8/15/2023 0 Comments Klenow fragment ligation![]() ![]() Of the polymerases studied, we observed that Klenow fragment was the most efficient at reading through the triazole linkage, with ca. As a positive control, we also conducted primer extension reactions using the template 7 that contained only natural phosphodiester linkages. Several DNA polymerases were examined and the ratio of 5 to 6 was used as an indicator of the translocation efficiency through the triazole linkage. The “stalled” product 5 and the full length product 6 were easily resolved by HPLC and identified on the basis of their molecular weights. ![]() ![]() With oligos 3 and 4 in hand, we set out to assess various DNA polymerases for their ability to read through the triazole linkage. The advantage of this approach was the removal of the triazole-linked template strand, which simplified subsequent LCMS analysis of the primer extension products. ![]() Therefore, we designed the scheme shown in Fig. We could not rule out incomplete denaturation as the source of fluorescent signal in high MW bands. Preliminary results using denaturing gel electrophoresis and a fluorescently labeled primer gave inconsistent results. We sought to quantify the read-through yield and determine the proportion of the “stalled” product, if present. An inefficient read-through process would lead to over-weighting of the few “lucky” sequences that were successfully extended and/or amplified early in the PCR. For library encoding purposes, the most efficient possible read-through is required, as the sampling depth of the selection output will be directly dependent on the read-through efficiency. Previous work had qualitatively shown that triazole-containing oligonucleotides could be amplified by PCR and that a triazole-containing plasmid could be translated in living bacteria 17, 18 or human cells 19 direct quantitation of read-through efficiency was not reported. Chemical ligation using CuAAC might offer more flexibility in terms of ligation conditions and sequence design, since CuAAC is a famously robust reaction and sticky ends would not be needed. Based on these results and our prior experience with Cu-catalyzed alkyne-azide cycloaddition (CuAAC) of oligos 20, we wondered whether a readable chemical ligation strategy might offer some advantages over the current enzymatic methods. Recent reports from Brown, El-Sagheer and Tavassolli have demonstrated that oligonucleotides containing a triazole linkage in place of a phosphodiester are competent substrates for PCR and thus could provide a “readable” encoding sequence 16, 17, 18, 19. Since that time, the various groups operating DNA-recorded technology have reported enzymatic construction of the DNA both ligase- and polymerase-based methods have been described 14, 15. They showed how iterative ligation could be employed to create an oligonucleotide that encodes successive combinatorial steps. In 1995, however, Kinoshita and Nishigaki introduced the concept of the enzymatic ligation of encoding oligonucleotide “tags” 13. In the original reports of DNA-encoding from the early 1990’s, the encoding DNA was built using solid-phase oligonucleotide synthesis with phoshoramidite building blocks 11, 12. Encoding strategies can be divided into two categories: DNA-directed methods, in which synthetic chemistry is programmed by DNA complementarity and DNA-recording methods, in which the encoding oligonucleotide is built during the library synthesis, so that the synthetic history of each molecule is recorded in its DNA strand.ĭNA-recording approaches require the iterative construction of both the chemical library members and the encoding oligonucleotide. Due to advances in the throughput of DNA-sequencing, DNA-encoding allows the interrogation of vast numbers of compounds, exceeding by orders of magnitudes the capacity of traditional “one compound per well” screening approaches 5, 6, 7, 8, 9, 10. In the past decade, DNA-encoding of small molecule libraries has emerged as an attractive strategy for the discovery of novel ligands to biological targets 1, 2, 3, 4. ![]()
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