Efficient Preparation of Large‐Sized Rings of Single‐Stranded DNA through One‐Pot Ligation of Multiple Fragments

Abstract

Circular single‐stranded DNA (c‐ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c‐ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one‐pot ligation method to efficiently prepare large c‐ssDNA. By ligating several short fragments of linear single‐stranded DNA (l‐ssDNA) in one‐pot by using T4 DNA ligase, longer l‐ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large‐sized single‐stranded c‐ssDNAs (e.g., 200‐nt) were successfully synthesized in high selectivity and yield.

Stepwise Strategy for One‐Pot Synthesis of Single‐Stranded DNA Rings from Multiple Short Fragments

“…In the “simple one‐pot strategy” in Scheme 1A, [11] however, all the short ssDNA fragments and the splints are added into a reaction vessel at the beginning of reaction. With this method, the reaction never proceeds in a stoichiometric fashion, and the amounts of target rings produced are limited, as described below.…”

“…In order to produce large c‐ssDNAs more easily without size‐limitation, we recently developed an alternative strategy in which multiple short linear ssDNA fragments (30–60 nt) as starting materials are convergently connected and cyclized in situ to c‐ssDNA (Scheme 1A) [11] . In a reaction vessel, all of these short fragments were mixed with all the splints before the treatment with T4 DNA ligase.…”

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis

“…At present, researchers have significantly improved the circularization efficiency by reducing MgCl 2 concentration [31], using the terminal hairpin of the ssDNA substrate [32] or frozen/lyophilization/cyclization (FLC) [33]. Sui et al reported a one-pot ligation method to efficiently prepare longer circular ssDNA (>90 nt) [34], but several splints were needed, and the yield was not high enough in some cases. In our new approach presented here, each ssDNA circle was synthesized by using two fragments, which could hybridize with each other to form two nicks (Figure 2A).…”

“…Recently, we have successfully prepared stable non-modified Z-DNA (LR-chimera involving the left-handed part and the right-handed one) under physiological ionic conditions (e.g., 10 mM MgCl 2 ) using two complementary 74~111 bp long circular single-stranded DNA (ssDNA) [30]. The circularization of ssDNA is a critical step, which needs to be improved [31][32][33][34]. In addition, the LR-chimera we prepared is difficult to attach onto the surface of the probe, because it has no ssDNA part for further hybridization.…”

“…This method is simple to operate and ca realize the quantitative analysis of Z-DNA binding under a variety of ion conditions. In this study, we developed a new approach to efficiently prepare circular ssDNAs without using splints (an extra short ssDNA to draw close two ends of the precursor ssDNA to form a nick) [34]. After hybridizing two DNA fragments to form two nicks, circular ssDNA can be obtained by nick sealing with T4 DNA ligase (Figure 2A).…”