Flamme Marie, Hanlon Steven, Marzuoli Irene, Püntener Kurt, Sladojevich Filippo, Hollenstein Marcel
Institut Pasteur, Université de Paris Cité, CNRS UMR3523, Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, 28, rue du Docteur Roux, 75724 Paris Cedex 15, Paris, France.
Pharmaceutical Devision, Synthetic Molecules Technical Development, F. Hoffmann-La Roche Ltd, 4070, Basel, Switzerland.
Commun Chem. 2022 Jun 1;5(1):68. doi: 10.1038/s42004-022-00685-5.
Chemically modified oligonucleotides have advanced as important therapeutic tools as reflected by the recent advent of mRNA vaccines and the FDA-approval of various siRNA and antisense oligonucleotides. These sequences are typically accessed by solid-phase synthesis which despite numerous advantages is restricted to short sequences and displays a limited tolerance to functional groups. Controlled enzymatic synthesis is an emerging alternative synthetic methodology that circumvents the limitations of traditional solid-phase synthesis. So far, most approaches strived to improve controlled enzymatic synthesis of canonical DNA and no potential routes to access xenonucleic acids (XNAs) have been reported. In this context, we have investigated the possibility of using phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and locked nucleic acid (LNA) oligonucleotides. Phosphate is ubiquitously employed in natural systems and we demonstrate that this group displays most characteristics required for controlled enzymatic synthesis. We have devised robust synthetic pathways leading to these challenging compounds and we have discovered a hitherto unknown phosphatase activity of various DNA polymerases. These findings open up directions for the design of protected DNA and XNA nucleoside triphosphates for controlled enzymatic synthesis of chemically modified nucleic acids.
化学修饰的寡核苷酸已成为重要的治疗工具，近期mRNA疫苗的问世以及多种小干扰RNA（siRNA）和反义寡核苷酸获得美国食品药品监督管理局（FDA）批准就反映了这一点。这些序列通常通过固相合成获得，尽管固相合成有诸多优点，但仅限于短序列，并且对官能团的耐受性有限。可控酶促合成是一种新兴的替代合成方法，可规避传统固相合成的局限性。到目前为止，大多数方法致力于改进经典DNA的可控酶促合成，尚未报道获得异源核酸（XNA）的潜在途径。在此背景下，我们研究了使用磷酸作为瞬时保护基团进行DNA和锁核酸（LNA）寡核苷酸可控酶促合成的可能性。磷酸在天然系统中广泛存在，我们证明该基团具有可控酶促合成所需的大多数特性。我们设计了通向这些具有挑战性化合物的稳健合成途径，并且发现了多种DNA聚合酶迄今未知的磷酸酶活性。这些发现为设计用于化学修饰核酸可控酶促合成的受保护DNA和XNA核苷三磷酸开辟了方向。
