Dmitry M. Kolpashchikov

Binary Probes for Nucleic Acid Analysis

Over the last four decades nucleic acid hybridization techniques have been widely used for the detection of specific nucleic acid sequences. In this approach a 15-nucleotide or longer DNA or RNA strand (the probe) forms complementary duplexes with the analyzed nucleic acid (analyte). Since the development of the first hybridization-based procedures by Hall and Spiegeleman1 and by Bolton and McCarty,2 oligoand polynucleotide probes have become routinely used as laboratory tools for nucleic acid analysis. The examples of such techniques include Southern3 and Northern4 blots, fluorescent in situ hybridization,5 and DNA microarrays.6 Furthermore, the introduction of real-time detection approaches, such as molecular beacon (MB) probes,7,8 has enabled fast assays, in which the fluorescence change is detected immediately after probe hybridization, thus avoiding the need to separate the probe-analyte hybrid from the excess of the unbound probe. However, low selectivity of the probe-analyte hybridization creates complications in the analysis of single-base differences between two polynucleotides. Indeed, a 15-nucleotide-long probe has a similar affinity to a fully complementary analyte and to an analyte containing a single noncomplementary base.9

Split DNA Enzyme for Visual Single Nucleotide Polymorphism Typing

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 pointof-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 hybridizationbased 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 R and â exist predominantly in the dissociated form (at certain concentrations and buffer conditions), while assembling in a G-quadruplex 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 substitution at the MAPT locus was shown to be associated with the risk of Alzheimer’s disease.6 Therefore, the analyte binding arms of the probe were tailored to recognize the major allele rs242557-G (Figure 1B,C). Figure 2 demonstrates the change of light absorption of the solution containing binary DNA peroxidase probe when 3-3′diaminobenzidine tetrahydrochloride (DAB) was used as an oxidizable substrate. The solution turned brown in the presence of 1 μM Figure 1. Design of the binary DNA peroxidase for SNP analysis: A: parent peroxidase-like DNA enzyme; B: binary DNA peroxidase probe; and C: the probe forms active peroxidase upon hybridizing to the abutting positions of the analyte. The enzyme catalyzes oxidation of a colorless substrate to colored products. The triethylene glycol linkers are shown as dashed lines in panel C. The SNP site in the analyte sequence is underlined.

A binary deoxyribozyme for nucleic acid analysis.

