Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K)

A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson-Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host-guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs

Controlling platinum, ruthenium, and osmium reactivity for anticancer drug design

The main task of the medicinal chemist is to design molecules that interact specifically with derailed or degenerating processes in a diseased organism, translating the available knowledge of pathobiochemical and physiological data into chemically useful information and structures. Current knowledge of the biological and chemical processes underlying diseases is vast and rapidly expanding. In particular the unraveling of the genome in combination with, for instance, the rapid development of structural biology has led to an explosion in available information and identification of new targets for chemotherapy. The task of translating this wealth of data into active and selective new drugs is an enormous, but realistic, challenge. It requires knowledge from many different fields, including molecular biology, chemistry, pharmacology, physiology, and medicine and as such requires a truly interdisciplinary approach. Ultimately, the goal is to design molecules that satisfy all the requirements for a candidate drug to function therapeutically. Therapeutic activity can then be achieved by an understanding of and control over structure and reactivity of the candidate drug through molecular manipulation

Electron scattering from molecules and molecular aggregates of biological relevance

In this Topical Review we survey the current state of the art in the study of low energy electron collisions with biologically relevant molecules and molecular clusters. We briefly describe the methods and techniques used in the investigation of these processes and summarise the results obtained so far for DNA constituents and their model compounds, amino acids, peptides and other biomolecules. The applications of the data obtained is briefly described as well as future required developments

Structure of duplex DNA containing the cisplatin 1,2-{Pt(NH3)2}2+-d(GpG) crosslink at 1.77 Å resolution

We report the 1.77-Å resolution X-ray crystal structure of a dodecamer DNA duplex with the sequence 5′-CCTCTGGTCTCC-3′ that has been modified to contain a single engineered 1,2-cis-{Pt(NH3)2}2+-d(GpG) cross-link, the major DNA adduct of cisplatin. These data represent a significant improvement in resolution over the previously published 2.6-Å structure. The ammine ligands in this structure are clearly resolved, leading to improved visualization of the cross-link geometry with respect to both the platinum center and to the nucleobases, which adopt a higher energy conformation. Also better resolved are the deoxyribose sugar puckers, which allow us to re-examine the global structure of platinum-modified DNA. Another new feature of this model is the location of four octahedral [Mg(H2O)6]2+ ions associated with bases in the DNA major groove and the identification of 124 ordered water molecules that participate in hydrogen-bonding interactions with either the nucleic acid or the diammineplatinum(II) moiety.National Cancer Institute (U.S.) (grant CA034992)David H. Koch Institute for Integrative Cancer Research at MIT (Koch Fund Fellowship

Direct Observation of Multiple Tautomers of Oxythiamine and their Recognition by the Thiamine Pyrophosphate Riboswitch

Structural diversification of canonical nucleic acid bases and nucleotide analogues by tautomerism has been proposed to be a powerful on/off switching mechanism allowing regulation of many biological processes mediated by RNA enzymes and aptamers. Despite the suspected biological importance of tautomerism, attempts to observe minor tautomeric forms in nucleic acid or hybrid nucleic acid-ligand complexes have met with challenges due to the lack of sensitive methods. Here, a combination of spectroscopic, biochemical, and computational tools probed tautomerism in the context of an RNA aptamer-ligand complex; studies involved a model ligand, oxythiamine pyrophosphate (OxyTPP), bound to the thiamine pyrophosphate (TPP) riboswitch (an RNA aptamer) as well as its unbound nonphosphorylated form, oxythiamine (OxyT). OxyTPP, similarly to canonical heteroaromatic nucleic acid bases, has a pyrimidine ring that forms hydrogen bonding interactions with the riboswitch. Tautomerism was established using two-dimensional infrared (2D IR) spectroscopy, variable temperature FTIR and NMR spectroscopies, binding isotope effects (BIEs), and computational methods. All three possible tautomers of OxyT, including the minor enol tautomer, were directly identified, and their distributions were quantitated. In the bound form, BIE data suggested that OxyTPP existed as a 4′-keto tautomer that was likely protonated at the N1′-position. These results also provide a mechanistic framework for understanding the activation of riboswitch in response to deamination of the active form of vitamin B1 (or TPP). The combination of methods reported here revealing the fine details of tautomerism can be applied to other systems where the importance of tautomerism is suspected.National Institutes of Health (U.S.) (Grant CA080024)National Institutes of Health (U.S.) (Grant CA26731)National Institutes of Health (U.S.) (Grant ES002109)National Institutes of Health (U.S.) (Grant ES007020)National Science Foundation (U.S.) (Grant CHE-1212557)Massachusetts Institute of Technology. Center for Environmental Health Sciences (National Institutes of Health (U.S.) Center Grant P30-ES002109)Massachusetts Institute of Technology. Laser Biomedical Research Center (National Institutes of Health (U.S.) Center Grant P41-EB015871)National Institutes of Health (U.S.) (Traineeship T32 ES007020)Massachusetts Institute of Technology (Poitras Pre-Doctoral Fellowship

Quantum Mechanical Studies of DNA and LNA

Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the electrostatic potentials were compared among model oligonucleotides, and it was observed that small structural modifications induce global changes in the molecular structure and surface potentials. Since ligand structure and electrostatic potential complementarity with a receptor is a determinant for the bonding pattern between molecules, minor chemical modifications may have profound changes in the interaction profiles of oligonucleotides, possibly leading to changes in pharmacological properties. The QM modeling data can be used to understand earlier observations of antisense oligonucleotide properties, that is, the observation that small structural changes in oligonucleotide composition may lead to dramatic shifts in phenotypes. These observations should be taken into account in future oligonucleotide drug discovery, and by focusing more on non RNA target interactions it should be possible to utilize the exhibited property diversity of oligonucleotides to produce improved antisense drugs

Tetranucleotides as a scaffold for diporphyrin arrays

The incorporation of porphyrin-substituted nucleosides into tetranucleotides using phosphoramidite chemistry on solid support is reported. Both diphenyl and tetraphenyl porphyrin nucleosides were used as building blocks. This method allows the synthesis of chiral homo- and heteroporphyrinic arrays, where the composition and thus the physical properties of the array can be modulated simply by reprogramming the DNA synthesizer. The porphyrin arrays are initially isolated in the free-base form. Remetallation to give the zinc-porphyrins can be achieved using standard procedures in solution. The UV-vis spectra of the arrays are reproducible by a superposition of the absorbance spectra of the individual porphyrins, indicating an undisturbed electronic ground state of the porphyrins in the arrays. The same is true for the steady-state emission spectra of the homoporphyrinic arrays, which are not influenced by the presence of the nucleotide strand. In the mixed porphyrin arrays, large differences in the excited-state properties compared to an equimolar mixture of the building blocks are observed by means that the emission of the diphenyl porphyrin moiety is quenched to a large extent, and the overall emission is dominated by the tetraphenyl porphyrin. The covalent connection of the porphyrins via the DNA-derived backbone therefore substantially alters the excited-state and energy-transfer properties of mixed porphyrin systems. The circular dichroism (CD) spectra show induced negative cotton effects in the region of the porphyrin B-band absorption, which is due to the attachment of the chromophores to the chiral oligonucleotide backbone. Addition of a complementary tetra-adenosine did not alter any of the spectroscopic properties, neither in chloroform nor in acetonitrile solutions. Therefore, it can be concluded that no duplex is formed, which is corroborated by 1H NMR spectroscop

Molecular principles underlying dual RNA specificity in the Drosophila SNF protein

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A′ protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A′ or U2 stem-loop IV and U2A′, SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2A′ immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2A′ can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts