High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays

Abstract

DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5′→3′ synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5′ end and with free 3′ hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3′→5′ synthesis.

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase

“… 42 We have previously measured the coupling efficiency of most non-RNA phosphoramidites for light-directed synthesis to be ∼99.9%, including DMTr-dT in its role as capping agent; G being the exception at 97–98%. 41 , 43 − 46 After synthesis and deprotection, the surface-bound oligonucleotides serve as initiator sequences for dT homopolymer extension with TdT polymerase. The efficiency of polymerization was evaluated by hybridization to the extension product.…”

“…Our results, which encompass the enzymatic extension of all 1364 possible sequence permutations of mono- up to pentamers for each of several nucleic acid chemistries, are based on the use of nucleic acid photolithography for the massively parallel synthesis of initiator strands on a common surface. 40 We have recently expanded the toolbox of light-sensitive DNA phosphoramidites used in photolithographic synthesis beyond the standard 3′ → 5′ (“forward”) direction, 41 and we are using this chemical diversity to investigate the activity of the TdT polymerase on a variety of initiators, from DNA oligonucleotides with accessible 3′ or 5′-OH groups (from “reverse” or “forward” DNA synthesis, respectively), to RNA-like nucleic acids with 2′O-methyl RNA (2′OMe-RNA), to mirror-image ( l -)DNA primer strands with a terminal 5′-OH. We also examined the potential of non-nucleosidic molecules to act as initiators for TdT-mediated enzymatic synthesis by preparing polymers of hexaethylene glycol (HEG) linkers.…”

Multi-level patterning nucleic acid photolithography

“…Traditionally, nanobiotechnology has relied almost exclusively on DNA, but inclusion of other building blocks in the synthesis toolbox results in greatly expanded functionality with modest additional synthesis complexity due to the shared coupling chemistry. The additional functionality originates from greater base-pairing options, such as that introduced by alphabet extensions (dNaM/d5SICS 40 or Hachimoji DNA 41 ), from manipulation of duplex stability through nucleic acid sugar modifications (LNA 42 or UNA 43 ), from RNA 44 and other natural but non-canonical nucleic acids, from reverse synthesis (5′→3′) 45 and structural modifiers including spacers and symmetric or asymmetric branching phosphoramidites, and from other engineered constructs with heterogeneous functionalities, such as mirror-image nucleic acids 46 , fluorescent phosphoramidites 47 , and binary-encoding phosphoramidites 48 . The use of photolithography in this first level of patterning allows near arbitrary synthesis flexibility and monomer choice, with only near-UV-absorbing modifications, such as pyrene and phenanthrene 49 possibly disallowed due to absorption spectrum overlap with the photolabile protecting groups.…”

How to copy and paste DNA microarrays

“…Since we make use of spPCR, a perfect unidirectional orientation of the DNA molecules with a 5′ (surface) to 3′ direction can be ensured. Other photolithographic methods are also available with which such an orientation can be achieved, although a 5′-3′ orientation is not standard 19,20 . The DNA density on the spot is mainly limited by the number of primers attached to the surface, the size of the polymerase and the number of spPCR cycles used.…”