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Cell Oct 2022The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However,...
The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
Topics: RNA, Guide, CRISPR-Cas Systems; CRISPR-Cas Systems; Endonucleases; Base Pairing; Nucleotides; Gene Editing
PubMed: 36306733
DOI: 10.1016/j.cell.2022.09.026 -
Nature Nov 2017The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in... (Comparative Study)
Comparative Study
The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
Topics: Adenosine Deaminase; Base Pairing; CRISPR-Associated Proteins; Cell Line, Tumor; DNA; DNA Cleavage; Gene Editing; Genome, Human; HEK293 Cells; Humans; Models, Molecular; Polymorphism, Single Nucleotide
PubMed: 29160308
DOI: 10.1038/nature24644 -
Journal of Chemical Theory and... Nov 2022Hoogsteen (HG) base pairs have a transient nature and can be structurally similar to Watson-Crick (WC) base pairs, making their occurrence and thermodynamic stability...
Hoogsteen (HG) base pairs have a transient nature and can be structurally similar to Watson-Crick (WC) base pairs, making their occurrence and thermodynamic stability difficult to determine experimentally. Herein, we employed the restrain-free-energy perturbation-release (R-FEP-R) method to calculate the relative free energy of the WC and HG base pairing modes in isolated and bound DNA systems and predict the glycosyl torsion conformational preference of purine bases. Notably, this method does not require prior knowledge of the transition pathway between the two end states. Remarkably, relatively fast convergence was reached, with results in excellent agreement with experimental data for all the examined DNA systems. The R-REP-R method successfully determined the stability of HG base pairing and more generally, the conformational preference of purine bases, in these systems. Therefore, this computational approach can help to understand the dynamic equilibrium between the WC and HG base pairing modes in DNA.
Topics: Base Pairing; DNA; Thermodynamics; Molecular Conformation; Purines; Nucleic Acid Conformation
PubMed: 36201305
DOI: 10.1021/acs.jctc.2c00848 -
Biochemistry Jun 2019Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four...
Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four additional letters (dP, dZ, dS, and dB). Their analysis demonstrates that the additional letters do not perturb the structure or stability of the base pairs formed between the natural letters and, remarkably, that the new base pairs, dP-dZ and dS-dB, behave virtually identically to the natural base pairs. This unprecedented result convincingly demonstrates that the thermodynamic and structural behavior previously thought to be the purview of only natural DNA is in fact not unique and can be imparted to suitably designed synthetic components. In addition, the first evidence that the eight-letter DNA can be transcribed into RNA by a mutant RNA polymerase is presented, paving the way for the transfer of more information from one biopolymer to another. Along with others working to develop unnatural DNA base pairs for both in vitro and in vivo applications, this work represents an important step toward the expansion of the genetic alphabet, a central goal of synthetic biology, and has profound implications for our understanding of the molecules and forces that can make life possible.
Topics: Base Pairing; DNA; DNA-Directed RNA Polymerases; Nucleotides; Synthetic Biology
PubMed: 31117391
DOI: 10.1021/acs.biochem.9b00274 -
Cold Spring Harbor Perspectives in... Dec 2018RNA molecules are folded into structures and complexes to perform a wide variety of functions. Determination of RNA structures and their interactions is a fundamental... (Review)
Review
RNA molecules are folded into structures and complexes to perform a wide variety of functions. Determination of RNA structures and their interactions is a fundamental problem in RNA biology. Most RNA molecules in living cells are large and dynamic, posing unique challenges to structure analysis. Here we review progress in RNA structure analysis, focusing on methods that use the "cross-link, proximally ligate, and sequence" principle for high-throughput detection of base-pairing interactions in living cells. Beginning with a comparison of commonly used methods in structure determination and a brief historical account of psoralen cross-linking studies, we highlight the important features of cross-linking methods and new biological insights into RNA structures and interactions from recent studies. Further improvement of these cross-linking methods and application to previously intractable problems will shed new light on the mechanisms of the "modern RNA world."
Topics: Base Pairing; Gene Expression Regulation; Nucleic Acid Conformation; RNA
PubMed: 30510063
DOI: 10.1101/cshperspect.a034926 -
Biophysical Journal Oct 2020Genetic information is encoded in the DNA double helix, which, in its physiological milieu, is characterized by the iconical Watson-Crick nucleo-base pairing. Recent NMR...
