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The binding site of a specific aminoglycoside binding RNA molecule
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.
Biochemistry
|May 16, 1998
Summary
This study identifies the tobramycin binding site on the J6f1 RNA aptamer. Key structural elements, including nucleotide bases and bulges, are crucial for high-affinity tobramycin binding.
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Aptamer Technology
Background:
- A previously developed RNA aptamer derivative, J6f1, binds tobramycin with high affinity (Kd ≈ 5 nM).
- J6f1 demonstrates significant selectivity for tobramycin over similar aminoglycosides.
Purpose of the Study:
- To delineate the specific binding site and molecular interactions between the J6f1 RNA aptamer and tobramycin.
- To understand the structural requirements for high-affinity and stoichiometric aminoglycoside binding.
Main Methods:
- Chemical interference assays
- Chemical modification studies
- Site-directed mutagenesis
Main Results:
- Tobramycin recognition by J6f1 involves direct contacts with nucleotide bases, not the phosphate backbone.
- The binding site is located within the stem-loop region of J6f1.
- Both the 3-nucleotide and 1-nucleotide bulges are essential for high-affinity binding.
Conclusions:
- The J6f1 RNA aptamer utilizes specific nucleotide bases and bulges for selective tobramycin recognition.
- Bulges likely facilitate conformational changes, enabling tobramycin access to the binding site.