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

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A genomic selection strategy to identify accessible and dimerization blocking targets in the 5'-UTR of HIV-1 RNA. / Jakobsen, Martin R; Damgaard, Christian K; Andersen, Ebbe S; Podhajska, Anna; Kjems, Jørgen.

In: Nucleic Acids Research, Vol. 32, No. 7, 2004, p. e67.

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@article{7117eb0c4efc475d97a3d84dece52509,
title = "A genomic selection strategy to identify accessible and dimerization blocking targets in the 5'-UTR of HIV-1 RNA",
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.",
keywords = "5' Untranslated Regions, Base Sequence, Binding Sites, DNA, Viral, Dimerization, Gene Library, Genome, Viral, Genomics, HIV Long Terminal Repeat, Molecular Sequence Data, Nucleic Acid Conformation, Oligoribonucleotides, Antisense, RNA, Viral, Selection, Genetic",
author = "Jakobsen, {Martin R} and Damgaard, {Christian K} and Andersen, {Ebbe S} and Anna Podhajska and J{\o}rgen Kjems",
year = "2004",
doi = "10.1093/nar/gnh064",
language = "English",
volume = "32",
pages = "e67",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "7",

}

RIS

TY - JOUR

T1 - A genomic selection strategy to identify accessible and dimerization blocking targets in the 5'-UTR of HIV-1 RNA

AU - Jakobsen, Martin R

AU - Damgaard, Christian K

AU - Andersen, Ebbe S

AU - Podhajska, Anna

AU - Kjems, Jørgen

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

KW - 5' Untranslated Regions

KW - Base Sequence

KW - Binding Sites

KW - DNA, Viral

KW - Dimerization

KW - Gene Library

KW - Genome, Viral

KW - Genomics

KW - HIV Long Terminal Repeat

KW - Molecular Sequence Data

KW - Nucleic Acid Conformation

KW - Oligoribonucleotides, Antisense

KW - RNA, Viral

KW - Selection, Genetic

U2 - 10.1093/nar/gnh064

DO - 10.1093/nar/gnh064

M3 - Journal article

VL - 32

SP - e67

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 7

ER -