Precise DNA Cloning via PAMless CRISPR-SpRYgests

While restriction enzymes (REs) remain the gold-standard for manipulating DNA in vitro, they have notable drawbacks including a dependence on short binding motifs that constrain their ability to cleave DNA substrates. Here we overcome limitations of REs by developing an optimized molecular workflow that leverages the PAMless nature of a CRISPR-Cas enzyme named SpRY to cleave DNA at practically any sequence. Using SpRY for DNA digests (SpRYgests), we establish a method that permits the efficient cleavage of DNA substrates at any base pair. We demonstrate the effectiveness of SpRYgests using more than 130 gRNAs, illustrating the versatility of this approach to improve the precision of and simplify several cloning workflows, including those not possible with REs. We also optimize a rapid and simple one-pot gRNA synthesis protocol, which reduces cost and makes the overall SpRYgest workflow comparable to that of RE digests. Together, SpRYgests are straightforward to implement and can be utilized to improve a variety of DNA engineering applications.

A framework for mutational signature analysis based on DNA shape parameters

The mutation risk of a DNA locus depends on its oligonucleotide context. In turn, mutability of oligonucleotides varies across individuals, due to exposure to mutagenic agents or due to variable efficiency and/or accuracy of DNA repair. Such variability is captured by mutational signatures, a mathematical construct obtained by a deconvolution of mutation frequency spectra across individuals. There is a need to enhance methods for inferring mutational signatures to make better use of sparse mutation data (e.g., resulting from exome sequencing of cancers), to facilitate insight into underlying biological mechanisms, and to provide more accurate mutation rate baselines for inferring positive and negative selection. We propose a conceptualization of mutational signatures that represents oligonucleotides via descriptors of DNA conformation: base pair, base pair step, and minor groove width parameters. We demonstrate how such DNA structural parameters can accurately predict mutation occurrence due to DNA repair failures or due to exposure to diverse mutagens such as radiation, chemical exposure, and the APOBEC cytosine deaminase enzymes. Furthermore, the mutation frequency of DNA oligomers classed by structural features can accurately capture systematic variability in mutagenesis of >1,000 tumors originating from diverse human tissues. A nonnegative matrix factorization was applied to mutation spectra stratified by DNA structural features, thereby extracting novel mutational signatures. Moreover, many of the known trinucleotide signatures were associated with an additional spectrum in the DNA structural descriptor space, which may aid interpretation and provide mechanistic insight. Overall, we suggest that the power of DNA sequence motif-based mutational signature analysis can be enhanced by drawing on DNA shape features.

Epidemiological investigation for brucellosis in dogs of Thrissur

India is endemic to bovine brucellosis, and there is a high potential for transmission of disease from ruminants to dogs. A total of 18 bitches belonging to five different breeds at different stage of abortion (30 days to 65 days of gestation) were selected for this study. Majority of them were showing abortion (88.89 per cent) at 45 to 65 days of the gestation. Microscopic examination of Stamp stained smear obtained from the aborted foetal stomach contents revealed red coccobacillary organisms suggestive of Brucella spp.in three cases. RBPT on paired sera samples on day of presentation and three weeks after abortion showed agglutination within four minutes in five out of 18 female dogs. DNA extracted from the aborted tissues of a RBPT positive Labrador dog yielded amplicons of 193 base pair specific for Brucella spp. on PCR. The results obtained from this study stress the need for screening dogs for canine brucellosis in the current brucellosis surveillance and control programmes.

1H NMR chemical exchange techniques reveal local and global effects of oxidized cytosine derivatives

