Structure–activity relationships of the ATP cofactor in ligase-catalysed oligonucleotide polymerisations

Yi, Lei; Hili, Ryan

doi:10.1039/c6ob02792j

Cited by 11 publications

(12 citation statements)

References 21 publications

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“…To this end, we developed Ligasecatalyzed OligOnucleotides PolymERization (LOOPER), which employs T4 DNA ligase to catalyze the DNA-templated polymerization of functionalized 5′-phosphorylated oligonucleotide fragments (Figure 3). [20][21][22][23][24][25] The functional group present on the oligonucleotide fragment is attached via the Hoogsteen face on one of the nucleobases within the oligonucleotide fragment (Figure 4). As the polymerization method employs codons, rather than single nucleotides, the theoretical maximum number of unique modifications that can be incorporated for a codon length n using a standard genetic code is 4 n .…”

Section: Ligase-catalyzed Oligonucleotide Polymerization (Looper)mentioning

confidence: 99%

Expanding the Chemical Diversity of DNA

Guo

Kong

et al. 2018

Synlett

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Nucleic acid polymers can be evolved to exhibit desired properties, including molecular recognition of a molecular target and catalysis of a specific reaction. These properties can be readily evolved despite the dearth of chemical diversity available to nucleic acid polymers, especially when compared to the rich chemical complexity of proteins. Expansion of nucleic acid chemical diversity has therefore been an important thrust for improving their properties for analytical and biomedical applications. Herein, we briefly describe the current state-of-the-art for the sequence-defined incorporation of modifications throughout an evolvable nucleic acid polymer. This includes contributions from our own lab, which have expanded the chemical diversity of nucleic acid polymers closer to the level observed in proteinogenic polymers.1 Introduction2 Polymerase-Catalyzed Synthesis of Modified Nucleic Acid Polymers3 Ligase-Catalyzed Oligonucleotide Polymerization (LOOPER)4 LOOPER with Small Modifications5 LOOPER with Large Modifications6 Evolution of Aptamers Derived from LOOPER Libraries7 Outlook

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Section: Ligase-catalyzed Oligonucleotide Polymerization (Looper)mentioning

confidence: 99%

Expanding the Chemical Diversity of DNA

Guo

Kong

et al. 2018

Synlett

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“…As such, recent efforts have been made to increase the selectivity of DNA ligases including T4 DNA ligase. [ 9,10,13–16 ] One strategy reported by Jang et al . utilized modified bases at the 5′‐phosphate end of a ligating probe strand to greatly enhance the selectivity of T4 DNA ligase for SNPs at the adjacent 3′‐hydroxy probe terminus.…”

Section: Introductionmentioning

confidence: 99%

“…[9][10][11][12] As such, recent efforts have been made to increase the selectivity of DNA ligases including T4 DNA ligase. [9,10,[13][14][15][16] One strategy reported by Jang et al utilized modified bases at the 5 0 -phosphate end of a ligating probe strand to greatly enhance the selectivity of T4 DNA ligase for SNPs at the adjacent 3 0 -hydroxy probe terminus. [13] Based on the authors' observations and supporting MD simulations, they proposed that a bulky non-hydrogen bonding modified nucleobase improved selectivity by increasing the distance between the template and the ligating strands.…”

Section: Introductionmentioning

confidence: 99%

Enhanced mismatch selectivity of T4 DNA ligase far above the probe: Target duplex dissociation temperature

et al. 2020

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T4 DNA ligase is a widely used ligase in many applications; yet in single nucleotide polymorphism analysis, it has been found generally lacking owing to its tendency to ligate mismatches quite efficiently. To address this lack of selectivity, we explored the effect of temperature on the selectivity of the ligase in discriminating single base pair mismatches at the 3′‐terminus of the ligating strand using short ligation probes (9‐mers). Remarkably, we observe outstanding selectivities when the assay temperature is increased to 7 °C to 13 °C above the dissociation temperature of the matched probe:target duplexes using commercially available enzyme at low concentration. Higher enzyme concentration shifts the temperature range to 13 °C to 19 °C above the probe:target dissociation temperatures. Finally, substituting the 5′‐phosphate terminus with an abasic nucleotide decreases the optimal temperature range to 7 °C to 10 °C above the matched probe:target duplex. We compare the temperature dependence of the T4 DNA ligase catalyzed ligation and a nonenzymatic ligation system to contrast the origin of their modes of selectivity. For the latter, temperatures above the probe:target duplex dissociation lead to lower ligation conversions even for the perfect matched system. This difference between the two ligation systems reveals the uniqueness of the T4 DNA ligase's ability to maintain excellent ligation yields for the matched system at elevated temperatures. Although our observations are consistent with previous mechanistic work on T4 DNA ligase, by mapping out the temperature dependence for different ligase concentrations and probe modifications, we identify simple strategies for introducing greater selectivity into SNP discrimination based on ligation yields.

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“…14 More recently, our lab has developed the Ligase-catalysed OligOnucleotide PolymERisation (LOOPER) to incorporate greater chemical diversity into nucleic acid polymers (Figure 1). [15][16][17][18][19][20] The method relies upon the DNA ligase-catalysed DNA-templated polymerisation of a library of short oligonucleotides, each equipped with a unique functional group. As the method employs codons, rather than single nucleotides, the number of modifications that can be incorporated throughout a nucleic acid polymer is dependent on the codon set size.…”

Section: Introductionmentioning

confidence: 99%