Synthesis and “DNA Interlocks” Formation of Small Circular Oligodeoxynucleotides

Abstract

Circular oligodeoxynucleotides (c-ODNs) have their particular characteristics in topological properties. However, different from oligoribonucleotides, enzymatic synthesis of small c-ODNs is still challenging using conventional methods. Herein, we successfully achieved highly efficient cyclization of linear single-stranded ODNs using T4 DNA ligase simply through the frozen/ lyophilization/cyclization (FLC) method. We successfully shortened the cyclization length of linear ODNs to 20 nt (l-ODN 20) with up to 63% yield, which was never achieved before through normal enzymatic methods. With the efficient FLC method, we further developed "DNA interlocks" which were intercross-linked with multiple c-ODNs using the one-pot FLC method. This FLC strategy provides a powerful, cheap, and convenient method to synthesize small c-ODNs for studying DNA nanotechnology and paves the way to achieve future deciphering of c-ODN functions.

Circular Antisense Oligonucleotides for Specific RNase-H-Mediated microRNA Inhibition with Reduced Off-Target Effects and Nonspecific Immunostimulation

“…According to our previous reported frozen/lyophilization/ cyclization (FLC) method, two different sizes of circular AMOs (30 mer and 38 mer) targeting miR 21 were first synthesized, as shown in Figures and S1. c-ODN 30 and c-ODN 38 were first used as latent miR 21 inhibitors to further explore their binding properties and templated RNase-H-mediated miRNA digestion.…”

“…Among more than 48,860 different mature microRNAs (miRBase), miR 21 is one of the most well-known and most studied onco-miRNAs, which is upregulated in many cancers. − In addition, miR 21 also plays an important role in cardiovascular disease, immune response, and organ fibrosis. , miR 222 is another common onco-miRNA, which significantly increases cell proliferation. ,− Previous studies have applied antisense microRNA oligonucleotides against miR 21 and miR 222 to reduce tumors in vivo . − However, few effective studies were investigated to reduce their possible off-target effects. Due to the specific topology of circular oligonucleotides, they have been reported to regulate and/or evaluate oligonucleotide confirmation and gene functions. − Herein, we reported a new strategy to develop small circular AMOs and specifically mediate microRNA inhibition. The circular topology of circular AMOs is expected to greatly reduce general off-target effects of ODNs as well as decrease their nonspecific immunological responses and interspecies crossing.…”

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

“…For efficient preparation of an LR-chimera consisting of two circular ssDNAs, we first developed a new circularization approach to completely suppress the polymeric byproducts (Figure 2). 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.…”

“…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.…”

Mechanical Flexibility of DNA: A Quintessential Tool for DNA Nanotechnology

“…DNA nanotechnology has exploited the flexibility-based cyclization of DNA fragments to construct fascinating DNA origami nanostructures. These include circular interlocks, single-and multiple-ring DNA catenanes, Borromean rings, and rotaxanes [202][203][204][205]. Circular functional nucleic acids have been employed as rolling circle amplification templates, aptamers, enzymes two-input logic gates, biosensors and have proved to be a cornerstone for the advancement of DNA nanotechnology [206].…”