Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups

Abstract

Nucleoside triphosphates having a 3′-ONH2 blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3′-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3′-ONH2 group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3′-ONH2 blocking group in “next generation sequencing”.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…Some of these practical applications, including the TUNEL assay and the functional labeling of DNA oligonucleotides, are already quite mature with kits available from commercial suppliers. On the other hand, the development of DNA synthesizers based on the TdT‐mediated polymerization of reversible terminators is not quite as mature as other applications but is a rapidly developing area with a lot of industrial potential . The diversification of the chemistry of nucleotides as well as the identification of other template‐independent polymerases such as Pol θ will certainly contribute to further broaden the realm of possible applications.…”

“…Another promising 3′‐ O ‐blocking group is the 3′‐ONH 2 unit ( 11 in Scheme ). This blocking group is of interest because of its small size (comparable to the size of a OCH 3 group), ease of removal (with a simple sodium nitrite treatment), and good recognition by DNA polymerases . Lastly, the 3′‐ O ‐azidomethyl and 3′‐ O ‐allyl ( 12 and 13 in Scheme , respectively) moieties have also been proposed as transient protecting groups for the development of potent reversible terminators …”

“…This blocking group is of interest because of its small size (comparable to the size of aO CH 3 group), ease of removal (with as imple sodium nitrite treatment), and good recognition by DNA polymerases. [110] Lastly, the 3'-O-azidomethyl [111] and 3'-O-allyl [112] (12 and 13 in Scheme 1, respectively)m oietiesh ave also been proposed as transientp rotecting groups for the development of potent reversible terminators. [104] In template-free enzymatic DNA synthesis, it is still ab ig challenge to achieveq uantitative coupling efficiencies by using 3'-O-blockedd NTPs.Recently,anew way for the de novo DNA synthesis has been described, and it makes use of aT dT-dNTP conjugate.…”

Rapid incorporation kinetics and improved fidelity of a novel class of 3′-OH unblocked reversible terminators

“…Eliminating TTP contamination from the 3′- O -amino-TTP stock proved difficult as these nucleotides comigrate by RP–HPLC, consistent with that noted by Benner (44). As 3′- O -amino-TTP is incorporated by many commercially-available DNA polymerases (32), we devised a solution-based, kinetic mop-up assay using Therminator DNA polymerase by incubating the reaction at 65°C for 1 min. This is based on the premise that the incorporation rate of TTP is faster than that of 3′- O -amino-TTP and short incubation times would effectively remove much of the contaminating natural nucleotide.…”

Synthesis of O-Amino Sugars and Nucleosides

“…Various chemically modified oligonucleotides have also been reported, with an increasing number showing up as drug candidates for oligonucleotide therapies [6,7,8,9]. Among them, O -amino nucleosides have been prepared for the development of human UDP-GlcNAc 4-epimerase inhibitors [10], modified oligonucleotides [11] or for next generation sequencing [12]. N-O, N -oxyamide or N -alkoxycarbamate-linked oligonucleotides have also been synthesized as oligonucleotides mimics [13,14,15,16,17].…”