High-Selectivity Single-Nucleotide Variant Capture Technology Based on the DNA Reaction Network

Abstract

The extremely low abundance of circulating tumor DNA in blood samples has limited the development of liquid biopsy techniques for the early diagnosis of major diseases. In this study, we demonstrate a DRN-based screening technique, SCREEN, which achieves the specific capture and enrichment of low abundance SNV nucleic acid samples without selective amplification. The SCREEN technique achieved a 108-fold increase in the abundance of single-nucleotide variant (SNV) nucleic acids from highly homologous mixtures (from 0.01% to 1.08%) and has been shown to significantly increase the abundance of SNV nucleic acids from 0.1% to 51% further through two rounds of capture. As a highly effective pre-enrichment technique, SCREEN has demonstrated the ability to enhance NGS in detecting an ultralow abundance SNV nucleic acid powerfully and has high compatibility with existing molecular diagnostic methods.

Foldback-crRNA-Enhanced CRISPR/Cas13a System (FCECas13a) Enables Direct Detection of Ultrashort sncRNA

“…Although the oligonucleotide addition system can detect ultrashort sncRNAs, adding an oligonucleotide increases the complexity of the experiment, and the detection performance is not satisfactory. Similar to the method of head-to-tail connection of crRNA and tracrRNA in the CRISPR/Cas9 system to form sgRNA, we directly synthesized complete foldback crRNA architecture (Figure A; the head-to-tail of crRNA and the oligonucleotide). − The foldback crRNA reduces reagent consumption and solves the structural instability caused by the “annealing–denaturation” dynamics at the crRNA–oligonucleotide hybrid terminal. − Further, the FCECas13a system was used for detecting target miRNA720 derivatives with different lengths, and the detection performance was compared in detail with standard Cas13a systems and oligonucleotide addition systems (Figures B and S1). Unlike the oligonucleotide addition system, the chain length of the target RNA was the major factor affecting the stability of the crRNA–target RNA hybrid in the FCECas13a system.…”

“…A 25 μL reaction mixture contained the following reagents: 2.5 μL of 10x reaction buffer (100 mM tris-HCl, pH 8.0; 500 mM KCl; and 15 mM MgCl 2 ), 5 μL of 5 μM RNA reporter, 2.5 μL of 10 U/μL enzyme inhibitor, and 5 μL of 25 nM LwaCas13a nuclease. Then, 2.5 μL of 10 nM miRNA720 derivatives with different lengths (14,16,18,20,22, 24, 26, and 28 nt) and 5 μL of the 5 nM corresponding foldback crRNA sequence (42,40,38,36,34,32,30, and 28 nt, respectively) were added to the above reaction system. Finally, all reaction systems were incubated for 30−60 min at 25 °C in a real-time fluorescence quantitative PCR instrument, and fluorescence readings were measured every 30 s.…”

“…For the FCECas13a assay system of miRNA720, the foldback crRNA with 39 nt spacer sequences can be divided into three parts: the upstream 17 nt sequences used to recognize the 17 nt miRNA720, the middle 11 nt sequences used to extend the spacer region to 28 nt, and the downstream 11 nt was complementary to the middle 11 nt extended region by foldback of itself. Similarly, the length of the foldback crRNA (42,40,38,36,34,32,30, and 28 nt) changes as the length of the miRNA720 derivative (14,16,18,20,22,24,26, and 28 nt) changes. A 25 μL reaction mixture contained the following reagents: 2.5 μL of 10x reaction buffer (100 mM tris-HCl, pH 8.0; 500 mM KCl; and 15 mM MgCl 2 ), 5 μL of 5 μM RNA reporter, 2.5 μL of 10 U/μL enzyme inhibitor, and 5 μL of 25 nM LwaCas13a nuclease.…”

Simulation-Guided Rational Design of DNA Probe for Accurate Discrimination of Single-Nucleotide Variants Based on “Hill-Type” Cooperativity

“…It enables design of toehold-exchange probes with good performance for single-nucleotide variant analysis. Many researchers made efforts to develop such toehold-exchange probes, including a single-toehold probe, a double-stranded DNA probe, , competitive compositions, , a kinetic amplification probe, an exonuclease-aided circuitry, − and the SCREEN technique. , Enriching the toolbox of toehold-exchange probes with an alternative mechanism can improve the performance for single-nucleotide variant analysis. Presently, there are also several DNA probes with cooperative, dual-target response, , but these probes’ specificity may be mainly due to the sequestration method or the kinetic obstacle.…”