Comparing two K-category assignments by a K-category correlation coefficient

Predicted assignments of biological sequences are often evaluated by Matthews correlation coefficient. However, Matthews correlation coefficient applies only to cases where the assignments belong to two categories, and cases with more than two categories are often artificially forced into two categories by considering what belongs and what does not belong to one of the categories, leading to the loss of information. Here, an extended correlation coefficient that applies to K-categories is proposed, and this measure is shown to be highly applicable for evaluating prediction of RNA secondary structure in cases where some predicted pairs go into the category “unknown” due to lack of reliability in predicted pairs or unpaired residues. Hence, predicting base pairs of RNA secondary structure can be a three-category problem. The measure is further shown to be well in agreement with existing performance measures used for ranking protein secondary structure predictions. Server and software is available at http://rk.kvl.dk/     

浅谈2020年诺贝尔化学奖：通向未来的基因编辑

The 2020 Nobel Prize in chemistry was awarded to two female scientists, Jennifer Doudna and Emmanuelle Charpentier, to recognize their seminal contribution to the invention of CRISPR technology for genome editing. CRISPR system enables new generation of gene editing through RNA-based recognition of double-stranded DNA. Empowered by its high efficiency, accuracy and programmability, CRISPR technology has revolutionized modern biology, and has been widely applied in basic research, gene therapy, animal and plant breeding. Here, we briefly introduce the discovery of CRISPR system and the scientific stories behind, and discuss the on-going development and future directions of many gene-editing related technologies.     

Acid-base and metal-ion binding properties of the RNA dinucleotide uridylyl-(5'-->3')-[5']uridylate (pUpU3-)

It is well known that Mg2+ and other divalent metal ions bind to the phosphate groups of nucleic acids. Subtle differences in the coordination properties of these metal ions to RNA, especially to ribozymes, determine whether they either promote or inhibit catalytic activity. The ability of metal ions to coordinate simultaneously with two neighboring phosphate groups is important for ribozyme structure and activity. However, such an interaction has not yet been quantified. Here, we have performed potentiometric pH titrations to determine the acidity constants of the protonated dinucleotide H2(pUpU)-, as well as the binding properties of pUpU3- towards Mg2+, Mn2+, Cd2+, Zn2+, and Pb2+. Whereas Mg2+, Mn2+, and Cd2+ only bind to the more basic 5'-terminal phosphate group, Pb2+, and to a certain extent also Zn2+, show a remarkably enhanced stability of the [M(pUpU)]- complex. This can be attributed to the formation of a macrochelate by bridging the two phosphate groups within this dinucleotide by these metal ions. Such a macrochelate is also possible in an oligonucleotide, because the basic structural units are the same, despite the difference in charge. The formation degrees of the macrochelated species of [Zn(pUpU)]- and [Pb(pUpU)]- amount to around 25 and 90 %, respectively. These findings are important in the context of ribozyme and DNAzyme catalysis, and explain, for example, why the leadzyme could be selected in the first place, and why this artificial ribozyme is inhibited by other divalent metal ions, such as Mg2+.     

Stable isotope labeling for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay analysis of ribonucleic acids

Matrix-assisted laser desorption/ionization mass spectrometry is a powerful analytical tool for the structural characterization of oligonucleotides and nucleic acids. Here we report the application of stable isotope labeling for the simplified characterization of ribonucleic acids (RNAs). An (18)O label is incorporated at the 3'-phosphate of oligoribonucleotides during the enzymatic processing of intact RNAs. As implemented, a buffer solution containing a 50 : 50 mixture of H(2)O and (18)O-labeled H(2)O is used during endonuclease digestion. Upon digestion, characteristic doublets representative of the isotopic distribution of oxygen are noted for those products that contain 3'-phosphate groups. This approach is used to distinguish readily endonuclease digestion products from incomplete digestion products and non-specific cleavage products. In addition, RNase digestion products containing the characteristic isotopic doublet can be selected for further characterization by post-source decay (PSD) analysis. PSD products carrying the 3'-phosphate group will appear as a doublet, thereby simplifying fragment ion assignment.     

