Improved measurement of absolute mRNA quantity without reverse transcription.

Gene expression studies using microarrays have provided important insights into understanding the mechanisms of transcriptional regulation in a variety of biological and disease phenomena. In a previous study, we developed Photo-DEAN, a universal-microarray-based RNA quantification method that enabled reverse transcription-free multiplex measurement of the absolute amount of RNA. Photo-DEAN promotes high-throughput and bias-less transcriptome analysis without the need for common controls or additional complicated normalization steps. In this study, we empirically identified two conditions (individual specificity and uniform duplex stability) necessary for in silico design of probe sequences, allowing the Photo-DEAN method to accurately measure the absolute amount of target RNA in total RNA. We then demonstrated that using the modified probe design conditions, the Photo-DEAN method successfully measured the absolute amount of pgi mRNA spiked into E. coli total RNA. The measurement was performed at five different sites in the coding region of pgi mRNA, exhibiting no significant site dependence. Theoretical considerations suggested that probe sequences longer than the previously used 30-bases better satisfy the necessary design conditions.

Influence of thermodynamically unfavorable secondary structures on DNA hybridization kinetics.

Nucleic acid secondary structure plays an important role in nucleic acid-nucleic acid recognition/hybridization processes, and is also a vital consideration in DNA nanotechnology. Although the influence of stable secondary structures on hybridization kinetics has been characterized, unstable secondary structures, which show positive ΔG° with self-folding, can also form, and their effects have not been systematically investigated. Such thermodynamically unfavorable secondary structures should not be ignored in DNA hybridization kinetics, especially under isothermal conditions. Here, we report that positive ΔG° secondary structures can change the hybridization rate by two-orders of magnitude, despite the fact that their hybridization obeyed second-order reaction kinetics. The temperature dependence of hybridization rates showed non-Arrhenius behavior; thus, their hybridization is considered to be nucleation limited. We derived a model describing how ΔG° positive secondary structures affect hybridization kinetics in stopped-flow experiments with 47 pairs of oligonucleotides. The calculated hybridization rates, which were based on the model, quantitatively agreed with the experimental rate constant.

A multiplex RNA quantification method to determine the absolute amounts of mRNA without reverse transcription.

We have developed a highly sensitive microarray-based method that determines the absolute amounts of mRNA in a total RNA sample in a multiplex manner without reverse transcription. This direct mRNA measurement promotes high-throughput testing and reduces bias in transcriptome analyses. Furthermore, quantification of the absolute amount of mRNA allows transcriptome analysis without common controls or additional, complicated normalization. The method, called Photo-DEAN, was validated using chemically synthesized RNAs of known quantities and mouse liver total RNA samples. We found that the absolute amounts of mRNA were successfully measured without the cDNA synthesis step, with a sensitivity of 15 zmol achieved in 7 h.

Multiplex cDNA quantification method that facilitates the standardization of gene expression data.

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h.

Experiments and simulation models of a basic computation element of an autonomous molecular computing system.

Autonomous DNA computers have been attracting much attention because of their ability to integrate into living cells. Autonomous DNA computers can process information through DNA molecules and their molecular reactions. We have already proposed an idea of an autonomous molecular computer with high computational ability, which is now named Reverse-transcription-and-TRanscription-based Autonomous Computing System (RTRACS). In this study, we first report an experimental demonstration of a basic computation element of RTRACS and a mathematical modeling method for RTRACS. We focus on an AND gate, which produces an output RNA molecule only when two input RNA molecules exist, because it is one of the most basic computation elements in RTRACS. Experimental results demonstrated that the basic computation element worked as designed. In addition, its behaviors were analyzed using a mathematical model describing the molecular reactions of the RTRACS computation elements. A comparison between experiments and simulations confirmed the validity of the mathematical modeling method. This study will accelerate construction of various kinds of computation elements and computational circuits of RTRACS, and thus advance the research on autonomous DNA computers.

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2'-deoxyuridine.

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology.

Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay.

A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost.

Coupled equilibrium model of hybridization error for the DNA microarray and tag-antitag systems.

