Cheulhee Jung

“Illusionary” Polymerase Activity Triggered by Metal Ions: Use for Molecular Logic‐Gate Operations

In recent years, an intense interest has grown in the interactions of nucleic acids with metal ions. Examples of such novel interactions include the specific binding of aptamers with metal ions and selective incorporation of metal ions as cofactors to promote the catalytic activities of nucleic acid enzymes (deoxyribozymes or ribozymes). Furthermore, certain metal ions, such as Hg, Ag, Cu, Ni, and Co, specifically bind to nucleosides or ligandosides to form metal-ion-mediated base pairs. This nonnatural base paring is stabilized by coordination of the metal ions to the nucleosides in a manner that is different from natural hydrogen bonding between complementary nucleosides. The novel interaction of nucleic acids with metal ions has recently been utilized for the construction of molecular-scale logic gates, which are essential for the development of molecular-scale computers and other computational devices. Most representatively, deoxyribozymes have been employed to build logic gates based on the fact that their catalytic activities can be regulated by the presence of specific metal ions. However, these kinds of logic gates typically rely on relatively complicated designs for gate switching and frequently require the involvement of RNA or chimeric DNA as an operational substrate. In addition to leading to high construction costs, this phenomenon ultimately requires complex operational features for controlling the system as a result of the susceptibility of RNA molecules to degradation. From this perspective, it would be highly desirable to develop molecular-scale logic gates that operate in a more simple and cost-effective manner. The results of the investigation described below have led to a new, simple strategy for construction of molecular-scale logic gates that are based on the “illusionary” polymerase activity at the mismatched site triggered by metal ions. As illustrated in Scheme 1, the underlying principle for operation of this system relies on specific interactions between metal ions (Hg or Ag) and the respective mismatched base pairs (thymine–thymine (T– T) or cytosine–cytosine (C–C)). Forward (F) and reverse (R) primers were designed to form T–T (Scheme 1a) or C–C (Scheme 1b) mismatches with template DNA at its 3’ end. The mismatched primers cannot be extended in the absence of the respective metal ions, because the terminal mismatching stalls action of the polymerase enzyme at the 3’ end, thus preventing the elongation reaction promoted by the polymerase. However, in the presence of Hg or Ag ions, the terminal T or C base at the 3’ end of the primer can form an nonnatural but stable T-Hg-T or C-Ag-C base pair with template DNA. This stabilization induces the polymerase activity and, as a consequence, amplification products are formed (Scheme 1). This activity is termed illusionary polymerase activity herein because it is derived from the illusion of DNA polymerase that the metal-ion-mediated base pair is perfectly matched. Figure 1 shows gel electrophoresis images of the products obtained from PCR mixtures containing F/R primers with terminal mismatched T (Figure 1a) or C base (Figure 1 b) at the 3’ end. As envisioned, use of the T–T and C–C mismatched primers results in formation of gel bands that correspond to amplification products only when the respective Hg and Ag ions are present (lane 3 in Figure 1a,b). In contrast, employment of perfectly matched primers results in the generation of amplification products regardless of whether or not the respective metal ions are present (lane 1, 4 in Figure 1a,b). Importantly, the fact that no significant difference is seen between the band intensities for systems with and without the metal ions indicates that the metal ions do not have an adverse effect on the polymerase reaction. To further support the proposal that the metal ions induce polymerase activity, melting curve analyses were performed for the extension products obtained from the T–T and C–C Scheme 1. Illustration of polymerase activity triggered by metal ions. a) Extension of T–T mismatched primer in the presence of Hg ions. b) Extension of C–C mismatched primer in the presence of Ag ions.

A gold nanorod-based optical DNA biosensor for the diagnosis of pathogens

A novel optical biosensor for detecting target DNA, utilizing gold nanorods (GNRs) as molecular probes is demonstrated. This sensor is based on simultaneous biorecognition-mediated hybridization of target DNA in a sandwich type manner with two different capture probe DNA sequences modified separately with identical sets of GNRs, which leads to aggregation of GNRs. The hybridization induced aggregation as revealed by TEM analysis, promotes the modulation of surface plasmon resonance of GNRs, which forms the basis of complementary target DNA detection from the Chlamydia trachomatis pathogen. Thermally induced reversible dissociation of hybridized DNA is demonstrated by melting analysis. The present sensing strategy is successfully demonstrated by detecting PCR amplified C. trachomatis pathogen gene isolated from human urine sample in a concentration range of 0.25-20 nM. Furthermore, this sensor displays excellent specificity by discriminating the target DNA versus other non-specific pathogenic genes.

