Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region

Yong Wang, Keita Hamasaki, Robert R. Rando

Research output: Contribution to journalArticle

155 Citations (Scopus)

Abstract

Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM. RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50. The binding is driven by a strongly negative enthalpic term. Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 μM range. The binding of aminoglycosides to this construct is thus not highly selective. A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 μM. Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding. The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).

Original languageEnglish
Pages (from-to)768-779
Number of pages12
JournalBiochemistry
Volume36
Issue number4
DOIs
Publication statusPublished - 1997 Jan 28
Externally publishedYes

Fingerprint

Aminoglycosides
Decoding
RNA
Molecules
Anisotropy
Framycetin
rev Gene Products
Fluorescence
Paromomycin
Anti-Bacterial Agents
Tobramycin
Fluorescent Dyes
Stoichiometry
Arginine
Experiments
Chemical activation
Derivatives

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region. / Wang, Yong; Hamasaki, Keita; Rando, Robert R.

In: Biochemistry, Vol. 36, No. 4, 28.01.1997, p. 768-779.

Research output: Contribution to journalArticle

@article{a46519f5d29a4aba9afa0785ff22694e,
title = "Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region",
abstract = "Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM. RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50. The binding is driven by a strongly negative enthalpic term. Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 μM range. The binding of aminoglycosides to this construct is thus not highly selective. A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 μM. Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding. The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).",
author = "Yong Wang and Keita Hamasaki and Rando, {Robert R.}",
year = "1997",
month = "1",
day = "28",
doi = "10.1021/bi962095g",
language = "English",
volume = "36",
pages = "768--779",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "4",

}

TY - JOUR

T1 - Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region

AU - Wang, Yong

AU - Hamasaki, Keita

AU - Rando, Robert R.

PY - 1997/1/28

Y1 - 1997/1/28

N2 - Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM. RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50. The binding is driven by a strongly negative enthalpic term. Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 μM range. The binding of aminoglycosides to this construct is thus not highly selective. A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 μM. Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding. The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).

AB - Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM. RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50. The binding is driven by a strongly negative enthalpic term. Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 μM range. The binding of aminoglycosides to this construct is thus not highly selective. A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 μM. Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding. The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).

UR - http://www.scopus.com/inward/record.url?scp=0031051477&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031051477&partnerID=8YFLogxK

U2 - 10.1021/bi962095g

DO - 10.1021/bi962095g

M3 - Article

VL - 36

SP - 768

EP - 779

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 4

ER -