Abstract
Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the RuAAC-catalyzed synthesis of a novel 1,5-disubstituted triazole (TL2) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL2 has superior replication kinetics to these analogues and is accurately replicated by polymerases. Derived structure–biocompatibility relationships show that linker length and the orientation of a hydrogen bond acceptor are critical and provide further guidance for the rational design of artificial biocompatible nucleic acid backbones.
Highlights
In search of the ideal nucleic acid triazole linkage, we recently developed a 1,5-disubstituted 1,2,3-triazole internucleoside linkage which was prepared by RuII-catalyzed azide−alkyne cycloaddition (RuAAC) (TL3; Figure 1).[38]
We directly compare the effect of RuAAC vs CuIcatalyzed azide−alkyne cycloaddition (CuAAC) to form TLs from the same precursors (TL2 vs Template 5 (TL5)), and we study the ability of oligonucleotides containing these TLs to form duplexes with a DNA or RNA target in comparison to reported triazoles TL1, TL3, TL4, and TL6.38 we test how efficiently polymerases are able to read through TLs 1−7 in replication templates
A profound understanding of the structure− biocompatibility relationship among different TLs is crucial for their successful application in biochemical and biological systems
Summary
The replacement of natural phosphodiester (PO) bonds in DNA or RNA by artificial internucleoside linkages can generate remarkable biomimetic oligonucleotides (ONs) with applications as therapeutics,[1] xenobiotic genetic polymers,[2−4] aptamers,[3,5−7] and synthetic genes.[8−10] Biological integrity and favorable biophysical properties are critical, and good hybridization properties, mismatch discrimination, and compatibility with certain enzymes (e.g., RNase H) play crucial roles for antisense oligonucleotides.[11−15] Other applications of backbone-modified, bioactive oligonucleotides include modified CRISPR-Cas[9] systems,[16] and compatibility with the cellular gene replication and expression machinery. In contrast to the previous primer extension experiment with Klenow fragment, replication through TL5 using hot-start Taq polymerase mainly resulted in truncated products from termination directly before or after the triazole (FL-replicons 5*; Figure 4A,B). Across the Klenow and hot-start Taq primer extension assays, TL2 and TL4 stand out as being the only TLs resulting in the clean formation of the expected full-length product without any detectable mutations or truncated fragments observed on gel or by MS. For 5-bond TL1, whose number of bonds is closest to the natural PO, inefficient read-through by hot-start Taq was observed confirming previous reports.[17,19] TL1 is mutagenic and results in single point deletions around the triazole when read by Klenow fragment or Taq polymerases.[19] The read-through efficiencies of 6-bond TLs 2−4 gave different results in the primer extension assay. The proposed molecular requirements agree with our previously described model, which was derived from read-through compatibility tests of a range of backbone linkages including amides, phosphoro(di)thioates, phosphoramidates, and squaramides.[18,19,24,25] It is important to emphasize that the observed read-through compatibilities are dependent on the polymerase and similar polymerase dependencies were reported for other PO mimics.[18,19]
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