Simple and Universal Platform for Logic Gate Operations Based on Molecular Beacon Probes

A new platform technology is herein described with which to construct molecular logic gates by employing the hairpin-structured molecular beacon probe as a basic work unit. In this logic gate operation system, single-stranded DNA is used as the input to induce a conformational change in a molecular beacon probe through a sequence-specific interaction. The fluorescent signal resulting from the opening of the molecular beacon probe is then used as the output readout. Importantly, because the logic gates are based on DNA, thus permitting input/output homogeneity to be preserved, their wiring into multi-level circuits can be achieved by combining separately operated logic gates or by designing the DNA output of one gate as the input to the other. With this novel strategy, a complete set of two-input logic gates is successfully constructed at the molecular level, including OR, AND, XOR, INHIBIT, NOR, NAND, XNOR, and IMPLICATION. The logic gates developed herein can be reversibly operated to perform the set-reset function by applying an additional input or a removal strand. Together, these results introduce a new platform technology for logic gate operation that enables the higher-order circuits required for complex communication between various computational elements.

Direct colorimetric diagnosis of pathogen infections by utilizing thiol-labeled PCR primers and unmodified gold nanoparticles

We describe here a greatly simplified colorimetric detection method to identify PCR-amplified nucleic acids. Our method relies on the PCR product having thiol group at one end, which is generated by employing thiolated PCR primer. After PCR amplification reaction, unmodified gold nanoparticles (AuNPs) are added into the reaction tube followed by the addition of NaCl to induce the aggregation of AuNPs. The PCR products strongly bind to the surface of AuNPs through the interaction of the terminal thiol groups and the long chain of DNA which has abundant negative charges enhances the electrostatic and steric repulsion among AuNPs, which consequently leads to the prevention of the salt-induced aggregation. As a result, the color of AuNPs remains red in the presence of the PCR-amplified nucleic acids, while the AuNPs change its color from red to blue due to the salt-induced aggregation in the absence of the PCR products. This simple but very efficient colorimetric strategy was successfully demonstrated by diagnosing Chlamydia infection using a real human urine sample. Since the results can be clearly seen with the naked eye without any complicated step such as surface modification of AuNPs or PCR product purification, this method can be easily applied to point-of-care diagnosis.

Gold nanoparticle embedded silicon nanowire biosensor for applications of label-free DNA detection

Gold nanoparticle (GN) embedded silicon nanowire (SiNW) configuration was proposed as a new biosensor for label-free DNA detection to enhance the sensitivity. The electric current flow between two terminals, a source and a drain electrode, were measured to sense the immobilization of probe oligonucleotides and their hybridization with target oligonucleotides. The complementary target oligonucleotide, breast cancer DNA with 1 pM, was sensed. In addition, its sensing mechanism and limit of detection (LOD) enhancement was investigated through simulation. The results support that the LOD can be improved by reducing the SiNW doping concentration. This emerging architecture combined nanostructure of spherical GN and SiNW has high potential as a label-free biosensor due to its facile fabrication process, high thermal stability, immobilization efficiency with a thiol-group in a self-assembled monolayer (SAM), and improved sensitivity.

A Polydiacetylene Microchip Based on a Biotin–Streptavidin Interaction for the Diagnosis of Pathogen Infections

A micropatterned polydiacetylene (PDA) chip, utilizing the unique fluorogenic property of PDA and a specific biotin-streptavidin (STA) interaction, is constructed to detect pathogen infections. To construct the PDA chip, biotin-modified diacetylene liposomes are immobilized on aldehyde glass and conjugated with STA, followed by UV irradiation to polymerize the STA-functionalized diacetylene liposomes. Genomic DNA of a model pathogen, Chlamydia trachomatis, is isolated from human samples and biotin-labeled target DNA is obtained through PCR amplification using biotin-11-dUTP. Owing to the stimulus caused by the biotin-STA interaction, the biotinylated DNA induces an intense fluorescence signal on the immobilized PDA. By using this strategy, it is possible to diagnose Chlamydia infections by applying DNA samples from several nonhealthy humans to a single PDA chip. The results of this study serve as the basis for a new strategy for fluorogenic PDA microarray-based diagnosis of pathogen infections.

Label-free DNA detection with a nanogap embedded complementary metal oxide semiconductor.

A nanogap embedded complementary metal oxide semiconductor (NeCMOS) is demonstrated as a proof-of-concept for label-free detection of DNA sequence. When a partially carved nanogap between a gate and a silicon channel is filled with charged biomolecules, the gate dielectric constant and charges are changed. When the gate oxide thickness reduces, the threshold voltage is significantly affected by a change of the charges, whereas it is scarcely influenced by a change of the dielectric constant. In the case of DNA, those two factors act on the threshold voltage oppositely in an n-channel NeCMOS but collaboratively in a p-channel NeCMOS because of the negative charges of DNA. Hence, a p-channel NeCMOS with a thin gate oxide is more attractive for DNA detection because it enhances the shift of threshold voltage; that is, it improves the sensitivity of DNA detection. In addition, the shift of threshold voltage according to the nanogap length is also investigated and the longer nanogap shows more shift of the threshold voltage.

