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. 2004 Apr 23;32(7):e67.
doi: 10.1093/nar/gnh064.

A genomic selection strategy to identify accessible and dimerization blocking targets in the 5'-UTR of HIV-1 RNA

Martin R Jakobsen  1 , Christian K Damgaard, Ebbe S Andersen, Anna Podhajska, Jørgen Kjems
Affiliations

A genomic selection strategy to identify accessible and dimerization blocking targets in the 5'-UTR of HIV-1 RNA

Martin R Jakobsen et al. Nucleic Acids Res. .

Abstract

Defining target sites for antisense oligonucleotides in highly structured RNA is a non-trivial exercise that has received much attention. Here we describe a novel and simple method to generate a library composed of all 20mer oligoribonucleotides that are sense- and antisense to any given sequence or genome and apply the method to the highly structured HIV-1 leader RNA. Oligoribonucleotides that interact strongly with folded HIV-1 RNA and potentially inhibit its dimerization were identified through iterative rounds of affinity selection by native gel electrophoresis. We identified five distinct regions in the HIV-1 RNA that were particularly prone to antisense annealing and a structural comparison between these sites suggested that the 3'-end of the antisense RNA preferentially interacts with single-stranded loops in the target RNA, whereas the 5'-end binds within double-stranded regions. The selected RNA species and corresponding DNA oligonucleotides were assayed for HIV-1 RNA binding, ability to block reverse transcription and/or potential to interfere with dimerization. All the selected oligonucleotides bound rapidly and strongly to the HIV-1 leader RNA in vitro and one oligonucleotide was capable of disrupting RNA dimers efficiently. The library selection methodology we describe here is rapid, inexpensive and generally applicable to any other RNA or RNP complex. The length of the oligonucleotide in the library is similar to antisense molecules generally applied in vivo and therefore likely to define targets relevant for HIV-1 therapy.