Simple, sequence-specific, and sensitive methods for DNA/RNA analysis are required for the rapid diagnosis of infection and genetic diseases, genome study, mRNA monitoring in living cells as well as environmental and forensic applications. The aim of this work is to introduce a new approach for the design of a highly selective probe for nucleic-acid detection based on deoxyribozyme molecules. The formation of at least 15–20 nucleotide-long hybrids between probe and analyte is required to uniquely define a specific fragment in a nucleic acid mixture of the genome size. Hybrids of such length are too stable to be sensitive to base mispairing since a single mismatch unit results in a relatively small energetic penalty.[1] Conventional techniques that use buffers with low ionic strength, denaturing agents, or elevated temperatures do not always lead to the desirable selectivity.[1, 2] One approach to improve selectivity of nucleicacid hybridization was realized with conformationally constrained probes, such as molecular beacons (MBs).[3, 4] MBs are oligo-eoxyribonucleotide hairpins with a fluorophore and quencher conjugated to the opposite ends of the oligomer. Binding to complementary nucleic acids switches MBs to the elongated conformation and increases their fluorescence. MBs distinguish mismatches over a wider temperature range than unconstrained probes do, because the stem–loop structure stabilizes the probe–analyte dissociated state.[4] Alternatively, the specificity of nucleic-acid recognition can be increased by splitting the probe into two halves.[5] Binary probes are more selective than conventional probes because each relatively short hybrid (7–10 nucleotides) is extremely sensitive to single-base mispairings. Here, a binary probe based on a deoxyribozyme that contains structural constrains is designed for the recognition of single-base substitutions in 20-mer DNA analytes, at room temperature. Deoxyribozymes, or DNA enzymes, are catalytic oligodeoxyribonucleotides derived by in vitro selection.[6] The advantages offered by catalytic DNAs include high chemical stability, low cost for synthesis, biocompatibility, and ease of structural prediction and modification. This relatively new class of catalytic molecules has been considered as a promising biochemical tool for nucleic-acid detection.[7] One particular attraction of DNA enzyme-based probes is their potentially improved sensitivity due to the catalytic amplification of the positive signal. Therefore, binary probes based on deoxyribozymes promise to be both highly selective and sensitive. Deoxyribozyme E6, which was selected earlier by Breaker and Joyce,[8] was chosen in this work as a model for proof-of-concept experiments. Deoxyribozyme E6 (Figure 1A) is a Mg2+-dependent DNA enzyme that recognizes DNA substrate with a single embedded ribonucleotide, and hydrolyzes the RNA phosphodiester bond with a catalytic rate of ~0.01 min−1.[8] It has been shown that E6 is able to cleave a fluorophore- and quencher-labeled substrate with approximately the same rate.[7b] This fluorescence-based approach has been used as the most convenient method for monitoring E6 catalytic activity. E6 contains a variable stem–loop, which serves only a structural function and is not directly involved in catalysis; this allowed the design of the binary probe. Figure 1 Design of the binary deoxyribozyme probe. A) Structures of the parent deoxyribozyme E6.[8] B) Binary deoxyribozyme biE6. C) Scheme for fluorescent detection of the analyte-dependent catalytic activity of biE6. The dithymidine linkers are shown in lower ... Deoxyribozyme E6 was divided into two fragments (biE6a and biE6b), the inessential AAG loop was removed, stem 1 was elongated to six nucleotides, and the analyte-binding arms were added to each half with dithymidine linkers (Figure 1 B). Structural constraints in the form of two pentanucleotide stems (stem 2 and 3) were introduced in the analyte-binding arms to further increase the selectivity of the binary probe.[5e] The reporter substrate (F substrate; Figure 1C) used in the study was complementary to the substrate-binding arms of the deoxyribozyme and contained a fluorophore and quencher at its 5’- and 3’-ends, respectively. When the nucleic-acid analyte was added, the two subunits of the enzyme cooperatively hybridized to the complementary region of the analyte and re-formed the deoxyribozyme catalytic core (Figure 1C). The active enzyme cleaved the reporter substrate; this led to higher fluorescence (Figure 1 C). It was found that addition of 80 nm A20 DNA analyte to a solution of biE6 and F substrate triggered an approximately four-times increase in fluorescence after 1 h incubation (Figure 2 A, graph 2). The rate of fluorescence increase was about the same when 80 nm E6 was incubated with F substrate (graph 4). Therefore, 80 nm A20 generated about 80 nm active biE6 in solution according to the suggested scheme (Figure 1 C). At the same time, biE6 activity was not observed in the absence of A20 analyte (graph 1). Polyacrylamide gel electrophoresis (PAGE) of the reaction mixtures revealed cleavage of F substrate in reaction mixtures 2 and 4 (Figure 2B, lanes 2 and 4), but not in control samples 1 and 3 (lanes 1 and 3). The observed intensities of the cleavage product in samples 2 and 4 were about the same; this was in good agreement with the fluorescent data (Figure 2A). These results prove the suggested model for analyte-dependent binary deoxyribozyme activation (Figure 1 C), and indicate that hybridization of the analyte-binding arms to A20 does not significantly reduce the catalytic activity of the core. Figure 2 Binary deoxyribozyme cleaves fluorogenic substrate only in the presence of A20 DNA analyte. F substrate (200 nm) and biE6 (100 nm, each strand) were incubated in the absence (sample 1) or presence (sample 2) of A20 DNA analyte (80 nm) in MgCl2 (50 mm ... To investigate the selectivity of biE6 the probe was incubated with single-base substituted analogues of A20 analyte (Table 1). It was found that the binary deoxyribozyme distinguished nineteen mutants out of the twenty tested (column 3). The discrimination factors (DFs) for eleven of the oligodeoxyribonucleotides were significantly higher than 3 (marked bold in Table 1). Taking into account that a signal:-background ratio (S/B) >3–4 is considered acceptable for fluorescence-based assays,[9] I conclude that biE6 might be practically useful for the detection of at least some of these eleven mutations. In comparison, the conventional molecular-beacon approach did not provide such high selectivity: MB-A20 (FAM–5’-CTCGCACCC ACTCTCTCCATGCGAG–dabcyl), an anti-A20 molecular beacon, distinguished true target from only 14 single-base substituted oligonucleotides with low DFs (Table 1, last column). These results indicate that biE6 might be an efficient single nucleotide polymorphism typing tool even at room temperature. Table 1 Discrimination factors (DFs)[a] for oligodeoxyribonucleotides that differed from A20 by a single nucleotide. In order to determine the sensitivity limit of the probe, biE6 was incubated with various concentrations of A20 analyte in the presence of F substrate. The ratios of the fluorescence intensities and background fluorescence as a function of the logarithm of analyte concentration are shown in Figure 3. In the presence of 1 nm analyte, the probe-generated fluorescence was more than three-times greater than the background (S/B 3.9). At the same time, the probe demonstrated high selectivity: 100 nm A20-4 gave a S/B ratio (1.9) that did not exceed the threshold of 3 (graph 2). In comparison, MB-A20 detected A20 only at a concentration of 20 nm (graph 3), while 100 nm A20-4 generated a detectable signal (S/B ~3; Figure 3, graph 4). Thus, in the experiments with A20 and A20-4, biE6 was at least 20-times more sensitive and more selective than MB-A20. Figure 3 Sensitivity of biE6 (graphs 1 and 2) in comparison to MB-A20 (graphs 3 and 4). Relative fluorescence intensities at 517 nm are represented as functions of A20 (graphs 1 and 3) and A20-4 (graphs 2 and 4) concentrations; S/B is signal:background ratio. Although binary hammerhead ribozymes[10] and binary deoxyribozyme ligases[11] have been reported, to the best of my knowledge, biE6 is the first split DNAzyme designed for fluorescent detection of specific nucleic acids. Excellent selectivity at room temperature is the main advantage of this probe. The binary construct based on E6 deoxyribozyme can detect 1 nm analyte. At the same time, split probes designed on the platforms of more efficient enzymes, such as DNAzymes recently obtained by Li and colleagues,[12] can potentially improve the sensitivity of this approach. The probe promises to be relatively inexpensive for multiplex analysis, since it requires synthesis of only two short unmodified DNA strands for each new analyte sequence while the double labeled reporter substrate is universal and can be utilized efficiently in bulk amounts. All these advantages raise the hope that further development of the technique will deliver a highly selective, sensitive, and inexpensive PCR-free method for nucleic-acid analysis.

