Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection)

Abstract

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2–4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.

Rapid Synthesis of Propargyl‐γ‐Modified Peptide Nucleic Acid Monomers for Late‐Stage Functionalization of Oligomers

“…The same chemistry was performed independently with L-BocÀ SerÀ OH and D-BocÀ SerÀ OH, yielding respectively L-4 and D-4. Despite using the same conditions as previously reported by our group on a similar substrate ( t Bu in lieu of propargyl), [38] chiral HPLC analysis of the reaction products showed this transformation to be less robust with variability across different batches (92-98 % e.e.). Gratifyingly, a simple crystallization protocol of the secondary amines as a mandelic acid salt afforded enantiomerically pure L-4 and D-4 (> 99 % ee; Scheme 2).…”

“…[61] Application of this method enabled the preparation of serine modified backbone with > 99 % ee. [38] Concurrently, Ly and co-workers used a similar approach to obtain Fmocprotected PNA monomers from FmocÀ Lys(Alloc)À OH with 95-97 % ee. [62] The synthesis of the desired PNA backbone modified at the γ position with a propargyl methylene ether side chain commenced with BocÀ SerÀ OH (Scheme 2) which is inexpensive for either enantiomer (D or L).…”

“…These include enhancement of cell permeability, [24,27,[33][34][35] enabling transfection with neg-+ Authors have contributed equally to this work. atively charged PNA, [36] improving dsDNA invasion, [37,38] enabling gene editing, [22,39,40] improving miR inhibition, [41] biasing secondary structure, [42] empowering templated chemical reactions, [43,44] tagging molecules within combinatorial libraries, [45] aptamers embracing the dual nature of PNA (peptide and nucleic acids), [46] enabling hybridization chain reactions (HCR), [47] biosupramolecular engineering, [48 -50] induced-fit switching [51] probes and empowering bimodal hybridization. [52] During our study on PNA-based HCR, [47] we had found that the enhanced solubility imparted by a miniPEG side chain at the γ-position [21] was critical for the success of HCR and outperformed the simpler hydroxymethylene substituent (side chain of Ser).…”

Design and Synthesis of Artificial Nucleobases for Sequence‐Selective DNA Recognition within the Major Groove

“…[27] Artificial nucleobases have recently also be incorporated into a pseudo-complementary G : C base pair mixed sequence dsDNA invasion and were applied in diagnostics. [28] Nevertheless, artificial oligonuclotides have been exploited in other Scheme 1. Examples of previously reported artificial nucleobases recognizing the base pairs of DNA via hydrogen bonds.…”

“…Artificial nucleobases have recently also be incorporated into a pseudo‐complementary G : C base pair mixed sequence dsDNA invasion and were applied in diagnostics [28] . Nevertheless, artificial oligonuclotides have been exploited in other fields due to their high fidelity in hybridization, e. g. as a powerful platform to program assemblies and networks with logic‐gated responses [29] …”