Chemical ligation as a method for the assembly of double-stranded nucleic acids: Modifications and local structure studies

A new procedure was developed as an alternative to the enzymatic assembly of natural and modified double-stranded DNAs using chemical reagent (chemical ligation). BrCN was suggested as an efficient coupling reagent, which induces superfast reactions in DNA duplexes. The physicochemical properties and the structure of new types of DNA duplexes, which are the substrates for chemical ligation, with breaks in phosphodiester chains, including concatemers, were studied. Chemical ligation was applied to prepare biologically active 17–200 base-pair double-stranded DNAs and DNA-RNA block-copolymers, to incorporate various modifications into DNA duplexes including pyrophosphate and phosphoramidate unnatural internucleotide bonds. The unique possibilities of this approach were demonstrated in the development of methods for circularization of oligodeoxy ribonucleotides and assembly of branched DNAs. The structural-kinetic concept of chemicalligation was created and the relationship between the reactivity of interacting groups and sequence-dependent local conformation of the ligation site in B-DNA was established. The lesser efficiency of chemical ligation of RNA fragments in comparison to that of DNA analogs was demonstrated and rationalized. This approach was used as a sensitive monitor of a stable double helix formation and third-strand binding to a DNA duplex.Translated from Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1889–1911, August, 1996.     

新化学发光试剂ITCI的合成及其性能研究

合成一种新化学发光试剂异硫氰酸异鲁米诺（ＩＴＣＩ），研究其化学发光及标记酵母ＲＮＡ的性能。标记反应条件温和快速，１克酵母ＲＮＡ可与１．５×１０－５ｍｏｌＩＴＣＩ结合。建立了测定ＩＴＣＩ和ＲＮＡ的化学发光分析方法，线性范围分别为１．０×１０－１０～１．０×１０－７ｍｏｌ／Ｌ和４．０～×１０－９～２．０×１０－７ｇ／ｍＬ，检测限分别为６．６×１０－１１ｍｏｌ／Ｌ和８．０×１０－１０ｇ／ｍＬ。     

Structural Aspects of Nucleic Acid Analogs and Antisense Oligonucleotides

Hybridization of complementary oligonucleotides is essential to highly valuable research tools in many fields including genetics, molecular biology, and cell biology. For example, an antisense molecule for a particular segment of sense messenger RNA allows gene expression to be selectively turned off, and the polymerase chain reaction requires complementary primers in order to proceed. It is hoped that the antisense approach may lead to therapeutics for treatment of various diseases including cancer. Areas of active research in the antisense field focus on the mechanisms of cellular uptake of antisense molecules and their delivery to specific cell sites, an improved understanding of how these molecules inhibit the production of proteins, as well as the optimization of the chemical stability of antisense molecules and the thermodynamic stability of the duplexes they form with the mRNA targets. The last two issues in particular have prompted chemists to launch an extensive search for oligonucleotide analogs with improved binding properties for hybridization with RNA and higher resistance toward nuclease degradation. During the last years this research has resulted in a flurry of new chemical analogs of DNA and RNA with modifications in the sugar–phosphate backbone as well as in the nucleobase sites. However, to date little effort has been directed toward uncovering the exact origins of the gain or loss in stability when nucleic acid analogs bind to RNA. Although large amounts of thermodynamic data have been collected, the structural perturbations induced by the modifications in hybrid duplexes are only poorly understood. For many modified oligonucleotides the compatibility of protection, coupling, and deprotection chemistry with standard DNA and RNA synthesis protocols makes it now possible to generate modified nucleic acid fragments or mixed oligonucleotides containing modifications at selected sites in quantities suitable for three-dimensional structure investigations. Such studies should reveal the structural origins of the observed changes in affinity and specificity of binding for particular modifications and may guide the development of second-and third-generation antisense molecules. In addition, the availability of a previously unimaginable variety of modified building blocks and the investigation of their structures provides the basis for a deeper understanding of the native DNA and RNA structures. This contribution will summarize the results of X-ray crystallographic structure determinations of modified nucleic acid fragments conducted in our laboratory during the last three years and the insights gained from them.     

Backbone extended pyrrolidine PNA (bepPNA): a chiral PNA for selective RNA recognition

Synthesis of cationic, chiral PNA analogues with an extra atom in the backbone (bepPNA) is reported. The (2S,4S) geometry of the pyrrolidine ring, and an additional carbon atom in the backbone of homopyrimidine-bepPNAs resulted in the optimization of the inter-nucleobase distance, such that selective binding to complementary RNA over DNA was observed in the triplex mode. It was evident from circular dichroism studies that oligomers with mixed aminoethylglycyl-bep (aeg-bep) repeating units, and also bepPNA with homogeneous backbone attained structures quite different from those of aegPNA₂:RNA/DNA complexes. The bepPNA, when incorporated in a duplex forming mixed purine-pyrimidine sequence, also showed a preference for binding complementary RNA over DNA.