Bridging the Two Worlds: A Universal Interface between Enzymatic and DNA Computing Systems

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in the way we control electronic computers. Enzyme-based computational systems can respond to a great variety of small molecule inputs. They have the advantage of signal amplification and highly specific recognition. DNA computing systems are most often controlled by oligonucleotide inputs/outputs and are capable of sophisticated computing as well as controlling gene expressions. Here, we developed an interface that enables communication of otherwise incompatible nucleic-acid and enzyme-computational systems. The enzymatic system processes small molecules as inputs and produces NADH as an output. The NADH output triggers electrochemical release of an oligonucleotide, which is accepted by a DNA computational system as an input. This interface is universal because the enzymatic and DNA computing systems are independent of each other in composition and complexity.

Four-way junction formation promoting ultrasensitive electrochemical detection of microRNA.

MicroRNAs (miRNAs) represent a class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. Here, we endeavored to develop a DNA four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive miRNA analysis. The developed sensor can be operated within the dynamic range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36 molecules per sample), without PCR amplification. Furthermore, the 4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy human and chronic lymphocyitc leukemia (CLL) patient serum, and the results were validated by qPCR analysis.

Enzyme-assisted binary probe for sensitive detection of RNA and DNA

The new enzyme-assisted assay for DNA/RNA detection provides real-time fluorescent signal readout along with low limit of detection and high discrimination power toward a single-base substitution. Requiring only two new unmodified DNA oligonucleotides for the detection of each new analyte, the assay is an efficient tool for low-cost analysis of multiple analytes.

Molecular logic gates for DNA analysis: detection of rifampin resistance in M.tuberculosis DNA

Elementary, Dr. Watson! A combination of YES and OR logic gates was applied to differentiate between DNA sequences of wild-type and rifampin-resistant (Rif(r)) Mycobacterium tuberculosis (Mtb) in a multiplex real-time fluorescent assay.

A single molecular beacon probe is sufficient for the analysis of multiple nucleic acid sequences.

Molecular beacon (MB) probes are dual‐labeled hairpin‐shaped oligodeoxyribonucleotides that are extensively used for real‐time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real‐time assays and improve the accuracy of single‐nucleotide polymorphism genotyping.

Real‐Time SNP Analysis in Secondary‐Structure‐Folded Nucleic Acids

Hybridization of two complementary nucleic acid strands is extensively used in the analysis of specific DNA/RNA sequences in real-time PCR, DNA microarrays and the techniques for RNA monitoring in living cells. The design of the hybridization probes is based on A-T and G-C complementarity and may seem straightforward. However, single-stranded DNA and RNA analytes often form stable secondary structures under assay conditions. The analysis of such folded analytes is often complicated since a region of interest may be involved in intramolecular hybridization and become inaccessible for hybridization with a probe. This complication severely limits sensitivity and creates and insurmountable obstacle for the detection of single nucleotide differences between two analytes.[1] This study demonstrates an approach that allows analysis of single nucleotide polymorphisms (SNPs) in folded analytes in real time at room temperature.

Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors.

Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and selectivity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose, or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a non-specific organometallic receptor. The method is general and enables broad clinical use of aptamers for detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.