Tandem repeat sequences are abundant in the human genome and transcriptome, where mononucleotide repeats (MNRs), particularly poly(A) and poly(T/U) repeats, are the most common. While some MNRs are apparently non-functional, other MNR sequences have significant roles in molecular processes and disease. For example, poly(A) tails of mRNA molecules aid in translation and nuclear export, while also protecting mRNA from enzymatic degradation. Scientists take advantage of the poly(A) tails of mRNA in order to isolate and detect mRNA using complementary oligonucleotides. For example, fluorescently labeled oligo(T) DNA or peptide nucleic acid (PNA) probes are used to image mRNA localization in cells via fluorescence in situ hybridization (FISH). Unfortunately, the yield of high purity MNR oligonucleotides is often quite low due to synthesis and purification challenges that arise when the same base is inserted several times in a row using solid-phase methods.<br/><br/>The Bowman Laboratory recently developed a scalable approach using thiol-ene click reactions to efficiently polymerize monomers with pendant nucleobases to form MNR oligonucleotides called “click nucleic acids” (CNAs). In the current work, oligo(T) CNAs were synthesized and microscale thermophoresis was used to characterize the dissociation constants of CNA, PNA, and DNA binding to complementary oligonucleotides under conditions commonly used for FISH. For example, CNA and PNA, but not DNA, were able to hybridize in buffers containing up to 80% organic solvent (DMSO or formamide) with minimal dependence on ionic strength (Fig. 1). While the dissociation constants for CNA-DNA hybridization were generally an order of magnitude higher than those for PNA-DNA hybridization, depending on the conditions, the low cost and high efficiency of oligonucleotide synthesis via click-chemistry make CNA probes attractive for biosensing and imaging of MNR-containing DNA and RNA.

Click nucleic acids as low-cost detection probes for mononucleotide repeat sequences

2019 Fall National Meeting

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