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The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.

ABSTRACT: Double-stranded (ds)DNA, not dsRNA, has an ability to form a homologous complex with single-stranded (ss)DNA or ssRNA of homologous sequence. D-loops and homologous triplexes are homologous complexes formed with ssDNA by RecA/Rad51-family homologous-pairing proteins, and are a key intermediate of homologous (genetic/DNA) recombination. R-loop formation independent of transcription (R-loop formation in trans) was recently found to play roles in gene regulation and development of mammals and plants. In addition, the crRNA-Cas effector complex in CRISPR-Cas systems also relies on R-loop formation to recognize specific target. In homologous complex formation, ssDNA/ssRNA finds a homologous sequence in dsDNA by Watson-Crick base-pairing. crRNA-Cas effector complexes appear to actively melt dsDNA to make its bases available for annealing to crRNA. On the other hand, in D-loop formation and homologous-triplex formation, it is likely that dsDNA recognizes the homologous sequence before the melting of its double helix by using its intrinsic molecular function depending on CH₂ at the 2'-position of the deoxyribose, and that the major role of RecA is the extension of ssDNA and the holding dsDNA at a position suitable for homology search. This intrinsic dsDNA function would also play a role in R-loop formation. The dependency of homologous-complex formation on 2'-CH₂ of the deoxyribose would explain the absence of homologous complex formation by dsRNA, and dsDNA as sole genome molecule in all cellular organisms.

SUBMITTER: Shibata T

PROVIDER: S-EPMC7677664 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.

Shibata Takehiko T Iwasaki Wakana W Hirota Kouji K

Computational and structural biotechnology journal 20201104

Double-stranded (ds)DNA, not dsRNA, has an ability to form a homologous complex with single-stranded (ss)DNA or ssRNA of homologous sequence. D-loops and homologous triplexes are homologous complexes formed with ssDNA by RecA/Rad51-family homologous-pairing proteins, and are a key intermediate of homologous (genetic/DNA) recombination. R-loop formation independent of transcription (R-loop formation in <i>trans</i>) was recently found to play roles in gene regulation and development of mammals an ...[more]

PMID: 33294131

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Project description:BackgroundDNA is a carrier of biological information. The hybridization process, the formation of the DNA double-helix from single-strands with complementary sequences, is important for all living cells. DNA microarrays, among other biotechnologies such as PCR, rely on DNA hybridization. However, to date the thermodynamics of hybridization is only partly understood. Here we address, experimentally and theoretically, the hybridization of oligonucleotide strands of unequal lengths, which form a bulged loop upon hybridization. For our study we use in-house synthesized DNA microarrays.ResultsWe synthesize a microarray with additional thymine bases in the probe sequence motifs so that bulged loops occur upon target hybridization. We observe a monotonic decrease of the fluorescence signal of the hybridized strands with increasing length of the bulged loop. This corresponds to a decrease in duplex binding affinity within the considered loop lengths of one to thirteen bases. By varying the position of the bulged loop along the DNA duplex, we observe a symmetric signal variation with respect to the center of the strand. We reproduce the experimental results well using a molecular zipper model at thermal equilibrium. However, binding states between both strands, which emerge through duplex opening at the position of the bulged loop, need to be taken into account.ConclusionsWe show that stable DNA duplexes with a bulged loop can form from short strands of unequal length and they contribute substantially to the fluorescence intensity from the hybridized strands on a microarray. In order to reproduce the result with the help of equilibrium thermodynamics, it is essential (and to a good approximation sufficient) to consider duplex opening not only at the ends but also at the position of the bulged loop. Although the thermodynamic parameters used in this study are taken from hybridization experiments in solution, these parameters fit our DNA microarray data well.

Project description:After the COVID-19 pandemic, messenger RNA (mRNA) has revolutionized traditional vaccine manufacturing. With the increasing number of RNA-based therapeutics, valuable new scientific insights into these molecules have emerged. One fascinating area of study is the formation of double-stranded RNA (dsRNA) during in vitro transcription (IVT) which is considered a significant impurity, as it has been identified as a major trigger in the cellular immune response pathway. Therefore, there is a growing importance placed to develop and optimize purification processes for the removal of this by-product. Traditionally, efforts have primarily focused on mRNA purification after IVT through chromatographic separations, with anion exchange and reverse phase chromatography emerging as effective tools for this purpose. However, to the best of our knowledge, the influence and significance of the quality of the linearized plasmid have not been thoroughly investigated. Plasmids production involves the growth of bacterial cultures, bacterial harvesting and lysis, and multiple filtration steps for plasmid DNA purification. The inherent complexity of these molecules, along with the multitude of purification steps involved in their processing, including the subsequent linearization and the less-developed purification techniques for linearized plasmids, often result in inconsistent batches with limited control over by-products such as dsRNA. This study aims to demonstrate how the purification process employed for linearized plasmids can impact the formation of dsRNA. Several techniques for the purification of linearized plasmids based on both, resin filtration and chromatographic separations, have been studied. As a result of that, we have optimized a chromatographic method for purifying linearized plasmids using monolithic columns with C4 chemistry (butyl chains located in the surface of the particles), which has proven successful for mRNAs of various sizes. This chromatographic separation facilitates the generation of homogeneous linearized plasmids, leading to mRNA batches with lower levels of dsRNA during subsequent IVT processes. This finding reveals that dsRNA formation is influenced not only by RNA polymerase and IVT conditions but also by the quality of the linearized template. The results suggest that plasmid impurities may contribute to the production of dsRNA by providing additional templates that can be transcribed into sequences that anneal with the mRNA molecules. This highlights the importance of considering the quality of plasmid purification in relation to dsRNA generation during transcription. Further investigation is needed to fully understand the mechanisms and implications of plasmid-derived dsRNA. This discovery could shift the focus in mRNA vaccine production, placing more emphasis on the purification of linearized plasmids and potentially saving, in some instances, a purification step for mRNA following IVT.

Dataset Information

The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.

Publications

The intrinsic ability of double-stranded DNA to carry out D-loop and R-loop formation.

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