Cyclization of secondarily structured oligonucleotides to single-stranded rings by using <i>Taq</i> DNA ligase at high temperatures

Cui, Yixiao; Han, Xutiange; An, Ran; Zhou, Gengxu; Komiyama, Makoto; Liang, Xingguo

doi:10.1039/c8ra02804d

Cited by 8 publications

(5 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The efficiency of the cyclisation reaction is sensitive to the length of the linear DNA precursor, and is inefficient if the nucleobase sequence leads to secondary structures. 25 , 26 In addition, the scale on which the cyclisation reaction can be carried out is limited by the cost of the enzyme. In principle these limitations could be circumvented by the use of chemical ligation ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

show abstract

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Such long complementary ends were believed to be necessary to make the T m of the corresponding duplexes (about 66–70 °C) comparable with (or slightly higher than) the ligation temperature. Accordingly, DNA fragments longer than 20 nt were ligated at 65 °C in Tth DNA ligase-based LCR, LDR, and other studies. − Only the cyclization of ssDNA was accomplished at 65 °C using an 18-nt-long splint (and thus 18 bp complementary portion) . However, unexpected findings in this paper showed that Tth DNA ligase has high ligation efficiency at 65–80 °C, even when the complementary portions of substrate DNAs are much shorter (7–10 nt).…”

Section: Discussionmentioning

confidence: 94%

“…31−36 Only the cyclization of ssDNA was accomplished at 65 °C using an 18nt-long splint (and thus 18 bp complementary portion). 37 However, unexpected findings in this paper showed that Tth DNA ligase has high ligation efficiency at 65−80 °C, even when the complementary portions of substrate DNAs are much shorter (7−10 nt). The "free ssDNA" ligation of Fss-γ 7 was successful even at 70 °C, which is much higher than the T m of the corresponding duplexes (around 39 °C, Table S4).…”

Section: ■ Discussionmentioning

confidence: 99%

Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures

et al. 2019

Self Cite

View full text Add to dashboard Cite

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7–10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45–70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.

show abstract

“…Recently, we reported that secondarily structured oligonucleotides can be successfully cyclized by using thermostable Taq DNA ligase at high temperatures (34). The ‘terminal hairpin strategy’ in this study is a significant extension in that secondary structures of DNA are successfully used as gate keeper to hold on intermolecular polymerization which is a significant problem for effective preparation of DNA rings.…”

Section: Discussionmentioning

confidence: 99%

“…The data were analyzed by Image Lab software to quantify the fluorescence emission from each band. The calculating methods of selectivity and yield were the same as our last papers (30,34). The conversion for the formation of DNA rings were calculated by the following equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}\begin{equation*}{\rm{Conversion}}\,\left( \% \right){\rm{ }} = {\rm{ }}(1 - \frac{R}{{P + C + R}}) \times 100,\end{equation*}\end{document}where C, P and R are the band intensities of circular single-stranded DNA ring, polymeric byproducts and remained single-stranded linear DNA (l-DNA), respectively.…”

Section: Methodsmentioning

confidence: 99%

OUP accepted manuscript

Cui

Han

et al. 2018

Nucleic Acids Research

View full text Add to dashboard Cite

When oligonucleotide bearing a hairpin near either its 3'- or 5'-end was treated with T4 DNA ligase, the intramolecular cyclization dominantly proceeded and its monomeric cyclic ring was obtained in extremely high selectivity. The selectivity was hardly dependent on the concentration of the oligonucleotide, and thus it could be added in one portion to the mixture at the beginning of the reaction. Without the hairpin, however, the formation of polymeric byproducts was dominant under the same conditions. Hairpin-bearing oligonucleotides primarily take the folded form, and the enzymatically reactive species (its open form) is minimal. As the result, the intermolecular reactions are efficiently suppressed due to both thermodynamic and kinetic factors. The 'terminal hairpin strategy' was applicable to large-scale preparation of a variety of DNA rings. The combination of this methodology with 'diluted buffer strategy', developed previously, is still more effective for the purpose. When large amount of l-DNA bearing a terminal hairpin (e.g. 40 μM) was treated in a diluted ligase buffer (0.1× buffer) with T4 DNA ligase, the DNA ring was prepared in 100% selectivity. Even at [l-DNA]0 = 100 μM in 0.1× buffer, the DNA ring was also obtained in pure form, simply by removing tiny quantity of linear byproducts by Exonuclease I.

show abstract

Cyclization of secondarily structured oligonucleotides to single-stranded rings by using Taq DNA ligase at high temperatures

Abstract: We have achieved the efficient preparation of single-stranded DNA rings from secondarily structured oligonucleotides.

Cited by 8 publications

References 40 publications

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

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

Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures

OUP accepted manuscript

Contact Info

Product

Resources

About