5-carboxycytosine (5caC) is a rare epigenetic modification found in nucleic acids of all domains of life. Despite its sparse genomic abundance, 5caC is presumed to play essential regulatory roles in transcription, maintenance and base-excision processes in DNA. In this work, we utilize nuclear magnetic resonance (NMR) spectroscopy to address the effects of 5caC incorporation into canonical DNA strands at multiple pH and temperature conditions. Our results demonstrate that 5caC has a pH-dependent global destabilizing and a base-pair mobility enhancing local impact on dsDNA, albeit without any detectable influence on the ground-state B-DNA structure. Measurement of hybridization thermodynamics and kinetics of 5caC-bearing DNA duplexes highlighted how acidic environment (pH 5.8 and 4.7) destabilizes the double-stranded structure by ≈10-20 kJ mol-1 at 37 °C when compared to the same sample at neutral pH. Protonation of 5caC results in a lower activation energy for the dissociation process and a higher barrier for annealing. Studies on conformational exchange on the µs time scale regime revealed a sharply localized base-pair motion involving exclusively the modified site and its immediate surroundings. By direct comparison with canonical and 5-formylcytosine (5fC)-edited strands, we were able to address the impact of the two most oxidized naturally occurring cytosine derivatives in the genome. These insights on 5caC's subtle sensitivity to acidic pH contribute to the long standing questions of its capacity as a substrate in base excision repair processes and its purpose as an independent, stable epigenetic mark.

Arginine Depletion in Human Cancers

Arginine is encoded by six different codons. Base pair changes in any of these codons can have a broad spectrum of effects including substitutions to twelve different amino acids, eighteen synonymous changes, and two stop codons. Four amino acids (histidine, cysteine, glutamine, and tryptophan) account for over 75% of amino acid substitutions of arginine. This suggests that a mutational bias, or “purifying selection”, mechanism is at work. This bias appears to be driven by C > T and G > A transitions in four of the six arginine codons, a signature that is universal and independent of cancer tissue of origin or histology. Here, we provide a review of the available literature and reanalyze publicly available data from the Catalogue of Somatic Mutations in Cancer (COSMIC). Our analysis identifies several genes with an arginine substitution bias. These include known factors such as IDH1, as well as previously unreported genes, including four cancer driver genes (FGFR3, PPP6C, MAX, GNAQ). We propose that base pair substitution bias and amino acid physiology both play a role in purifying selection. This model may explain the documented arginine substitution bias in cancers.

Quantum Surprises from the Watson-Crick and Hoogsteen G·C Nucleobase Pairs: A Comprehensive QM/QTAIM Investigation

In this study at the MP2/6-311++G(d,p)//B3LYP/6-311++G(d,p) level of theory in the isolated state it was revealed 14 novel physico-chemical mechanisms of the tautomerization of the G·C nucleotide base pairs in the Watson-Crick G·C(WC) / G*·C*(WC), reverse Watson-Crick G*·C*(rWC) / G·C*O2(rWC), Hoogsteen G*t·C*(H) / G*N7·C(H) or reverse Hoogsteen G*t·C*(rH) / G*tN7·C(rH) configurations into the wobble (wWC, wH) and reverse wobble (rwWC, rwН) base pairs: 1. G·C(WC)↔G·C*(rwWC), 2./3. G*·C*(WC)↔G·C*(rwWC)/G*N2·C*(rwWC), 4. G*·C*(rWC)↔G*·C(wWC), 5. G·C*O2(rWC)↔G·C*(wWC); 6./7./8./9. G*t·C*(H)↔G*t·C(rwН)/G*t·C*O2(wH)/G*t·C*O2(rwН)/G*tN7·C*(rwН)↔G*t·C*O2(rwН), 10. G*N7·C(H)↔G*t·C(wH) amino, 11./12. G*t·C*(rH)↔G*N7·C*(wН)/G*t·C(wН), 13. G*tN7·C(rH)↔G*tN7·C*(wН)↔G*t·C(wН) and 14. G*N7·C*(rwH)↔G*N7·C*(rwH) perp↔G-·C+(wH)↔G*t·C(rwН) reaction pathways. It was established that the presence in the base pair of the two anti-parallel neighboring H-bonds is a necessary and sufficient condition for the implementation of such transformations, since it enables intermolecular proton transfer between the bases inside the base pair. It was found out that these tautomeric transitions are controlled by the TSs with quasi-orthogonal structure, which are tight G+·C-/G-·C+ ion pairs, joined by at least two parallel intermolecular H-bonds, connected on a common negatively charged endocyclic N-/C- atoms – proton acceptor. All reaction pathways have been reliably confirmed. These transitions are accompanied by the changing of the mutual cis-orientation of the N9H and N1H glycosidic bonds of the bases on the trans-orientation and vice versa. These data complement the reported earlier mechanisms of the tautomerisations of the classical A·T and G·C DNA base pairs. Experimental verification of the novel G·C nucleobase pairs is looking as an attractive task for the future research.