Isothermal Target and Signaling Probe Amplification Method, Based on a Combination of an Isothermal Chain Amplification Technique and a Fluorescence Resonance Energy Transfer Cycling Probe Technology

An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and fluorescence resonance energy transfer cycling probe technology (FRET CPT), is described. In the new ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. Using the resulting dual target and signaling probe amplification system, even a single copy level of a target gene can be successfully detected and quantified under isothermal conditions.

Colorimetric SNP Genotyping Based on Allele-Specific PCR by Using a Thiol- Labeled Primer

chemistry. [7] However, these methods usually require multiple manipulations, high levels of technical expertise, and expensive instruments, all of which makes them unsuitable for pointof-care testing (POCT) applications. Consequently, a great incentive exists for the development of simple, rapid, and costeffective SNP genotyping methods that are suitable for POCT in facility-limited environments. Colorimetric methods fulfill most of the above requirements as they enable cost-effective and on-site detection without the need for sophisticated equipment. To date, a number of such methods for SNP detection that utilize, for example, metal nanoparticles, [8] peroxidase-mimicking DNAzymes, [9] and peptide nucleic acids (PNAs), [1] have been reported. Among these, gold nanoparticle (AuNP)-based approaches [10–12] have received great attention owing to their unique optical properties, robustness, and high surface areas, which make them ideally suited as signaling probes in colorimetric-detection platforms. [12] However, the requirement for modification of the AuNP surface with oligonucleotide probes and/or the need for precise temperature control during the assay significantly limits applications of AuNP-based methods. In recent studies, we developed a novel strategy based on unmodified AuNPs for the colorimetric detection of target nucleic acids that are amplified by thiol-labeled primers without time-consuming and complicated procedures. [13] We successfully demonstrated its diagnostic utility by reliably identifying Chlamydia trachomatis infection. Here, we have extended our previous strategy for nucleic acid detection by devising a novel method for SNP identification that incorporates a modified allele-specific PCR (ASPCR) method. In principle, ASPCR is a cost-effective procedure that characterizes SNPs based on differences between the PCR efficiency of allele-specific primers. [14] The primers consist of sequences adjacent to the polymorphic site that are complementary to the allelic variant and differ only in the terminal nucleotide at the 3’-end. [2] In the first step of our new assay procedure, ASPCR is performed with a thiolated primer. In the second step, AuNPs are mixed with the products and then subjected to salt-induced aggregation. The resulting color of the solution (i.e., red or blue) indicates the genotype at the SNP site because only the thiolated PCR products provide the AuNPs with a significant resistance to salt-induced aggregation and inhibit the red-to-blue color transition. The strategy we have devised for SNP identification is schematically illustrated in Figure 1 for the detection of a single base mutation. In the first step, ASPCR is performed separately in four different reaction tubes containing each of the four thiolated forward primers, which differ only in the terminal base at their 3’-ends (A, T, C and G). When the base at the 3’end of the forward primer is complementary to the antisense strand of the genomic target DNA, PCR amplification occurs and generates PCR amplicons labeled with a thiol group at the 5’-end of one strand. In contrast, the other three primers, which have a noncomplementary nucleotide at their 3’-ends, cannot be extended and thus they fail to generate PCR amplicons. The existence of the thiol-labeled PCR products is then colorimetrically determined by sequentially adding unmodified AuNPs and NaCl to each tube. When thiolated PCR amplicons are mixed with AuNPs, they bind to the surface of unmodified AuNPs through a strong gold–thiol interaction. The electrostatic repulsion between the AuNPs is enhanced as a consequence of the large number of negative charges on their surfaces due to the negatively charged phosphate backbone of the bound DNA. In addition, steric repulsion between the AuNPs is increased because the longer (compared to the diameter of AuNPs) DNA grafted onto the AuNPs surface forms a thick polymeric barrier that prevents the particles from approaching each other. [15] As a consequence of these “electrostatic/steric stabilization effects”, [16] salt-induced aggregation of AuNPs is significantly inhibited, and the solution remains red when salt is added. In contrast, in the absence of the thiol-labeled PCR amplicon, AuNPs undergo immediate aggregation upon salt addition; this results in a colorimetric transition from red to blue. By employing this strategy, the genotypes at mutation sites can be conveniently determined based on the color of the sample. To prove the conceptual basis of this novel strategy for SNP genotyping and to confirm the crucial role played by thiolated primers in the AuNPs-based colorimetric assay, ASPCR was per

Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay ☆

We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.