Fluorescent probing for RNA molecules by an unnatural base-pair system

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5′-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions

An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies

Highly specific unnatural base pair systems as a third base pair for PCR amplification

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds–Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds–Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds–Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds–Px pair system provides a framework for new biotechnology

STRUCTURE AND SPECTROSCOPIC CONSTANTS OF FeCN: AN AB INITIO MOLECULAR ORBITAL STUDY

Author Institution: Department of Chemistry, Faculty of Science, Ochanomizu University; National Institute of Materials and Chemical Research, 1-1 HigashiFeCN is one of the possible candidates for an Fe-bearing interstellar molecule. Since no experimental data is available, prediction of its structure and spectroscopic constants by ab initio molecular orbital (MO) calculations has been requested. The ground state FeCl is known to be $^{6}\Delta$, and Cl and CN are known to show a similar chemical behavior. Hence, we assumed $^{6}\Delta$ state for the ground state FeCN. Wachters' all-electron basis set augmented with Bauschlicher's f-GTO, [8s.6p,4d,1f], for Fe and Dunning's cc-pVTZ basis sets for C and N have been employed. Active space for the MR-SDCI calculations has been selected to include Fe 3d and 4s as well as $CN \pi$ and $\pi^{\ast}$ orbitals obtained by the full valence MC-SCF MO calculations. Three dimentional potential energy surface has been calculated at the MR-SDCI level, and analyzed by the Mills' 2nd order perturbation theory. The $^{6}\Delta$ FeCN is predicted to be a linear molecule with Fe-C and CN bond lengths of 2.066 and 1.169 \AA, respectively, yielding $B_{e}, D_{J}$, and $B_{0}$ to be 3667.9, 0.0012, and 3676.1 MHz. Anharmonic vibrational frequencies $\nu_{1}, \nu_{2}$, and $\nu_{3}$ are predicted to be 2158, 185, and $450 cm^{-1}$, respectively

THEORETICAL PREDICTION OF THE SPECTROSCOPIC CONSTANTS OF FeS: AN AB INITIO MOLECULAR ORBITAL STUDY

Author Institution: Department of Chemistry Faculty of Science, Ochanomizu University; Department of Chemistry Faculty of Science, National Institute for Advanced Interdisciplinary ResearchEnergy levels of nearly degenerated $^{5} \Delta$ and $^{5}\Sigma$ states have been studied by the MR-SDCI + Q/Roos-ANO (or various combinations of other basis sets) method with Breit-Pauli Hamiltonian for relativistic effects and spin-orbit coupling interaction corrections. The $^{5}\Sigma$ state has been predicted to be situated between $^{5}\Delta_{2}$ and $^{5}\Delta_{1}$ substates, and hence the ground state is $^{5}\Delta_{i}$. Spectroscopic constants for $^{5}\Delta$ state (and those for $^{5}\Sigma$ state) are predicted as follows: $r_{e}=2.2047$ (1.9963) \AA; $B_{0}=6043.2 (6217.1)$ MHz; $D_{0}=3.80 (3.64)$ kHz; $\nu=510.4 (543.9) cm^{-1}$; $\mu_{e}=5.92 (4.88)D$

Earliest histopathological changes in COVID-19 pneumonia with comprehensive gene expression analyses: A case series study

Aims. In COVID-19 pneumonia, early detection and appropriate treatment are essential to prevent severe exacerbation. Therefore, it is important to understand the initiating events of COVID-19 pneumonia. However, at present, the literature about early stage disease has been very limited. Here, we investigated the earliest histopathological changes and gene expression profiles associated with COVID-19 pneumonia. Methods and Results. We carefully examined 25 autopsied cases with different clinical courses. Dilation of capillaries and edematous thickening of the alveolar septa were found even in areas that macroscopically looked almost normal. Pneumocytes, histocytes/ macrophages, and vascular endothelial cells were immunohistochemically positive for tissue factor, which is an important early responder to tissue injuries. Comprehensive gene expression analyses revealed that those lesions presented differential profiles compared to those of control lungs and were associated with a significant upregulation of the lysosomal pathway. Conclusions. Alveolar capillary dilation and edematous thickening may be the earliest histopathological change detected in COVID-19 pneumonia. Intensive investigations of such lesions may lead to an understanding of the initiating event of not only COVID-19 pneumonia but also of general diffuse alveolar damage

Frequency of subclinical interstitial lung disease in COVID-19 autopsy cases: potential risk factors of severe pneumonia

Abstract Risk factors of severe coronavirus disease 2019 (COVID-19) have been previously reported; however, histological risk factors have not been defined thus far. The aim of this study was to clarify subclinical hidden interstitial lung disease (ILD) as a risk factor of severe pneumonia associated with COVID-19. We carefully examined autopsied lungs and chest computed tomography scanning (CT) images from patients with COVID-19 for interstitial lesions and then analyzed their relationship with disease severity. Among the autopsy series, subclinical ILD was found in 13/27 cases (48%) in the COVID-19 group, and in contrast, 8/65 (12%) in the control autopsy group (p = 0.0006; Fisher’s exact test). We reviewed CT images from the COVID-19 autopsy cases and verified that subclinical ILD was histologically detectable in the CT images. Then, we retrospectively examined CT images from another series of COVID-19 cases in the Yokohama, Japan area between February–August 2020 for interstitial lesions and analyzed the relationship to the severity of COVID-19 pneumonia. Interstitial lesion was more frequently found in the group with the moderate II/severe disease than in the moderate I/mild disease (severity was evaluated according to the COVID-19 severity classification system of the Ministry of Health, Labor, and Welfare [Japan]) (moderate II/severe, 11/15, 73.3% versus moderate I/mild, 108/245, 44.1%; Fisher exact test, p = 0.0333). In conclusion, it was suggested that subclinical ILD could be an important risk factor for severe COVID-19 pneumonia. A benefit of these findings could be the development of a risk assessment system using high resolution CT images for fatal COVID-19 pneumonia