A generic sandwich-type biosensor with nanomolar detection limits.

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.

Recognition of poly(C) DNA and RNA sequences in mammalian cells is achieved by a subfamily of the KH (hnRNP K homology) domain-containing proteins known as poly(C)-binding proteins (PCBPs). To reveal the molecular basis of poly(C) sequence recognition, we have determined the crystal structure, at 1.7-A resolution, of PCBP2 KH1 in complex with a 7-nucleotide DNA sequence (5'-AACCCTA-3') corresponding to one repeat of the human C-rich strand telomeric DNA. The protein-DNA interaction is mediated by the combination of several stabilizing forces including hydrogen bonding, electrostatic interactions, van der Waals contacts, and shape complementarities. Specific recognition of the three cytosine residues is realized by a dense network of hydrogen bonds involving the side chains of two conserved lysines and one glutamic acid. The co-crystal structure also reveals a protein-protein dimerization interface of PCBP2 KH1 located on the opposite side of the protein from the DNA binding groove. Numerous stabilizing protein-protein interactions, including hydrophobic contacts, stacking of aromatic side chains, and a large number of hydrogen bonds, indicate that the protein-protein interaction interface is most likely genuine. Interaction of PCBP2 KH1 with the C-rich strand of human telomeric DNA suggests that PCBPs may participate in mechanisms involved in the regulation of telomere/telomerase functions.

Effects of Cationic Residues and Base Sequence in Nucleic Acid Binding of Histone-Derived Antimicrobial Peptides

Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence-specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast-in essence, digital-signal that enables sensitive, single-molecule sensing of the cross-linked duplexes.

Multiplexed isothermal nucleic acid amplification

Several computational methods based on stochastic context-free grammars have been developed for modeling and analyzing functional RNA sequences. These grammatical methods have succeeded in modeling typical secondary structures of RNA and are used for structural alignment of RNA sequences. However, such stochastic models cannot sufficiently discriminate member sequences of an RNA family from non-members and hence detect non-coding RNA regions from genome sequences. A novel kernel function, stem kernel, for the discrimination and detection of functional RNA sequences using support vector machines (SVM) is proposed. The stem kernel is a natural extension of the string kernel, specifically the all-subsequences kernel, and is tailored to measure the similarity of two RNA sequences from the viewpoint of secondary structures. The stem kernel examines all possible common base-pairs and stem structures of arbitrary lengths, including pseudoknots between two RNA sequences and calculates the inner product of common stem structure counts. An efficient algorithm was developed to calculate the stem kernels based on dynamic programming. The stem kernels are then applied to discriminate members of an RNA family from nonmembers using SVM. The study indicates that the discrimination ability of the stem kernel is strong compared with conventional methods. Further, the potential application of the stem kernel is demonstrated by the detection of remotely homologous RNA families in terms of secondary structures. This is because the string kernel is proven to work for the remote homology detection of protein sequences. These experimental results have convinced us to apply the stem kernel to find novel RNA families from genome sequences.

ABSTRACTCurrently, systems for the detection of nucleic acid sequences, known as DNA-chips, are getting lots of attention. Such systems usually involve either an enzymatic or chemical labelling reaction as part of the detection process. The next generation of DNA-chips aims at a labelfree, fully electronic readout system. Several new approaches to signal generation that avoid a labelling step have been developed in recent years. Besides other surface sensitive measurements the possibility of electrochemical impedance and field-effect measurements for the detection of biomolecules have been discussed. The fully electronic detection of charged biomolecules based on the field-effect principle offers a labelfree method, which combines the unique sensitivity and selectivity of biomolecular recognition reactions with an electronic chip-based readout. In this approach one type of molecules is fixed at a surface and the biomolecular reaction with complementary molecules is detected by change in the drain-source current of the transistor. This change can occur by a change of the interface capacitance of the transistor gate or by change of the surface potential during adsorption of the molecules. At the moment a complete theoretical description of the detection principle is still under discussion. However, the fully electronic readout of biomolecular reactions offers a unique principle for the construction of many different sensors for bioassays. We are working on an approach to detect the hybridization of DNA sequences using electrolyte-oxide-semiconductor field-effect transistor (EOSFET) arrays. This method allows direct and in situ detection of specific DNA sequences without any labelling.

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L(-1) and an assay range spanning four orders of magnitude (5 pmol L(-1)-50 nmol L(-1)) with a coefficient of variation </=5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.

