Towards the enzymatic synthesis of phosphorothioate containing LNA oligonucleotides

Flamme, Marie; Hanlon, Steven P.; Iding, Hans; Puentener, Kurt; Sladojevich, Filippo; Hollenstein, Marcel

doi:10.1016/j.bmcl.2021.128242

Cited by 25 publications

(40 citation statements)

References 46 publications

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“…The triphosphate of this Z base (dZTP) is compatible with enzymatic synthesis ( 42 , 81–83 ) and recently it was reported to completely alleviate the pH dependency of poly-pyrimidine TFOs. ( 42 ) Therefore, expansion of the SNI-PEX protocol to include dZTPs and artificial backbones—such as locked nucleic acids or phosphorothioate nucleotides ( 84 )—should unlock the potential of this AMN-TFO technology as a targeted therapy with increased cellular uptake and enhance stability against degradation in biological systems.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

McGorman

Fantoni

O'Carroll

et al. 2022

Nucleic Acids Research

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Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators—DPQ and DPPZ—and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.

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Section: Discussionmentioning

confidence: 99%

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

McGorman

Fantoni

O'Carroll

et al. 2022

Nucleic Acids Research

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“…10−15 Since extensive studies have shown that TdT inherently incorporates several nucleotides within 1 s and can elongate the synthetic DNA to several kilobases, 16−18 the superior synthesis length and speed are far beyond the reach of the commercially available phosphoramidite technology. 19,20 A wide variety of nucleotide analogues have been incorporated into DNA by TdT, which has enabled numerous applications, 21 such as 3′ end-labeling of DNA/RNA, 22−24 immobilization of DNA, 25,26 stabilization of DNA, 27,28 conjugation of DNA to protein, 29,30 and inspirational de novo synthesis of DNA. 31 The major challenge in the application of TdT for the programmed synthesis of artificial DNA lies in controlling the polymerization of user-defined nucleotides.…”

Section: ■ Introductionmentioning

confidence: 99%

“…A wide variety of nucleotide analogues have been incorporated into DNA by TdT, which has enabled numerous applications, such as 3′ end-labeling of DNA/RNA, − immobilization of DNA, , stabilization of DNA, , conjugation of DNA to protein, , and inspirational de novo synthesis of DNA . The major challenge in the application of TdT for the programmed synthesis of artificial DNA lies in controlling the polymerization of user-defined nucleotides .…”

Section: Introductionmentioning

confidence: 99%

Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase

et al. 2022

ACS Catal.

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Template-free enzymatic approaches are considered the most promising solution for next-generation artificial DNA synthesis. However, the development of these technologies has been hampered by the lack of efficient enzymes specialized for stepwise nucleotide addition. By combining evolutionary analysis, high-throughput mutagenesis scanning, and rational design, we identified a terminal deoxynucleotidyl transferase from Zonotrichia albicollis (ZaTdT) and reshaped its catalytic cavity to better accommodate 3′-ONH2-modified nucleotides. The catalytic activity of the engineered ZaTdT for 3′-ONH2-dNTPs is 3 orders of magnitude higher than that of the commonly used mammalian TdT. The engineered ZaTdT enables highly efficient single-nucleotide extension of the growing oligonucleotide chain with an average stepwise yield of 98.7%, which makes it practical for de novo enzymatic DNA synthesis.

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“…On the other hand, there are the enzymatic approaches based on in vitro transcription using T7 RNA polymerases[ 8 , 9 , 10 , 11 ] or modifications using transferases. [ 12 , 13 , 14 , 15 , 16 , 17 ] Enzymatic methods allow synthesis essentially without size limitation; preparation of RNAs comprising 400 nt has been reported.…”

mentioning

confidence: 99%

Solid‐Phase‐Supported Chemoenzymatic Synthesis of a Light‐Activatable tRNA Derivative

2021

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Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of lightcontrollable RNA nucleotides into the tRNA Met sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.

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Towards the enzymatic synthesis of phosphorothioate containing LNA oligonucleotides

Cited by 25 publications

References 46 publications

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications

Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase

Solid‐Phase‐Supported Chemoenzymatic Synthesis of a Light‐Activatable tRNA Derivative

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