Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation

Shivalingam, Arun; Tyburn, Agnes E. S.; El‐Sagheer, Afaf H.; Brown, Tom

doi:10.1021/jacs.6b11530

Cited by 29 publications

(77 citation statements)

References 61 publications

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“…Within DNA, isolated amides can slightly increase duplex stability with target RNA 24 while consecutive amides were reported to have minimal effects. 25 Amide-modified ONs are stable in serum 24 and the backbone is well tolerated in replication, transcription 26 and RNAi 27 but its application in the context of RNase H activation has not been reported.…”

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Consecutive 5′- and 3′-amide linkages stabilise antisense oligonucleotides and elicit an efficient RNase H response

et al. 2020

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Antisense oligonucleotides are now entering the clinic for hard-totreat diseases. New chemical modifications are urgently required to enhance their drug-like properties. We combine amide coupling with standard oligonucleotide synthesis to assemble backbone chimera gapmers that trigger an efficient RNase H response while improving serum life time and cellular uptake.Antisense oligonucleotides (ASOs) are short (B20mer) chemically modified oligomers that bind to their complementary RNA targets to modulate gene expression at the mRNA level. 1,2 Thus, ASOs can target proteins that are considered undruggable through conventional approaches. 3 As such, they hold enormous promise for hard-to-treat diseases as evidenced by a number of recently approved oligonucleotide (ON)-based drugs. [4][5][6] Chemical modifications are essential to improve the serum stability and pharmacodynamic properties of ASOs, as unmodified ONs are rapidly digested by nucleases in vivo 7,8 and suffer from poor cellular uptake and tissue distribution. 9 Whilst there have been considerable advances to modify ONs either at the nucleobase, sugar or backbone, a set of distinct chemical modifications to confer ideal drug-like properties has not yet been achieved. 10 Commonly used ribose modifications include 2 0 -F, 2 0 -OMe, 2 0 -O-(2-methoxyethyl) and locked nucleic acids, all of which have been shown to improve target affinity and serum stability. 11 The most commonly used phosphodiester (PO) mimic is the phosphorothioate (PS) linkage which is compatible with ribonuclease H (RNase H) activation, 12 a mechanism resulting in degradation of an mRNA upon formation of an ASO:mRNA heteroduplex. PS linkages also improve metabolic stability 13 and enhance pharmacodynamic properties through interactions with plasma proteins. 8,14,15 However, unspecific protein binding can contribute to the toxic potential of PS-ASOs 16,17 and the PS linkage is P-chiral resulting in a mixture of diastereomers (more than half a million isomers in Mipomersen 12,18 ). Moreover, inefficient cellular uptake remains a major challenge for ASO therapeutics. Therefore, the investigation of other artificial backbone linkages is urgently needed.Charge-neutral backbone modifications represent an interesting class of PO mimics. Among those, (thio)phosphonoacetate esters, 19 phosphotriesters 20 and alkyl phosphonates 21 can enhance cellular uptake by eliminating the PO negative charges. Moreover, incorporation of a single methylphosphonate can eliminate hepatotoxicity of PS-ASOs. 22 Phosphorodiamidate morpholino oligomers (PMOs) combine backbone and sugar modifications and enhance delivery through interactions with scavenger receptors, 23 but are not compatible with standard ON synthesis. All aforementioned PO mimics also suffer from increased steric complexity due to their P-chiral linkages.The absence of a chiral centre and the well-established solidphase peptide synthesis methods make the amide internucleoside linkage 24 (AM, Fig. 1A) a promising candidate for backbone surrogates. Within...

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Consecutive 5′- and 3′-amide linkages stabilise antisense oligonucleotides and elicit an efficient RNase H response

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“…The TCS ligation product contained a triazole linkage called Tz3 ( 22 ), which is the same linkage structure as TL DNA ( 19 ). A recent study demonstrated that Tz3-containing DNA serves as a better PCR substrate than DNA containing other types of triazole linking ( 22 ). However, primer extension assays performed in the study showed that the efficiency of read-through over Tz3 was low for both Taq DNA polymerase and Phusion DNA polymerase ( 22 ).…”

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confidence: 99%

“…Interestingly, the heteroduplex between natural DNA and analog DNAs, in which every phosphodiester bond is replaced with a five-bond triazole linkage, is extremely stable, in contrast to less-stable duplexes containing other analogs ( 19 ), demonstrating the impact of DNA backbone-periodicity on physical characteristics of DNA. Consistent with this notion, DNA analogs with five-bond triazole linking can be used as a primer for reverse transcription of mRNA ( 21 ), and have recently been shown to serve as a more efficient template for DNA polymerase than other DNA analogs ( 22 ). Of note, DNA containing a single triazole linkage in the middle of its sequence forms a less stable duplex with natural DNA than fully triazole-linked DNA does ( 22 ).…”

Section: Introductionmentioning

confidence: 92%

“…Consistent with this notion, DNA analogs with five-bond triazole linking can be used as a primer for reverse transcription of mRNA ( 21 ), and have recently been shown to serve as a more efficient template for DNA polymerase than other DNA analogs ( 22 ). Of note, DNA containing a single triazole linkage in the middle of its sequence forms a less stable duplex with natural DNA than fully triazole-linked DNA does ( 22 ).…”

Section: Introductionmentioning

confidence: 92%

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Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA

Miura

Fujino

Kogashi

et al. 2018

Nucleic Acids Research

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Next-generation sequencing of single-stranded DNA (ssDNA) is attracting increased attention from a wide variety of research fields. Accordingly, various methods are actively being tested for the efficient adaptor-tagging of ssDNA. We conceived a novel chemo-enzymatic method termed terminal deoxynucleotidyl transferase (TdT)-assisted, copper-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated ssDNA ligation (TCS ligation). In this method, TdT is used to incorporate a single 3′-azide-modified dideoxyribonucleotide onto the 3′-end of target ssDNA, followed by CuAAC-mediated click ligation of the azide-incorporated 3′-end to a 5′-ethynylated synthetic adaptor. This report presents the first proof-of-principle application of TCS ligation with its use in the preparation of a next-generation sequencing library.

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Squaramides and Ureas: A Flexible Approach to Polymerase‐Compatible Nucleic Acid Assembly

et al. 2020

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Joining oligonucleotides together (ligation) is a powerful means of retrieving information from the nanoscale. To recover this information, the linkages created must be compatible with polymerases. However, enzymatic ligation is restrictive and current chemical ligation methods lack flexibility. Herein, a versatile ligation platform based on the formation of urea and squaramide artificial backbones from minimally modified 3′‐ and 5′‐amino oligonucleotides is described. One‐pot ligation gives a urea linkage with excellent read‐through speed, or a squaramide linkage that is read‐through under selective conditions. The squaramide linkage can be broken and reformed on demand, while stable pre‐activated precursor oligonucleotides expand the scope of the ligation reaction to reagent‐free, mild conditions. The utility of our system is demonstrated by replacing the enzymatically biased RNA‐to‐DNA reverse transcription step of RT‐qPCR with a rapid nucleic‐acid‐template‐dependent DNA chemical ligation system, that allows direct RNA detection.

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Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation

Cited by 29 publications

References 61 publications

Consecutive 5′- and 3′-amide linkages stabilise antisense oligonucleotides and elicit an efficient RNase H response

Consecutive 5′- and 3′-amide linkages stabilise antisense oligonucleotides and elicit an efficient RNase H response

Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA

Squaramides and Ureas: A Flexible Approach to Polymerase‐Compatible Nucleic Acid Assembly

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