A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding.
This work describes the rational design, synthesis, and study of a ligand that selectively complexes CUG repeats in RNA (and CTG repeats in DNA) with high nanomolar affinity. This sequence is considered a causative agent of myotonic dystrophy type 1 (DM1) because of its ability to sequester muscleblind-like (MBNL) proteins. Ligand 1 was synthesized in two steps from commercially available compounds, and its binding to CTG and CUG repeats in oligonucleotides studied. Isothermal titration calorimetry studies of 1 with various sequences showed a preference toward the T-T mismatch (K(d) of 390 +/- 80 nM) with a 13-, 169-, and 85-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Binding and Job analysis of 1 to multiple CTG step sequences revealed high affinity binding to every other T-T mismatch with negative cooperativity for proximal T-T mismatches. The affinity of 1 for a (CUG)(4) step provided a K(d) of 430 nM with a binding stoichiometry of 1:1. The preference for the U-U in RNA was maintained with a 6-, >143-, and >143-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Ligand 1 destabilized the complexes formed between MBNL1N and (CUG)(4) and (CUG)(12) with IC(50) values of 52 +/- 20 microM and 46 +/- 7 microM, respectively, and K(i) values of 6 +/- 2 microM and 7 +/- 1 microM, respectively. These values were only minimally altered by the addition of competitor tRNA. Ligand 1 does not destabilize the unrelated RNA-protein complexes the U1A-SL2 RNA complex and the Sex lethal-tra RNA complex. Thus, ligand 1 selectively destabilizes the MBNL1N-poly(CUG) complex.
Arambula
Jonathan F
JF
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Ramisetty
Sreenivasa Rao
SR
Baranger
Anne M
AM
Zimmerman
Steven C
SC
D000166:Acridines / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014227:Triazines / Q000737:chemistry / Q000378:metabolism; D018911:Trinucleotide Repeats
D000166:Acridines; D008024:Ligands; C493663:MBNL1 protein, human; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D014227:Triazines; D012313:RNA; D004247:DNA; D012343:RNA, Transfer