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

Abstract

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.

Artificial nucleic acid backbones and their applications in therapeutics, synthetic biology and biotechnology

“…A combinatorial approach for the discovery of splint-templated chemical ligations has been reported to identify DNA-compatible reactions to ligate terminally functionalised ONs [57]. Moreover, the generation of artificial backbone mimics has been shown for bridging 5 0 -S-phosphorothioester linkages (Ps) [58], PA [59][60][61][62], AM [61], urea [63], SQAM [63], TL1 [64] and TL3 [61]. Indeed, copper-catalysed azide-alkyne cycloaddition (CuAAC) to form TL3 was reported for the assembly of whole genes [12,65] and long RNA [9,10] from azide and alkyne modified shorter ONs.…”

“…Similarly, phosphoramidate backbones can be read by DNA and RNA polymerases [ 59 , 61 ], and translated by ribosomes [ 60 ], and introduction of PA-modified genes can lead to the expression of their associated genetic information in bacteria [ 13 ]. Apart from gene synthesis, artificial backbones such as TL2, [ 31 ] urea [ 63 ] and SQAM [ 63 ] were reported as components of modified primers in PCR. In the case of SQAM, in situ template assembly by target-templated SQAM formation was utilised for RNA detection [ 63 ].…”

“…Apart from gene synthesis, artificial backbones such as TL2, [ 31 ] urea [ 63 ] and SQAM [ 63 ] were reported as components of modified primers in PCR. In the case of SQAM, in situ template assembly by target-templated SQAM formation was utilised for RNA detection [ 63 ]. Other examples include the construction of a functional hammerhead ribozyme [ 9 ] or bioactive single guide RNAs (sgRNAs) for gene editing using a split-and-click strategy to form TL3 (CRISPR–Cas9 activation, Figure 2D ) [ 10 ].…”

A New 1,5-Disubstituted Triazole DNA Backbone Mimic with Enhanced Polymerase Compatibility

“…In general, replication-competent artificial DNA backbones can be used for gene synthesis, , sequencing, or nucleic acid detection . A detailed study revealed several molecular characteristics of artificial backbones that are required for compatibility with DNA polymerases during replication .…”

“…A detailed study revealed several molecular characteristics of artificial backbones that are required for compatibility with DNA polymerases during replication . Such polymerase compatible artificial backbones comprise 5′- S -phosphorothioesters, phosphorothioates, disulfides, boranophosphates, phosphoramidates, ,, amides, , ureas, squaramides, and triazoles. ,,,− Among these, the triazole linkage (TL) represents a powerful and versatile chemical moiety that can be readily formed by the Cu I -catalyzed azide–alkyne cycloaddition (CuAAC) reaction, resulting in 1,4-disubstituted 1,2,3-triazoles . Examples include TL 1 , TL 4 , TL 5 , and TL 6 (Figure ).…”