Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation.

Jinfeng Chen,Ysobel R Baker,Asha Brown,Tom Brown,Afaf H El-Sagheer

doi:10.1039/c8sc02952k

Abstract

Cyclic oligonucleotides are valuable targets with a broad range of potential applications spanning molecular biology and nanotechnology. Of particular importance is their role as templates in the rolling circle amplification (RCA) reaction. We describe three different chemical cyclisation methods for the preparation of single-stranded cyclic DNA constructs. These chemical cyclisation reactions are cheaper to carry out than the enzymatic reaction, and more amenable to preparative scale purification and characterisation of the cyclic product. They can also be performed under denaturing conditions and are therefore particularly valuable for cyclic DNA templates that contain secondary structures. The resulting single-stranded cyclic DNA constructs contain a single non-canonical backbone linkage at the ligation point (triazole, amide or phosphoramidate). They were compared to unmodified cyclic DNA in rolling circle amplification reactions using φ-29 and Bst 2.0 DNA polymerase enzymes. The cyclic templates containing a phosphoramidate linkage were particularly well tolerated by φ-29 polymerase, consistently performing as well in RCA as the unmodified DNA controls. Moreover, these phosphoramidate-modified cyclic constructs can be readily produced in oligonucleotide synthesis facilities from commercially available precursors. Phosphoramidate ligation therefore holds promise as a practical, scalable method for the synthesis of fully biocompatible cyclic RCA templates. The triazole-modified cyclic templates generally gave lower and more variable yields of RCA products, a significant proportion of which were double-stranded, while the performances of the templates containing an amide linkage lie in between those of the phosphoramidate- and triazole-containing templates.

Highlights

Rolling circle ampli cation (RCA) is an isothermal enzymatic technique that is widely used to make very long single-stranded DNA and RNA.[1,2] The product contains a speci c, tandemrepeating sequence which is encoded by the complementary cyclic template.[3,4] This technique has been harnessed as a simple and powerful method of signal ampli cation in the development of sensitive detection methods for a variety of nucleic acid, protein, cellular and small molecule targets for 8110 | Chem
When incorporated into linear oligonucleotides these phosphodiester backbone analogues can be read through by DNA polymerases during linear copying and the polymerase chain reaction (PCR), with accurate transfer of genetic information.[28,32,35]. They could potentially be incorporated into cyclic rolling circle amplification (RCA) templates provided that they can be read through by the speci c DNA polymerases that are compatible with efficient RCA
The use of chemically modi ed templates in RCA is demanding because it requires the polymerase to accurately read through the modi ed linkage repeatedly in order to generate very long ampli cation products

Summary

Introduction

Rolling circle ampli cation (RCA) is an isothermal enzymatic technique that is widely used to make very long single-stranded DNA and RNA.[1,2] The product contains a speci c, tandemrepeating sequence which is encoded by the complementary cyclic template.[3,4] This technique has been harnessed as a simple and powerful method of signal ampli cation in the development of sensitive detection methods for a variety of nucleic acid, protein, cellular and small molecule targets for 8110 | Chem. The second study used a triazole-containing DNA template in an RCA-based uorogenic assay for microRNA, but the authors did not compare the efficiency of the RCA reaction with that of an unmodi ed template.[46] In this work we investigate amide and phosphoramidate chemical linkages (Fig. 1(ii)B and C), both of which can be formed by di-imide coupling chemistry.

Results

Conclusion