Biomedical subjects

Richard Ting

Publications and source records attributed to Richard Ting.

6 recordsLinked to original sources

PubMed2005

Arylfluoroborates and alkylfluorosilicates as potential PET imaging agents: high-yielding aqueous biomolecular 18F-labeling.

Organometalloid compositions of silicon and boron permit rapid, high-yielding, one-step radiolabeling of a covalently linked protein ligand (biotin) under aqueous conditions to give the corresponding alkyltetrafluorosilicates and aryltrifluoroborate salts. Biotin was chosen as a test ligand for protein targeting because of its quantitative interaction with avidin, which in turn allowed us to calculate fluoridation yields that approach 80-100%. The silicate was found to be moderately stable to hydrolysis, whereas the borate appears to be so stable that its hydrolytic decomposition was not readily measured. With the stability of both compounds ascertained, this work describes a novel and robust radiolabeling method that may find use in the development of positron emission tomography radiopharmaceuticals.

Boron↗

PubMed2004

Substrate specificity and kinetic framework of a DNAzyme with an expanded chemical repertoire: a putative RNaseA mimic that catalyzes RNA hydrolysis independent of a divalent metal cation.

This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 9(25)-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 9(25)-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5'-product terminating in a 2',3'-phosphodiester and a 3'-product terminating in a 5'-OH. Under single turnover conditions at 24 degrees C, 9(25)-11t displays a maximum first-order rate constant, k(cat), of 0.037 min(-1) and a catalytic efficiency, k(cat)/K(m), of 5.3 x 10(5) M(-1) min(-1). The measured value of k(cat) under catalyst excess conditions agrees with the value of k(cat) observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 9(25)-11t was gauged in terms of k(cat) values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease k(cat) values by a factor of 4 to 250, indicating that 9(25)-11t displays significant sequence specificity despite the lack of an apparent Watson-Crick base-pairing scheme for recognition.

Base Sequence↗

PubMed2004

Triggering DNAzymes with light: a photoactive C8 thioether-linked adenosine.

Herein we report evidence for a light-inducible DNAzyme. In so doing, we also disclose the synthesis and photochemical properties of a novel nucleoside: 8-(2-(4-imidazolyl)ethyl-1-thio)-2'-deoxyriboadenosine (d1). The light sensitivity of (d1) was evaluated via an examination of the photoinduced reactivation of DNAzyme 8-17E from an inactive form that contained a single nucleotide (d1) modification. Restoration of DNAzyme activity results from a photoinduced reversion of (d1) to unmodified deoxyadenosine. Deuterium studies indicate that water is the source of hydrogen in the C8-H product and not the alkylthio group, suggesting that reversion of (1) to adenosine is not a consequence of simple homolysis of the C8-S bond but of an unprecedented photochemical conversion. This adenosine, which affords significant control of catalytic reactivation of a DNAzyme, may find general use in photodecaging other biological systems.

Adenosine↗

PubMed2004

Covalent Schiff base catalysis and turnover by a DNAzyme: a M2+ -independent AP-endonuclease mimic.

A DNAzyme, synthetically modified with both primary amines and imidazoles, is found to act as a M2+ -independent AP lyase-endonuclease. In the course of the cleavage reaction, this DNAzyme forms a covalent Schiff base intermediate with an abasic site on a complementary oligodeoxyribonucleotide. This intermediate, which is inferred from NaCNBH3 trapping as well as cyanide inhibition, does not evidently accumulate because the second step, dehydrophosphorylative elimination, is fast compared to Schiff base formation. The 5'-product that remains linked to the catalyst hydrolyzes slowly to regenerate free catalyst. The use of duly modified DNAzymes to perform Schiff base catalysis demonstrates the value of modified nucleotides for enhancing the catalytic repertoire of nucleic acids. This work suggests that DNAzymes will be capable of catalyzing aldol condensation reactions.

Base Sequence↗

PubMed2004

High affinity DNAzyme-based ligands for transition metal cations - a prototype sensor for Hg2+.

Inspired by recent interest in DNAzymes as transition metal ion sensors, a survey of the effects of various transition metals on the intramolecular cleavage rate of an imidazole modified, M(2+)-independent, self-cleaving "9(25)-11" DNA is reported. In particular, 9(25)-11 activity was strongly inhibited by Hg(2+)(K(d)(APP)= 110 +/- 9 nM). It is postulated that the affinity and selectivity of 9(25)-11 for Hg(2+) stems from the fact that this synthetically modified DNAzyme contains imidazoles. This study demonstrates the utility of modified nucleotides in developing DNAzyme sensors for metals ions, especially those for which unmodified nucleic acids might not serve as inherently good ligands.

Base Pairing↗

PubMed2002

Toward an RNaseA mimic: A DNAzyme with imidazoles and cationic amines.

Site-specific RNA cleavage has received considerable attention over the years. Directed synthesis to append imidazoles or amines or both to oligonucleotides to target specific RNA cleavage represents an exciting avenue of research. However, to date catalysis by such synthetic constructs, particularly in terms of turnover, has been difficult to observe. This is the first report of a truly catalytic M2+-independent DNAzyme synthetically modified with imidazoles and cationic amines that would seem to mimic RNaseA. This work now demonstrates how synthetic organic chemistry, when merged with combinatorial selection, can result in a new class of DNAzymes that meets the ongoing synthetic challenges for developing relatively small biomimetic catalysts.

Amines↗