Selective Nucleic Acid Capture with Shielded Covalent Probes

Abstract

Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo).

Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression

“…To further characterize the binding of Pu27 to its target sequence and determine the mechanism by which this binding could alter c-MYC transcription, we took advantage of a UV activated cross-linker to covalently bind the DNA target sequence and the Pu27 encoding oligonucleotide [ 45 ]. The cross-linker [3-cyanovinylcarbazole ( CNV K = K)] is a photoactive nucleoside analogue originally described by Yoshimura et.al.…”

“…Pierce] that was incorporated at the position 1 = PuK1 or position 12 = PuK12 of the Pu27 nucleotide sequence (PuK1 and PuK12 were synthetized by IDT). The cross-linking protocol follows the methodology described by Dr. Pierce [ 45 ] with some modifications. The target sequences (1.8μM) were mixed or not with Pu27, PuK1 or PuK12 (3μM) in SSC buffer (150mM NaCl, 15mM trisodium citrate, pH7.0) and annealed for 5min at 95°C in heating block.…”

A branch-migration based fluorescent probe for straightforward, sensitive and specific discrimination of DNA mutations

“…In a more recent work by Zhang et al. , the theoretical DF for C:C mismatch was calculated to be >10 000 (23). However, the reported experimental results were far below this value.…”

Nucleic Acid Nanostructures for Chemical and Biological Sensing

“…Single nucleotide polymorphisms (SNPs) are the most common genetic variation in the human genome . DNA nanostructrues have recently been developed for highly specific detection of SNPs . Chen et al reported one such example based on conditionally fluorescent molecular probes ( Figure a).…”