Lanthanide Cofactors Accelerate DNA-Catalyzed Synthesis of Branched RNA

Abstract

Most deoxyribozymes (DNA catalysts) require metal ions as cofactors for catalytic activity, with Mg(2+), Mn(2+), and Zn(2+) being the most represented activators. Trivalent transition-metal ions have been less frequently considered. Rare earth ions offer attractive properties for studying metal ion binding by biochemical and spectroscopic methods. Here we report the effect of lanthanide cofactors, in particular terbium (Tb(3+)), for DNA-catalyzed synthesis of 2',5'-branched RNA. We found up to 10(4)-fold increased ligation rates for the 9F7 deoxribozyme using 100 μM Tb(3+) and 7 mM Mg(2+), compared to performing the reaction with 7 mM Mg(2+) alone. Combinatorial mutation interference analysis (CoMA) was used to identify nucleotides in the catalytic region of 9F7 that are essential for ligation activity with different metal ion combinations. A minimized version of the DNA enzyme sustained high levels of Tb(3+)-assisted activity. Sensitized luminescence of Tb(3+) bound to DNA in combination with DMS probing and DNase I footprinting results supported the CoMA data. The accelerating effect of Tb(3+) was confirmed for related RNA-ligating deoxyribozymes, pointing toward favorable activation of internal 2'-OH nucleophiles. The results of this study offer fundamental insights into nucleotide requirements for DNA-catalyzed RNA ligation and will be beneficial for practical applications that utilize 2',5'-branched RNA.

Lanthanide-dependent RNA-cleaving DNAzymes as metal biosensors

“…66 Aside from RNA-cleaving DNAzymes, lanthanides are also used in DNAzymes for DNA cleavage and RNA ligation. 67,68 We are interested in RNA-cleaving DNAzymes, since they are usually small in size and highly efficient and can be readily engineered into biosensors.…”

Functional Hallmarks of a Catalytic DNA that Makes Lariat RNA

“…CoMA is an efficient and reliable methodf or simultaneous analysiso fa ll possible point mutants of af unctionalD NA and has been used to identify the nucleotide requirementso fs everal deoxyribozymes. [9,14] The experimental strategy of CoMA consists of four steps. In the first step, four libraries of DNA mutantsa re synthesized by solid-phase synthesis.…”

“…[7] The formation of nonlinear2 ',5'-phosphodiester bonds with deoxyribozymes invites additional applicationso fD NA catalysts. [8] The deoxyribo-zyme 10DM24 was used under lanthanide-mediated rate-accelerating conditions [9] to site-specifically attach fluorescent dyes, spin-labels, biotin,o rc ross-linkers to biologically related RNA molecules via a2 ',5'-phosphodiester linkage with al abeled guanosine nucleotide. [10] The application of 2',5'-phosphodiester bond-formingd eoxyribozymes can be furthere xtended to the preparative synthesis of lariat and 2',5'-branched RNA molecules.…”

Debranchase-resistant labeling of RNA using the 10DM24 deoxyribozyme and fluorescent modified nucleotides

“…The synthesis of 2′,5′-branched RNAs at branch-sites other than adenosine had been reported using 10DM24 with 5′-triphosphorylated oligonucleotide substrates, 13 but the 10–100-fold slower reaction rates prevented efficient installation of a fluorescent single-nucleotide branch. Taking advantage of the recent findings that lanthanide ions accelerate DNA-catalyzed 2′,5′-phosphodiester bond formation, 14, 15 and that branch-sites flanked by purine nucleotides are preferred substrates for 10DM24, 6 GMP-branched RNAs with all four different branch sites were prepared in good yields using a combination of Mg 2+ and Tb 3+ . However, even in the optimal sequence context and reaction conditions, the single nucleotide 2′,5′-linkage at branchpoint adenosines was generated ~280-fold faster than at branchpoint cytidines (Fig.…”