Hachimoji DNA and RNA: A genetic system with eight building blocks

Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos

Articles Nearest-Neighbor Thermodynamics and NMR of DNA Sequences with Internal A‚A, C‚C, G‚G, and T‚T Mismatches †

ABSTRACT: Thermodynamic measurements are reported for 51 DNA duplexes with A‚A, C‚C, G‚G, and T‚T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37°C is G‚G > T‚T ≈ A‚A > C‚C. The observed stability trend for the closing Watson-Crick pair on the 5′ side of the mismatch is G‚C g C‚G g A‚T g T‚A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC‚GGC to +2.66 kcal/mol for ACT‚ ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of ∆G°3 7 ) 3.3%, ∆H°) 7.4%, ∆S°) 8.1%, and T M ) 1.1°C. The imino proton region of 1-D NMR spectra shows that G‚G and T‚T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases. DNA mismatches occur in vivo due to misincorporation of bases during replication (1), heteroduplex formation during homologous recombination (2), mutagenic chemicals (3, 4), ionizing radiation (5), and spontaneous deamination (6). Knowledge of the thermodynamics of DNA mismatches will be useful for elucidating the mechanisms of polymerase fidelity and mismatch repair efficiency. Moreover, thermodynamic parameters for mismatch formation are important for DNA secondary structure prediction (see http://sun2.science.wayne.edu/∼jslsun2 and http://mfold1.wustl.edu/∼mfold/dna/form1.cgi). Recent work has shown that triplet repeat sequences form transiently stable hairpins that contain like with like base mismatche

Mechanics of the IL2RA Gene Activation Revealed by Modeling and Atomic Force Microscopy

Transcription implies recruitment of RNA polymerase II and transcription factors (TFs) by DNA melting near transcription start site (TSS). Combining atomic force microscopy and computer modeling, we investigate the structural and dynamical properties of the IL2RA promoter and identify an intrinsically negative supercoil in the PRRII region (containing Elf-1 and HMGA1 binding sites), located upstream of a curved DNA region encompassing TSS. Conformational changes, evidenced by time-lapse studies, result in the progressive positioning of curvature apex towards the TSS, likely facilitating local DNA melting. In vitro assays confirm specific binding of the General Transcription Factors (GTFs) TBP and TFIIB over TATA-TSS position, where an inhibitory nucleosome prevented preinitiation complex (PIC) formation and uncontrolled DNA melting. These findings represent a substantial advance showing, first, that the structural properties of the IL2RA promoter are encoded in the DNA sequence and second, that during the initiation process DNA conformation is dynamic and not static

Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA

ABSTRACT: Thermodynamics of 22 oligonucleotides with internal single G‚A mismatches dissolved in 1 M NaCl were determined from absorbance versus temperature melting curves. These data, combined with five literature sequences, were used to derive nearest-neighbor thermodynamic parameters for seven linearly independent trimer sequences with internal G‚A mismatches and Watson-Crick flanking base pairs. The G‚A mismatch parameters predict ∆G°3 7 , ∆H°, ∆S°, and T M with average deviations of 4.4%, 7.4%, 8.0%, and 1.5°C, respectively. The nearest-neighbor parameters show that G‚A mismatch stability is strongly context dependent, and ∆G°3 7 ranges from +1.16 kcal/mol for TGA/AAT to -0.78 kcal/mol for GGC/CAG. In addition, one-dimensional 1 H NMR spectra show that the G‚A pairing geometry is pH and context dependent

Touchdown PCR for increased specificity and sensitivity in PCR amplification

Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs, increasing specificity, sensitivity and yield, without the need for lengthy optimizations and/or the redesigning of primers

Development of novel single-stranded nucleic acid aptamers against the pro-Angiogenic and metastatic enzyme heparanase (HPSE1)

Heparanase is an enzyme involved in extracellular matrix remodelling and heparan sulphate proteoglycan catabolism. It is secreted by metastatic tumour cells, allowing them to penetrate the endothelial cell layer and basement membrane to invade target organs. The release of growth factors at the site of cleaved heparan sulphate chains further enhance the potential of the tumour by encouraging the process of angiogenesis. This leads to increased survival and further proliferation of the tumour. Aptamers are single or double stranded oligonucleotides that recognise specific small molecules, peptides, proteins, or even cells or tissues and have shown great potential over the years as diagnostic and therapeutic agents in anticancer treatment. For the first time, single stranded DNA aptamers were successfully generated against the active heterodimer form of heparanase using a modified SELEX protocol, and eluted based on increasing affinity for the target. Sandwich ELISA assays showed recognition of heparanase by the aptamers at a site distinct from that of a polyclonal HPSE1 antibody. The binding affinities of aptamer to immobilised enzyme were high (7×107 to 8×107 M−1) as measured by fluorescence spectroscopy. Immunohistochemistry and immunofluorescence studies demonstrated that the aptamers were able to recognise heparanase with staining comparable or in some cases superior to that of the HPSE1 antibody control. Finally, matrigel assay demonstrated that aptamers were able to inhibit heparanase. This study provides clear proof of principle concept that nucleic acid aptamers can be generated against heparanase. These reagents may serve as useful tools to explore the functional role of the enzyme and in the future development of diagnostic assays or therapeutic reagents