DNA as a Recyclable Natural Polymer

Abstract

Nature has the ability of circularly re-using its components to produce the molecules and materials it needs. An example is the ability of most living organisms of digesting proteins they feed off into amino acids and then using such amino acids in the ribosomal synthesis of new proteins. Recently, it has been shown that such recycling of proteins can be reproduced outside living organisms. The key proteins' feature that allows for this type of recycling is their being sequence-defined polymers. Arguably, nature's most famous sequencedefined polymer is DNA. Here it is shown that it is possible, starting from sheared calf-DNA, to obtain all four nucleotides as monophosphate-nucleotides (dNMPs). These dNMPs are phosphorylated in a one-pot, multi-enzymes, phosphorylation reaction to generate triphosphate-nucleotides (dNTPs). Finally, dNTPs so achieved (with a global yield of ∼60%) are used as reagents for PCR (polymerase chain reaction) to produce target DNA strands, and for the diagnosis of targeted DNA by quantitative PCR (qPCR). This approach is an efficient, convenient, and environmentally friendly way to produce dNTPs and DNA through recycling according to the paradigm of a circular economy.

Recycling of Polymerase Chain Reaction (PCR) kits

“…The phosphorylation yield was relatively good (on average 93 ± 2%, Figure S2d), which is similar to our reported phosphorylation yield of dNMPs. 5 In the re-generated PCR kit, dNDPs residue was 5.94  0.04%, ATP/ADP residue was 12.23  0.02%, and dNTPs content was 81.81  0.02% (Figure 1c). We notice that a fraction of the dNTPs was not fully hydrolyzed and retained as 3mer-6mer oligonucleotides in the recycled PCR kit (Figure S3).…”

“…Next, the DNA fragments were hydrolyzed by a mixture of exonucleases to release dNMPs, as in the established method. 5 After the enzymatical depolymerization process, the amount of dNMPs in the recycled PCR kit was highly increased (Figure S2a, line 3). Further, the released dNMPs were phosphorylated to generate dNTPs (Scheme 1, step 3), as in the established one-pot phosphorylation method.…”

“…Further, the released dNMPs were phosphorylated to generate dNTPs (Scheme 1, step 3), as in the established one-pot phosphorylation method. 5 Briefly, the phosphorylation reaction was catalyzed by T4 NMP Kinase and E. coli S30 extract (rich in phosphotransferase), with acetyl-phosphate/ATP as dual-phosphate-donors. After phosphorylation, the dNMPs peaks almost disappeared, and the intensity of dNTPs peaks increased (Figure S2a, line 4).…”

“…2,3 Our group has recently reported a recycling approach for sequence-defined polymers (SDP) that we call NaCRe (nature-inspired circular-recycling system), we first implemented it onto proteins 4 and more recently onto DNA. 5 Briefly, NaCRe's concept is that in theory a random mixture of SDPs can be depolymerized into the constituent monomers that in turn are re-assembled in a new polymers that can potentially differ from any of the starting ones. This has been shown for proteins (SDPs made of amino acids) and for DNA SDPs made of nucleotides.…”

“…17 To the best of our knowledge, the de-polymerization and recycling of DNA materials had not been reported before our recent publication. 5 A PCR kit is basically a DNA polymerization and amplification tool. Briefly, PCR is a polymerase catalyzed, templated, temperature-controlled, precision DNA polymerization process.…”