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Figures

Figure 1
Figure 1
Construction of the RNA library. A pUC18 plasmid containing the HIV-1(LAI) leader sequence (+1–355) was digested partially with DNase I to create random fragments. A double-stranded oligonucleotide, containing a T7 promoter, a specific tag sequence (Tag A) and a MmeI restriction site, was ligated to the fragments. After MmeI digestion the DNA fragments were ligated to a downstream linker, containing a random two-nucleotides 3′-overhang and another tag sequence (Tag B). The RNA library was synthesized by standard run-off transcription using T7 RNA polymerase.
Figure 2
Figure 2
Selection of RNA oligonucleotides that bind to the HIV-1 leader. Radiolabelled HIV-1 leader RNA (+1–355) was incubated under dimerization conditions alone (lane 1) or in the presence of control oligonucleotides, CN1, known to stimulate dimerization (lane 2), DISSkripkin, known to inhibit dimerization (lane 3), or the selected RNA pools after 1–5 rounds of selection (lanes 4–8). The positions of dimeric (D) and monomeric (M) RNA species are indicated.
Figure 3
Figure 3
Position of the antisense oligonucleotides used in this study. (A) The selected target sites superimposed on the BMH secondary structure of the HIV-1 HXB2 subtype leader drawn according to Damgaard et al. (21). Thick lines denote the sequences complementary to the selected RNA oligonucleotides after five rounds of selection. The thin line denotes the sequence complementary to the PSI-specific oligonucleotides that was obtained frequently after the third round of selection, but only once after the fifth round (Table 1). The major structure elements are denoted (see text for details). (B) The annealing sites for DISupstream–, DISskripkin– and the selected DIS–oligonucleotides indicated by dotted, stippled and full lines respectively. (C) The annealing sites for PBSnat– and PBS–oligonucleotides.
Figure 3
Figure 3
Position of the antisense oligonucleotides used in this study. (A) The selected target sites superimposed on the BMH secondary structure of the HIV-1 HXB2 subtype leader drawn according to Damgaard et al. (21). Thick lines denote the sequences complementary to the selected RNA oligonucleotides after five rounds of selection. The thin line denotes the sequence complementary to the PSI-specific oligonucleotides that was obtained frequently after the third round of selection, but only once after the fifth round (Table 1). The major structure elements are denoted (see text for details). (B) The annealing sites for DISupstream–, DISskripkin– and the selected DIS–oligonucleotides indicated by dotted, stippled and full lines respectively. (C) The annealing sites for PBSnat– and PBS–oligonucleotides.
Figure 4
Figure 4
HIV-1 leader RNA dimerization assay. HIV-1 leader RNA (+1–355) was incubated under dimerization conditions in the absence (Control), in the presence of oligonucleotide CN1 (lane 2), DISskripkin–DNA (lane 3) or selected antisense DNA and RNA oligonucleotides as denoted above (see Table 2 for sequences). The dimerization efficiency, indicated below, was calculated as [dimer]/[dimer]+[monomer] from three independent experiments. The positions of dimeric (D) and monomeric (M) RNA species are indicated.
Figure 5
Figure 5
Pausing of reverse transcription by selected oligonucleotides. Binding of the selected oligonucleotides (Table 2) and their ability to block reverse transcription was investigated by a reverse transcription progressivity assay using an extended HIV-1 template (+1–444). A 5′-end-labelled DNA primer complementary to position 384–401 of the viral RNA was used to prime reverse transcription. A control without oligonucleotide (lane 1) and a sequencing ladder (lanes 7–10) are included.
Figure 6
Figure 6
Binding analysis of the selected oligonucleotides to HIV-1 RNA. Non-labelled HIV-1 leader RNA (+1–355) was incubated in the presence of the indicated radiolabelled oligonucleotides that include (A) DISupstream–RNA (lanes 1–5), DIS–RNA (lanes 6–10) and DISSkripkin–RNA (lanes 11–15), (B) DISSkripkin –DNA (lanes 2–6) and DIS–DNA (lanes 7–11) and (C) PBSnat–DNA (lanes 2–6) and PBS–DNA (lanes 7–11). See Table 2 for sequences. The samples were incubated for the indicated time period (min) before being subjected to native gel electrophoresis. The relative binding efficiency of labelled oligonucleotide to the HIV-1 leader RNA was calculated from three independent experiments as the sum of counts in the monomer and dimer bands normalized in each experiment to the maximum binding observed at the last time point. (A) The bands corresponding monomeric and dimeric RNA oligos were investigated by running the same samples in a 12% polyacrylamide native gel (lower autoradiogram). Radiolabelled HIV-1 RNA was included as a marker in panels B and C. The positions of dimeric (D) and monomeric (M) RNA species are indicated.
Figure 6
Figure 6
Binding analysis of the selected oligonucleotides to HIV-1 RNA. Non-labelled HIV-1 leader RNA (+1–355) was incubated in the presence of the indicated radiolabelled oligonucleotides that include (A) DISupstream–RNA (lanes 1–5), DIS–RNA (lanes 6–10) and DISSkripkin–RNA (lanes 11–15), (B) DISSkripkin –DNA (lanes 2–6) and DIS–DNA (lanes 7–11) and (C) PBSnat–DNA (lanes 2–6) and PBS–DNA (lanes 7–11). See Table 2 for sequences. The samples were incubated for the indicated time period (min) before being subjected to native gel electrophoresis. The relative binding efficiency of labelled oligonucleotide to the HIV-1 leader RNA was calculated from three independent experiments as the sum of counts in the monomer and dimer bands normalized in each experiment to the maximum binding observed at the last time point. (A) The bands corresponding monomeric and dimeric RNA oligos were investigated by running the same samples in a 12% polyacrylamide native gel (lower autoradiogram). Radiolabelled HIV-1 RNA was included as a marker in panels B and C. The positions of dimeric (D) and monomeric (M) RNA species are indicated.
Figure 6
Figure 6
Binding analysis of the selected oligonucleotides to HIV-1 RNA. Non-labelled HIV-1 leader RNA (+1–355) was incubated in the presence of the indicated radiolabelled oligonucleotides that include (A) DISupstream–RNA (lanes 1–5), DIS–RNA (lanes 6–10) and DISSkripkin–RNA (lanes 11–15), (B) DISSkripkin –DNA (lanes 2–6) and DIS–DNA (lanes 7–11) and (C) PBSnat–DNA (lanes 2–6) and PBS–DNA (lanes 7–11). See Table 2 for sequences. The samples were incubated for the indicated time period (min) before being subjected to native gel electrophoresis. The relative binding efficiency of labelled oligonucleotide to the HIV-1 leader RNA was calculated from three independent experiments as the sum of counts in the monomer and dimer bands normalized in each experiment to the maximum binding observed at the last time point. (A) The bands corresponding monomeric and dimeric RNA oligos were investigated by running the same samples in a 12% polyacrylamide native gel (lower autoradiogram). Radiolabelled HIV-1 RNA was included as a marker in panels B and C. The positions of dimeric (D) and monomeric (M) RNA species are indicated.
Figure 7
Figure 7
Time course for the disruption of preformed HIV-1 dimer into RNA monomers in the presence of DIS-specific oligonucleotides. One picomole of radiolabelled HIV-1 leader RNA was dimerized and challenged with 5 pmol DIS–RNA or DIS–DNA for 0, 30, 120 or 240 min at 37°C before native gel electrophoresis. The dimerization efficiency was calculated as [dimer]/[dimer]+[monomer] from three independent experiments.
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