In this work, a detailed coupled equilibrium model is presented for predicting the ensemble average probability of hybridization error per chip-hybridized input strand, providing the first ensemble average method for estimating postannealing microarray/TAT system error rates. Following a detailed presentation of the model and implementation via the software package NucleicPark, under a mismatched statistical zipper model of duplex formation, error response is simulated for both mean-energy and randomly encoded TAT systems versus temperature and input concentration. Limiting expressions and simulated model behavior indicate the occurrence of a transition in hybridization error response, from a logarithmically convex function of temperature for excess inputs (high-error behavior), to a monotonic, log-linear function of temperature for dilute inputs (low-error behavior), a novel result unpredicted by uncoupled equilibrium models. Model scaling behavior for random encodings is investigated versus system size and strand-length. Application of the model to TAT system design is also undertaken, via the in silico evolution of a high-fidelity 100-strand TAT system, with an error response improved by nine standard deviations over the performance of the mean random encoding.

DigiTag assay for multiplex single nucleotide polymorphism typing with high success rate.

As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost.

A simple classification of the volvocine algae by formal languages.

There are several explanations of why certain primitive multicellular organisms aggregate in particular forms and why their constituent cells cooperate with one another to a particular degree. Utilizing the framework of formal language theory, we have derived one possible simple classification of the volvocine algae-one of the primitive multicells-for some forms of aggregation and some degrees of cooperation among cells. The volvocine algae range from the unicellular Chlamydomonas to the multicellular Volvox globator, which has thousands of cells. The classification we use in this paper is based on the complexity of Parikh sets of families on Chomsky hierarchy in formal language theory. We show that an alga with almost no space closed to the environment, e.g., Gonium pectorale, can be characterized by PsFIN, one with a closed space and no cooperation, e.g., Eudorina elegans, by PsCF, and one with a closed space and cooperation, e.g., Volvox globator, by PslambdauSC. This classification should provide new insights into the necessity for specific forms and degrees of cooperation in the volvocine algae.

Systematic single base-pair substitution analysis of DNA binding by the cAMP receptor protein in cyanobacterium Synechocystis sp. PCC 6803.

The cAMP receptor protein SYCRP1 in cyanobacterium Synechocystis sp. PCC 6803 is a regulatory protein that binds to the consensus DNA sequence (5'-AAATGTGATCTAGATCACATTT-3') for the cAMP receptor protein CRP in Escherichia coli. Here we examined the effects of systematic single base-pair substitutions at positions 4-8 (TGTGA) of the consensus sequence on the specific binding of SYCRP1. The consensus sequence exhibited the highest affinity, and the effects of base-pair substitutions at positions 5 and 7 were the most deleterious. The result is similar to that previously reported for CRP, whereas there were differences between SYCRP1 and CRP in the rank order of affinity for each substitution.

Equilibrium analysis of the efficiency of an autonomous molecular computer.

In the whiplash polymerase chain reaction (WPCR), autonomous molecular computation is implemented in vitro by the recursive, self-directed polymerase extension of a mixture of DNA hairpins. Although computational efficiency is known to be reduced by a tendency for DNAs to self-inhibit by backhybridization, both the magnitude of this effect and its dependence on the reaction conditions have remained open questions. In this paper, the impact of backhybridization on WPCR efficiency is addressed by modeling the recursive extension of each strand as a Markov chain. The extension efficiency per effective polymerase-DNA encounter is then estimated within the framework of a statistical thermodynamic model. Model predictions are shown to provide close agreement with the premature halting of computation reported in a recent in vitro WPCR implementation, a particularly significant result, given that backhybridization had been discounted as the dominant error process. The scaling behavior further indicates completion times to be sufficiently long to render WPCR-based massive parallelism infeasible. A modified architecture, PNA-mediated WPCR (PWPCR) is then proposed in which the occupancy of backhybridized hairpins is reduced by targeted PNA(2)/DNA triplex formation. The efficiency of PWPCR is discussed using a modified form of the model developed for WPCR. Predictions indicate the PWPCR efficiency is sufficient to allow the implementation of autonomous molecular computation on a massive scale.