Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage

doi:10.1016/S0968-0896(01)00033-5

Bioorganic & Medicinal Chemistry

Volume 9, Issue 10, October 2001, Pages 2581-2587

https://doi.org/10.1016/S0968-0896(01)00033-5 Get rights and content

Abstract

The chemical ligation of DNA molecules can be mediated by terminal phosphorothioate displacement of a 5′ iodine. We have selected deoxyribozymes that can catalyze the formation of such phosphorothioester internucleotide linkages. The selected deoxyribozymes enhance the rate of ligation in part through the provision of a template that aligns the ligation junction and do not appear to require metal ions for catalysis.

Graphic

Introduction

DNA has been shown to be as competent a catalyst as RNA.⁹ However, there has to date been only a single selection of a deoxyribozyme ligase.² This DNA enzyme catalyzed the ligation of its 5′ hydroxyl to a target oligonucleotide bearing an activated 3′phosphorimidazolide, forming a new phosphodiester bond. The selected catalyst required divalent cations (Zn²⁺ or Cu²⁺) for its activity, suggesting that ligases selected from DNA pools are similar to those that have previously been selected from RNA pools, which in general have also used metals to catalyze the attack of hydroxyl moieties on activated phosphates (typically phosphoanhydrides) to form 2′-5′ or 3′-5′ phosphodiester linkages.1, 7, 11

We were interested in determining whether deoxyribozymes might also be capable of facilitating ligase reactions via chemistries not typically found in nucleic acids. To this end, we adapted a selection procedure to a novel ligation chemistry that had previously been developed by Kool and co workers.¹⁴ In this chemistry, a 5′ iodine is displaced by a 3′ phosphorothioate to form a bridging 5′ phosphorothioester linkage (Fig. 1a). While this chemistry has been shown to be very efficient for the ligation and circularization of oligonucleotides, it is relatively slow; ligation reactions typically require many hours to accumulate appreciable ligation products.¹⁶ Using in vitro selection we hoped to enhance the overall speed of the reaction via the selection of a catalyst.

After 11 rounds of selection and amplification we have identified deoxyribozymes that can catalyze the formation of this unnatural internucleotide linkage. Interestingly, the deoxyribozyme reaction has proven to be metal-independent.

Section snippets

5′IN90 pool construction

The pool was designed and synthesized according to previously reported methods.¹¹ In brief, synthesis was carried out in our laboratory on an Expedite 8909 DNA synthesizer (PE Biosystems, Foster City, CA) using standard phosphoramidite chemistry. All synthesis reagents were purchased from Glen Research (Sterling, VA). Primers containing 5′ I-dT were synthesized with ultramild amidites. The 3′ phosphorothioates were synthesized on 3′ phosphate-CPG by replacing the normal oxidizer with the

Selection of deoxyribozyme ligase

In order to select a deoxyribozyme ligase capable of enhancing the formation of a bridging 5′ phosphorothioester linkage we employed a strategy similar to that originally developed by Bartel and Szostak (1993) and successfully adapted by our lab¹¹ (Fig. 1b,c). The starting pool contained 90 random sequence positions. The 5′ constant region was designed to form a hairpin structure and contained a 5′I-dT. A phosphoramidite containing the iodinated residue was incorporated into a primer and

Acknowledgements

The authors with to thank Michael Robertson for his assistance with this project. This work was supported by a grant from the NASA Astrobiology Institute, NCC2-1055.

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Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage

Abstract

Introduction

Section snippets

5′IN90 pool construction

Selection of deoxyribozyme ligase

Acknowledgements

J. Mol. Biol.

Chem. Biol.

Curr. Opin. Struct. Biol.

J. Mol. Biol.

Curr. Opin. Chem. Biol.

Tetrahedron Lett.

Science

Nature