Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups

Abstract

A set of modified 2′-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

The Structure-Based Design of SARS-CoV-2 nsp14 Methyltransferase Ligands Yields Nanomolar Inhibitors

“…In the case of compound 8 bearing quinoline substituent, its analogue 11 with ethylene linker was synthesized via reduction of the ethynediyl bridge by catalytic hydrogenation on Pd/C. 25 Final deprotection of 7-substituted SAH analogues 4 – 11 under acidic conditions (TFA/water 9:1) afforded the target compounds 12 – 19 in very good to excellent yields (83–97%, Scheme 1 ).…”

Dependence of click-SELEX performance on the nature and average number of modified nucleotides

“…the replacement of the chemical modified EdU-residue in the DNA by EdU during the first extension cycle of the PCR reaction, which subsequently is used as template for the following PCR cycles. Although it has been shown previously, that fully and modified DNA templates can be amplified by PCR, 20,21 a reduction of the number of EdUs per strand was found to have no strong impact on the overall enrichment of binding species, although the occurrence of high copy number sequences, correlating with the number of unique sequences in the selected DNA populations, was found shifted to later selection cycles. Indeed, using an average of 5 EdUs per strand has become a standard in out lab for click-SELEX experiments.…”

Recent progress in non-native nucleic acid modifications

“…222,226 At these positions base modifications can often be incorporated using standard polymerases such as Vent (exo -) or KOD, including multiple consecutive incorporations. 227,228 Other sites such as position N4 of pyrimidines have been shown to accommodate modifications rather well and efficiently serve as substrates for polymerases. 229 Modifications can also be accommodated in the minor groove of DNA by altering dN*TPs at position 2 of purines without impairing contacts with polymerases necessary for enzymatic synthesis.…”