Gene assemblyviaone-pot chemical ligation of DNA promoted by DNA nanostructures

Abstract

A gene was obtained from 14 oligonucleotides self-assembled and chemically ligated in a DNA nanostructure.

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

“…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.T hrough the introduction of commercially available 5'-a nd 3'-amino groups,o ne-pot ligation can generate two different linkages:1)aurea linkage that multiple polymerases can read-through with excellent speed, or 2) as quaramide linkage whose formation can be reversed and offers accurate read-through under selective conditions.For the latter, the linkage can also be formed from stable pre-activated intermediates in mild buffered conditions in the absence of small-molecule reagents.T he diversity of this platform lends itself to many applications including DNA-encoded libraries, [2] aptamer proximity-ligation [39] and DNAnano-construct assembly. [40] Here we have demonstrated its use in RNAdetection, where it performs comparably to RT-qPCR while offering advantages,such as removing RNA polymerase bias, [21][22][23] faster speed of execution, and greater inherent target specificity.…”

“…For the latter, the linkage can also be formed from stable pre‐activated intermediates in mild buffered conditions in the absence of small‐molecule reagents. The diversity of this platform lends itself to many applications including DNA‐encoded libraries, aptamer proximity‐ligation and DNA nano‐construct assembly . Here we have demonstrated its use in RNA detection, where it performs comparably to RT‐qPCR while offering advantages, such as removing RNA polymerase bias, faster speed of execution, and greater inherent target specificity.…”

Chemistries for DNA Nanotechnology

“…Using a similar approach, the same group used 6helix bundle DNA nanostructures functionalized with azides and alkynes to direct the synthesis of a 762 nt gene from 14 oligonucleotides. 293 Synthesis of a smaller 365 nt gene from splint oligonucleotides by templated CuAAC had been demonstrated earlier by Kukwikila et al 294 Very recently, Dietz's lab described a platform for crosslinking of DNA origami by insertion of an additional pair of thymines at specific positions in DNA staple strands, thus enabling thymine dimerization upon exposure to 310 nm light (Figure 25). 295 These cross-linked structures remained stable in buffers containing 5 mM Mg 2+ at a wide range of temperatures with only minor degradation observed even at 90 °C, and the cross-linked structures also showed increased stability toward certain nucleases compared to the native structures.…”

“…When the number of alkyne and azide modifications was increased, the DNA structures showed improved stability toward heating as well as exonucleases (Figure C). Using a similar approach, the same group used 6-helix bundle DNA nanostructures functionalized with azides and alkynes to direct the synthesis of a 762 nt gene from 14 oligonucleotides . Synthesis of a smaller 365 nt gene from splint oligonucleotides by templated CuAAC had been demonstrated earlier by Kukwikila et al…”

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

“…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. This approach enables the precise, site-specific introduction of artificial backbones and other modifications, but is limited by the compatibility of the ligation reaction with terminally modified ONs under aqueous conditions.…”