Split DNA Enzyme for Visual Single Nucleotide Polymorphism Typing

Abstract

Single nucleotide polymorphisms (SNPs) are the most abundant forms of genetic variations in the human genome. Large-scale sequence analysis is needed for a population-based genetic risk assessment and diagnostic tests once a mutation has been identified. However, most of the methods for SNP screening require enzymatic manipulations such as endonuclease digestion, ligation or primer extension, and often separation of the resultant products. 1 These labor intensive and time-consuming procedures are some of the biggest impediments to moving SNP typing techniques to point-of-care settings, which require straightforward, inexpensive, and disposable detection formats. Toward the fulfilling of these requirements a probe for visual SNP detection was developed in this study.Binary probes for fluorimetric analysis of single nucleotide substitutions were developed earlier. 2 The probes demonstrate improved selectivity in comparison with conventional hybridization-based approaches, since the two parts of the probes form relatively short (7-10 nucleotide) duplexes with target sequences. These short hybrids are extremely sensitive to single nucleotide substitutions at room temperature and generate a high fluorescent signal only in the presence of the fully complementary targets. Binary probes do not require precise temperature control for SNP typing. 2d,e However, a fluorimeter is required for signal registration. To avoid the need for instruments for both SNP typing and signal readout, a binary probe that generates a visual output after hybridization to the target was designed in this study based on a peroxidase-like DNA enzyme.A hemin binding DNA aptamer ( Figure 1A) was obtained earlier by in vitro selection. 3 It was shown that in the presence of hemin it forms a guanine quartet, which demonstrates hydrogen peroxidase-like activity ~250 times greater than hemin alone. 4 This DNA enzyme was used for the design of allosterically regulated sensors for nucleic acids, AMP, and lysozyme that allow colorimetric or luminescent readouts. 5 To construct a binary probe, the sequence of the peroxidase-like DNA enzyme ( Figure 1A) was split into two halves, the deoxycytidine was removed, and the analyte binding arms were added to each half via triethylene glycol linkers ( Figure 1B). In the absence of nucleic acid analyte strands α and β exist predominantly in the dissociated form (at certain concentrations and buffer conditions), while assembling in a Gquadruplex structure and acquiring peroxidase activity when hybridized to the adjacent positions of the analyte ( Figure 1C). The active peroxidase catalyzes the oxidation of a colorless substrate to a colored product, which can be detected both visually and spectrophotometrically. DNA that is a part of the coding sequence for microtubule associated protein tau (MAPT) was chosen as a model analyte for this study. Hyplotype H1c carrying SNP rs242557 G to A

Toward Non-Enzymatic Ultrasensitive Identification of Single Nucleotide Polymorphisms by Optical Methods

“…As illustrated below, several non-enzymatic techniques provide advantages of improved repeatability, decreased cost and robustness. Based on the chemical nature of the utilized probe, these techniques can be divided into three groups: methods applying nano-and magnetic-particles , (1) aptamer-and protein-based assays [47][48][49][50][51][52], (2) and other techniques utilizing bright chemically modified nucleic acid analogues [53][54][55][56][57][58][59][60]…”

Beyond allostery: Catalytic regulation of a deoxyribozyme through an entropy-driven DNA amplifier

“…Zhang and Winfree have shown that single-nucleotide mutations, insertions, and deletions in the catalyst strand can result in 10-to 300-fold reductions in its efficiency in catalyzing strand exchange [17]. These reductions in signal observed with single-nucleotide mismatches are comparable to other well-developed DNA sensors [26][27][28][31][32][33]. Consistent with these findings, we observed that when a single nucleotide was changed in the toehold region (Figure 3, inset), the mutant Trigger did not yield any peroxidase activity above background even at 150 nM concentration ( Figure 3, sample #10~12).…”

“…We also demonstrate that an entropy-driven catalytic DNA circuit can function as a generic signal amplification module that allows lower concentrations of input molecules to control the production of higher concentrations of deoxyribozymes, and thus exceed the limit of stoichiometric allosteric control. The system is qualitatively sensitive to mismatches in the input nucleic acid, suggesting that the combined entropy-driven strand exchange and allosteric deoxyribozyme circuit might facilitate the future development of enzyme-free, point-of-care diagnostic assay system [26][27][28].…”

Visual Detection ofrpoBMutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

“…In the detection system, the initial tool for SNP analysis is the molecular-beacon probe (19); however, the requirement for the instrument and the individually fluorescence-labeled probe is definitely costly. The peroxidase DNAzyme was selected due to its robustness, its sensitivity, and its relatively low cost (3,12,14,25,26,27,32). Here, we apply the 3:1 split DNAzyme to detect target DNA.…”