC) protein of human immunodeficiency virus HIV‐1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N‐terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C‐C‐C/H‐C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

et al. 1994

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“…Secondly, triplexes containing a polypurine RNA strand and an antiparallel G,T‐containing strand have not yet been detected. The only triplex formed by a polypurine RNA tract with a clamp oligonucleotide containing a G,T‐fragment has a parallel orientation of this fragment with respect to RNA [5,24]. The results obtained in the present work show that our knowledge on the conformational options of RNA is still limited and that in particular cases RNA can behave unusually, such as to become involved in reverse Hoogsteen binding with a third strand.…”

Section: Discussionmentioning

confidence: 81%

Targeting of single‐stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides

Maksimenko

Волков

Bertrand

et al. 2000

European Journal of Biochemistry

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The aim of this work was to construct an anti‐messenger targeted to the pim‐1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single‐stranded targets of both DNA (5′‐CCCTCCTTTGAAGAA‐3′) and RNA type (5′‐CCCUCCUUUGAAGAA‐3′). The third, ‘Hoogsteen’ strand of the triplex was represented by G,T‐rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3′‐GGGAGGAAACTTCTTTT‐TTGTTGTTT‐TT‐GGTGGG‐5′, where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the ‘Hoogsteen’ strand switches from one strand of the Watson–Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti‐messenger (underlined). The portion of the ‘Hoogsteen’ sequence of the triplex between the two TT dinucleotides binds to the 3′ extremity of the target strand and runs parallel to it. The portion situated at the 5′ end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5′‐TTCTTCAAAGGAGGG‐3′^3′‐GGGTGGTTT‐T‐GTTGTT‐5′) the portion of the ‘Hoogsteen’ sequence that bound to the 3′ extremity of the target strand had to be antiparallel to it.

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“…Aptamers (Sullenger et al 1990;Ess, Hutton, and Aronow 1994), antigene nucleic acids (Volkmann, Dannull, and Moeling 1993;Grigoriev et al 1993), ribozymes (Koizumi, Kamiya, and Ohtsuka 1992;Kashani-Sabet et al 1994), and antisense oligonucleotides (Stein and Cheng 1993;Li and Huang 1997) are among the nucleic acid drugs with successful in-vitro and in-vivo applications. On the other hand, liposomes are considered an important class of vehicles with potential for use in the protection and delivery of nucleic acids to cells and tissues.…”

mentioning

confidence: 99%

Formation of supramolecular structures by negatively charged liposomes in the presence of nucleic acids and divalent cations

et al. 1998

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Cationic liposomes are being increasingly studied as delivery vehicles for bioactive agents such as DNA and other polynucleotides. The mechanism of interaction of DNA with liposomes and the organization of these interacting structures during and after the interaction are still poorly understood. Nucleic acids are known to induce aggregation and size enlargement of liposomes. In the case of phosphatidylcholine (PC) vesicles, these processes depend on the presence and concentration of divalent metal cations and the amount of cholesterol in the liposomes. In this study, anionic small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) composed of dicetylphosphate (DCP):PC:cholesterol at 2:7:1 molar ratios were prepared and incubated with the DNA (from wheat) and Ca(2+) (50 mM) at 25 degrees C with the aim of transferring the genetic material into the liposomes by inducing fusion of liposome-liposome aggregates created in the presence, and with the help, of DNA. The organization and the nature of the resultant liposome-DNA-Ca(2+) complexes were investigated by scanning tunneling microscopy (STM) and fluorescence microscopy. Observations of complexes with similar appearances with both SUV and MLV, as shown by two quite different microscopic approaches, prove that the resultant forms are real and not artifacts of the methodology used. At this stage it is not clear whether the detected complexes represent an intermediate state before fusion of liposomes which will lead to engulfing of the genomic material by the fused liposomes, or the final form. In either case the structures consisting of some adhered or semifused liposomes bearing the nucleic acid seem to be candidates as vehicles for in-vitro and in-vivo transfection.

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The polypurine tract, PPT, of HIV as target for antisense and triple-helix-forming oligonucleotides

Cited by 29 publications

References 22 publications

Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

Targeting of single‐stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides

Formation of supramolecular structures by negatively charged liposomes in the presence of nucleic acids and divalent cations

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