Pyrene-zipper array assembled via RNA duplex formation.

We describe a new strategy for multipyrene modification of RNA sequences to form unique structures of pyrene aromatic arrays, the "pyrene-zipper array", on duplex RNA that exhibits remarkably strong excimer fluorescence.

Nylon-oligomer degrading enzyme/substrate complex: catalytic mechanism of 6-aminohexanoate-dimer hydrolase.

We performed X-ray crystallographic analyses of 6-aminohexanoate-dimer hydrolase (Hyb-24DN), an enzyme responsible for the degradation of nylon-6, an industry by-product, and of a complex between Hyb-24DN-A(112) (S112A-mutant of Hyb-24DN) and 6-aminohexanoate-linear dimer (Ald) at 1.58 A and 1.4 A resolution, respectively. In Hyb-24DN, Asp181-O(delta) forms hydrogen bonds with Tyr170-O(eta), -two of the catalytic and binding amino acids, and a loop between Asn167 and Val177. This state is the so-called open form, allowing its substrate to bind in the space between the loop and catalytic residues. Upon substrate binding (in Hyb-24DN-A(112)/Ald complex), the loop is shifted 4.3 A at Tyr170-C(alpha), and the side-chain of Tyr170 is rotated. By the combined effect, Tyr170-O(eta) moves a total of 10.5 A, resulting in the formation of hydrogen bonds with the nitrogen of amide linkage in Ald (closed form). In addition, electrostatic interaction between Asp181-O(delta) and the amino group in Ald stabilizes the substrate binding. We propose here that the enzyme catalysis proceeds according to the following steps: (i) Ald-induced transition from open to closed form, (ii) nucleophilic attack of Ser112 to Ald and formation of a tetrahedral intermediate, (iii) formation of acyl enzyme and transition to open form, (iv) deacylation. Amino acid substitutions reducing the enzyme/Ald interaction at positions 181 or 170 drastically decreased the Ald-hydrolytic activity, but had very little effect on esterolytic activity, suggesting that esterolytic reaction proceeds regardless of conversion. Present models illustrate why new activity against the nylon oligomer has evolved in an esterase with beta-lactamase folds, while retaining the original esterolytic functions.

Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference.

Through binding and fluorescence studies of oligonucleotides covalently attached to a pyrene group via one carbon linker at the sugar residue, we previously found that pyrene-modified RNA oligonucleotides do not emit well in the single-stranded form, yet the attached pyrene emits with a significantly high quantum yield upon binding to a complementary RNA strand. In sharp contrast, similarly modified pyrene-DNA probes exhibit very weak fluorescence both in the double-stranded and single-stranded forms. The pyrene-modified RNA oligonucleotides therefore provide a useful tool for monitoring RNA hybridization. The purpose of this paper is to present the structural basis for the different fluorescence properties of pyrene-modified RNA/RNA and pyrene-modified DNA/DNA duplexes. The results of absorption, fluorescence anisotropy and circular dichroism studies all consistently indicated that the pyrene attached to the RNA duplex is located outside of the duplex, whereas the pyrene incorporated into the DNA duplex intercalates into the double helix. (1)H NMR measurements unambiguously confirmed that the pyrene attached to the DNA duplex indeed intercalates between the base pairs of the duplex. Molecular dynamics simulations support these differences in the local structural elements around the pyrene between the pyrene-RNA/RNA and the pyrene-DNA/DNA duplexes.

Pyrene aromatic arrays on RNA duplexes as helical templates.

RNA oligomers having multiple (2 to 4) pyrenylmethyl substituents at the 2'-O-sugar residues were synthesized. UV-melting studies showed that the pyrene-modified RNAs could form duplexes with complementary RNA sequences without loss of thermal stability. Absorption, fluorescence, and circular dichroism (CD) spectra revealed that the incorporated pyrenes projected toward the outside of A-form RNA duplexes and assembled in helical aromatic arrays along the minor grooves of the RNA duplexes. Results of computer simulations agreed with the assembled structures of the pyrenes. The helical pyrene arrays exhibited remarkably strong excimer fluorescence, which was dependent on the sequence contexts of RNA duplexes.