Circular DNA and DNA/RNA hybrid molecules as scaffolds for ricin inhibitor design.
Ricin Toxin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA tetra-loop portion of 28S eukaryotic ribosomal RNA. Truncated stem-loop versions of the 28S rRNA are RTA substrates. Here, we investigate circular DNA and DNA/RNA hybrid GAGA sequence oligonucleotides as minimal substrates and inhibitor scaffolds for RTA catalysis. Closing the 5'- and 3'-ends of a d(GAGA) tetraloop creates a substrate with 92-fold more activity with RTA (kcat/Km) than that for the d(GAGA) linear form. Circular substrates have catalytic rates (kcat) comparable to and exceeding those of RNA and DNA stem-loop substrates, respectively. RTA inhibition into the nanomolar range has been achieved by introducing an N-benzyl-hydroxypyrrolidine (N-Bn) transition state analogue at the RTA depurination site in a circular GAGA motif. The RNA/DNA hybrid oligonucleotide cyclic GdAGA provides a new scaffold for RTA inhibitor design, and cyclic G(N-Bn)GA is the smallest tight-binding RTA inhibitor (Ki = 70 nM). The design of such molecules that lack the base-paired stem-loop architecture opens new chemical synthetic approaches to RTA inhibition.
Sturm
Matthew B
MB
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Roday
Setu
S
Schramm
Vern L
VL
D003500:Cyclization; D004270:DNA, Circular / Q000737:chemistry*; D015195:Drug Design; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010091:Oximes / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D012276:Ricin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004270:DNA, Circular; D009841:Oligonucleotides; D010091:Oximes; D012313:RNA; D012276:Ricin