Chapter 13 - Nucleic Acid–Based Methods in the Detection of Foodborne Pathogens

DNA hybridization detection using less than 10-nm gap silicon nanogap structure

Detection of nucleic acid sequences in cervical tumors

Nucleotide and oligonucleotide derivatives as enzyme and nucleic acid targeted irreversible inhibitors, biochemical aspects

The Invader assay is a homogeneous, isothermal, signal amplification system for the quantitative detection of nucleic acids. The assay can directly detect either DNA or RNA without target amplification or reverse transcription. It is based on the ability of Cleavase enzymes to recognize as a substrate and cleave a specific nucleic acid structure generated through the hybridization of two oligonucleotides to the target sequence. The combination of sequence-specific oligonucleotide hybridization and structure-specific enzymatic cleavage results in a highly specific assay well suited for discriminating closely related gene sequences. This includes detection of single nucleotide polymorphisms directly from genomic DNA as well as highly homologous mRNAs in closely related gene families. Because Cleavase substrate recognition is structure, and not sequence dependent, cleavage and detection can be applied to virtually any DNA or RNA sequence.

We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5′ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5′ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5′ end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

Nucleic acids are essential biomolecules that encode all of the information necessary for life. Specific pairing of A with T (or U) and C with G during replication, transcription, and translation is the key to effective transmission of genetic information between generations, as well as accurate conversion of genetic information into protein sequence. Given the magnitude of the tasks orchestrated by the Watson-Crick base pairing, it is striking to consider that biological systems accomplish these tasks using only four nucleobases. Realizing the powerful nature of base-pair recognition, researchers have been inspired to ask the question of whether the genetic code can be artificially expanded to generate biological systems having novel functions. It was this question that led Alex Rich in 1962 to propose the concept of orthogonal base pairing between iso-G and iso-C and inspired Professor Steven A. Benner in the late 1980s to expand the genetic alphabet from four to six letters. Benner's early research focused on the development of new base pairs having hydrogen bonding patterns orthogonal to those in the canonical Watson-Crick base pairs. In 1994, Professor Eric T. Kool opened a new functional dimension with the creation of nonhydrogen bonding unnatural nucleobase surrogates. Expansion of the genetic alphabet has dramatically increased the functional potential of DNA, for example, by enabling site-directed oligonucleotide labeling and in vitro selections with oligonucleotides having increased chemical diversity. Translation of an expanded DNA alphabet into RNA is a challenging task, but one which has potential to give rise to semisynthetic organisms with increased biodiversity. This special issue highlights recent accomplishments at the interface of organic chemistry and molecular biology which hold promise to further expand the potential of nucleic acids having unnatural nucleobases. Specifically, the reports in this special issue focus on the synthesis of unnatural nucleobases and nucleic acid backbones, the exploration of their structure and duplex stabilizing ability, and the polymerase mediated replication and transcription of DNA containing unnatural nucleobases. T. Lonnberg and a coworker report the synthesis and study of a bis(pyrazolyl)purine ribonucleoside having increased hydrophobic surface area and the ability to form complex with metal ions. The hydrogen bonding pattern of this nucleoside makes it complementary to thymine and uridine. The authors demonstrate that the bis(pyrazolyl) nucleobase is capable of forming a Pd2+-mediated base pair with uridine in the monomeric state. When incorporated into an oligonucleotide, the bis(pyrazolyl) nucleobase stabilizes DNA duplexes when paired with thymine, but this stabilization appears to result from increased π-stacking interactions rather than metal complexation. These studies open the door to applications using unnatural nucleobases to increase the binding affinity of probes and therapeutics targeted at native DNA and RNA. Much effort has focused on the incorporation of unnatural nucleobases into native DNA and RNA, and the availability of nonnative backbones such as LNA, PNA, and GNA opens the door for further expansion of nucleic acid structure and function. C. Forster and coworkers evaluate the structural properties of “all LNA” duplexes and demonstrate that these duplexes have a relaxed helical structure unique from that of DNA and RNA. J. M. Heemstra and coworkers report an expedient synthetic route to the Fmoc-protected PNA backbone, which serves as a key intermediate in the synthesis of PNA monomers having unnatural nucleobases. Together, these studies facilitate the exploration of nucleic acids having both nonnative backbones and unnatural nucleobases, which is in turn anticipated to provide molecules having novel structural and functional properties. The ability to use native or engineered replication and transcription machinery with nucleic acids containing unnatural nucleobases is critical to many in vitro and in vivo applications. I. Hirao and coworkers describe a two-unnatural-base-pair system capable of incorporating unnatural nucleobases into DNA via polymerase chain reaction, then into RNA via T7 transcription. Using this system, they demonstrate sequence-specific incorporation of a biotinylated nucleobase into a 260-mer RNA sequence. Generating modified RNA of this length is not feasible using purely synthetic methods; thus this work significantly improves access to site-specifically labeled RNA sequences. Additionally, P. J. Beuning and a coworker review the use of native A and Y family DNA polymerases for replication of DNA containing unnatural nucleobases. This review serves as an excellent resource for researchers seeking to utilize unnatural nucleobases and provides significant insight into how polymerases deal with both synthetic and damaged DNA nucleobases. Collectively, these reports demonstrate the potential of unnatural nucleobases for applications in chemistry and biology and offer valuable tools for further exploring nucleic acid structural diversity.