Yulia V. Gerasimova

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.

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.

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.

Nucleic Acid Detection using MNAzymes

Deoxyribozymes are promising biotechnological tools. In a recent JACS article, Mokany et al. reported on the design of multi-component deoxyribozyme (MNAzyme) sensors based on 10-23 and 8-17 DNA enzymes. The sensors can detect down to 5 pM of a specific nucleic acid. The versatility of MNAzyme platform allows the design of catalytic cascades for signal amplification. This work is a step forward to PCR-free molecular diagnostics.

RNA-Cleaving Deoxyribozyme Sensor for Nucleic Acid Analysis: The Limit of Detection

Along with biocompatibility, chemical stability, and simplicity of structural prediction and modification, deoxyribozyme‐based molecular sensors have the potential of an improved detection limit due to their ability to catalytically amplify signal. This study contributes to the understanding of the factors responsible for the limit of detection (LOD) of RNA‐cleaving deoxyribozyme sensors. A new sensor that detects specific DNA/RNA sequences was designed from deoxyribozyme OA‐II [Chiuman, W.; Li, Y. (2006) J. Mol. Biol. 357, 748–754]. The sensor architecture allows for a unique combination of high selectivity, low LOD and the convenience of fluorescent signal monitoring in homogeneous solution. The LOD of the sensor was found to be ∼1.6×10−10 M after 3 h of incubation. An equation that allows estimation of the lowest theoretical LOD using characteristics of parent deoxyribozymes and their fluorogenic substrates was derived and experimentally verified. According to the equation, “catalytically perfect” enzymes can serve as scaffolds for the design of sensors with the LOD not lower than ∼2×10−15 M after 3 h of incubation. A new value termed the detection efficiency (DE) is suggested as a time‐independent characteristic of a sensors sensitivity. The expressions for the theoretical LOD and DE can be used to evaluate nucleic acid and protein enzymes for their application as biosensing platforms.

DNA nanotechnology for nucleic acid analysis: DX motif-based sensor

A light on the tiles: A sensor that fluoresces in the presence of specific nucleic acids was designed and characterized. The sensor uses a molecular beacon probe and three adaptor strands to form a five-stranded assembly, a DX-tile, with a specific analyte. This sensor is a highly selective and affordable tool for the real-time analysis of DNA and RNA.

Detection of bacterial 16S rRNA using a molecular beacon-based X sensor

We demonstrate how a long structurally constrained RNA can be analyzed in homogeneous solution at ambient temperatures with high specificity using a sophisticated biosensor. The sensor consists of a molecular beacon probe as a signal reporter and two DNA adaptor strands, which have fragments complementary to the reporter and to the analyzed RNA. One adaptor strand uses its long RNA-binding arm to unwind the RNA secondary structure. Second adaptor strand with a short RNA-binding arm hybridizes only to a completely complementary site, thus providing high recognition specificity. Overall the three-component sensor and the target RNA form a four-stranded DNA crossover (X) structure. Using this sensor, Escherichia coli16S rRNA was detected in real time with the detection limit of ~0.17 nM. The high specificity of the analysis was proven by differentiating Bacillus subtilis from E. coli 16S rRNA sequences. The sensor responds to the presence of the analyte within seconds.

Folding of 16S rRNA in a Signal‐Producing Structure for the Detection of Bacteria

Sixty-four DNA strands hybridize to 16S rRNA to form 32 deoxyribozyme catalytic cores that produce a fluorescent signal. The approach allows detection of 0.6 pM 16S rRNA, or about 3×10(4) bacterial cells in a PCR-free format.

Connectable DNA logic gates: OR and XOR logics.

Modern computer processors are based on semiconductor logic gates connected to each other in complex circuits. This study contributes to the development of a new class of connectable logic gates made of DNA in which the transfer of oligonucleotide fragments as input/output signals occurs upon hybridization of DNA sequences. The DNA strands responsible for a logic function form associates containing immobile DNA four-way junction structures when the signal is high and dissociate into separate strands when the signal is low. A basic set of logic gates (NOT, AND, and OR) was designed. Two NOT gates, two AND gates, and an OR gate were connected in a network that corresponds to an XOR logic function. The design of the logic gates presented here may contribute to the development of the first biocompatible molecular computer.