Near‐Quantitative Preparation of Short Single‐Stranded DNA Circles

Abstract

Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA nanostructures. However, it is challenging to prepare such ssDNA circles, particularly when the DNA size becomes very small (e.g. a 20 nucleotide (nt) long ssDNA circle). Often, such short ssDNA dominantly form concatemers (either linear or circular) due to intermolecular ligation, instead of forming monomeric ssDNA circles by intramolecular ligation. Herein, a simple method to overcome this problem by designing the complementary linker molecules is reported. It is demonstrated that ssDNA, as short as 16 nts, can be enzymatically ligated (by the commonly used T4 DNA ligase) into monomeric ssDNA circles at high concentration (100 μM) with high yield (97 %). This method does not require any special sequence, thus, it is expected to be generally applicable. The experimental protocol is identical to regular DNA ligation, thus, is expected to be user friendly for general chemists and biologists.

Circular Single-Stranded DNA: Discovery, Biological Effects, and Applications

“…(D) A new design of splint strands allows for the near-quantitative monomeric cyclization of single DNA strands by DNA T4 ligase. Reproduced from ref . Copyright 2023 The Author(s).…”

“…Until now, several significant approaches have been developed to improve the ligation efficiency. These include the utilization of a terminal hairpin strategy to promote dominant intramolecular cyclization, the implementation of frozen/lyophilization/cyclization method for highly efficient cyclization (Figure C), the design of a new splint strand Hm to facilitate cyclization (Figure D), and the application of one-pot ligation method for efficient production of large C-ODNs. , Creatively, Li et al have designed an intramolecular terminal hybridization strategy to optimize the ligation efficiency via CircLigase, a naturally thermostable RNA ligase 1 (Figure E). There is an unconventional method to prepare short CssDNA named “M1.3” (704 nt) .…”