Abstract
AbstractJoining 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.
Highlights
Oligonucleotides provide the ideal means of storing information at the molecular level
Ligation efficiency was dependent on pH and squarate methyl ester concentration
Our squaramide and urea ligation platforms expand the scope of polymerase-compatible nucleic acid assembly and crucially provide greater flexibility in this process than previously reported methods
Summary
Oligonucleotides provide the ideal means of storing information at the molecular level. All face potential drawbacks, including copper dependence (CuAAC), precursor handling (disulfide, PA, PS), slow ligation rates (PS), and poor read-through fidelity (disulfide) This leaves scope for artificial backbones that synergise all their advantageous properties, including excellent fidelity, reversibility (disulfide), and pre-activation (PA, PS). Related carbamate,[24,25,26] thiourea,[27] urea,[26] and squaramides[28] have been introduced into oligonucleotides by iterative coupling or as phosphoramidite dinucleotides, but neither their formation by templated ligation nor their read-through fidelity have been explored All of these backbones could be derived from unmodified or minimally modified oligonucleotides where the terminal 5’- or 3’-hydroxy groups are replaced by amino groups. The reagents (for example, 1,1’-carbonyldiimidazole (CDI) and squarate ester) used to generate these linkages become progressively less reactive upon each nucleophilic addition-elimination step,[29] potentially allowing oligonucleotides to be pre-activated prior to ligation with another strand
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