Genetic information is encoded in the DNA double helix, which, in its physiological milieu, is characterized by the iconical Watson-Crick nucleo-base pairing. Recent NMR relaxation experiments revealed the transient presence of an alternative, Hoogsteen (HG) base pairing pattern in naked DNA duplexes, and estimated its relative stability and lifetime. In contrast with DNA, such structures were not observed in RNA duplexes. Understanding HG base pairing is important because the underlying "breathing" motion between the two conformations can significantly modulate protein binding. However, a detailed mechanistic insight into the transition pathways and kinetics is still missing. We performed enhanced sampling simulation (with combined metadynamics and adaptive force-bias method) and Markov state modeling to obtain accurate free energy, kinetics, and the intermediates in the transition pathway between Watson-Crick and HG base pairs for both naked B-DNA and A-RNA duplexes. The Markov state model constructed from our unbiased MD simulation data revealed previously unknown complex extrahelical intermediates in the seemingly simple process of base flipping in B-DNA. Extending our calculation to A-RNA, for which HG base pairing is not observed experimentally, resulted in relatively unstable, single-hydrogen-bonded, distorted Hoogsteen-like bases. Unlike B-DNA, the transition pathway primarily involved base paired and intrahelical intermediates with transition timescales much longer than that of B-DNA. The seemingly obvious flip-over reaction coordinate (i.e., the glycosidic torsion angle) is unable to resolve the intermediates. Instead, a multidimensional picture involving backbone dihedral angles and distance between hydrogen bond donor and acceptor atoms is required to gain insight into the molecular mechanism.
Topics: Base Pairing; DNA; Hydrogen Bonding; Kinetics; Nucleic Acid Conformation; RNA
PubMed: 32946766
DOI: 10.1016/j.bpj.2020.08.031 -
Biophysical Journal Dec 2022In this perspective, we provide a summary of recent developments in self-assembling three-dimensional (3D) DNA crystals. Starting from the inception of this subfield, we... (Review)
Review
In this perspective, we provide a summary of recent developments in self-assembling three-dimensional (3D) DNA crystals. Starting from the inception of this subfield, we describe the various advancements in structure that have led to an increase in the diversity of macromolecular crystal motifs formed through self-assembly, and we further comment on the future directions of the field, which exploit noncanonical base pairing interactions beyond Watson-Crick. We then survey the current applications of self-assembling 3D DNA crystals in reversibly active nanodevices and materials engineering and provide an outlook on the direction researchers are taking these structures. Finally, we compare 3D DNA crystals with DNA origami and suggest how these distinct subfields might work together to enhance biomolecule structure solution, nanotechnological motifs, and their applications.
Topics: Nucleic Acid Conformation; DNA; Base Pairing; Nanotechnology
PubMed: 36004779
DOI: 10.1016/j.bpj.2022.08.019 -
Molecular Microbiology Feb 2017Dual-function sRNAs are a subgroup of small regulatory RNAs that act on the one hand as base-pairing sRNAs to inhibit or activate target gene expression and on the other... (Review)
Review
Dual-function sRNAs are a subgroup of small regulatory RNAs that act on the one hand as base-pairing sRNAs to inhibit or activate target gene expression and on the other hand as peptide-encoding mRNAs that function either in the same or in another metabolic pathway. Here, we review and compare the five currently known and intensively characterized dual-function sRNAs with regard to their two functions, their biological role, their evolutionary conservation and their requirements for RNA chaperones. Furthermore, we summarize the data available on five potential dual-function sRNAs, whose base-pairing function is well established whereas the role of their encoded peptides has not yet been elucidated. In addition, we provide three examples for RNAs with more than one function that do not fall into the above-mentioned category. With the application of RNAseq, peptidomics and transcriptomics it can be expected that the number of dual-function sRNAs will considerably increase within the next years, thus enhancing our knowledge on the regulatory potential of these RNAs.
Topics: Bacteria; Base Pairing; Gene Expression Regulation, Bacterial; Peptides; RNA, Antisense; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated
PubMed: 27750368
DOI: 10.1111/mmi.13558 -
Biochimie Nov 2011Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. tmRNA uses these two functions to release ribosomes stalled during... (Review)
Review
Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. tmRNA uses these two functions to release ribosomes stalled during translation and target the nascent polypeptides for degradation. This concerted reaction, known as trans-translation, contributes to translational quality control and regulation of gene expression in bacteria. tmRNA is conserved throughout bacteria, and is one of the most abundant RNAs in the cell, suggesting that trans-translation is of fundamental importance for bacterial fitness. Mutants lacking tmRNA activity typically have severe phenotypes, including defects in viability, virulence, and responses to environmental stresses.
Topics: Bacteria; Base Pairing; Gene Expression Regulation, Bacterial; Mutation; Protein Biosynthesis; RNA, Bacterial; Ribosomes; Stress, Physiological
PubMed: 21664408
DOI: 10.1016/j.biochi.2011.05.029 -
FEMS Microbiology Reviews May 2015The increasing numbers of characterized base-pairing small RNAs (sRNAs) and the identification of these regulators in a broad range of bacteria are allowing comparisons... (Review)
Review
The increasing numbers of characterized base-pairing small RNAs (sRNAs) and the identification of these regulators in a broad range of bacteria are allowing comparisons between species and explorations of sRNA evolution. In this review, we describe some examples of trans-encoded base-pairing sRNAs that are species-specific and others that are more broadly distributed. We also describe examples of sRNA orthologs where different features are conserved. These examples provide the background for a discussion of mechanisms of sRNA evolution and selective pressures on the sRNAs and their mRNA target(s).
Topics: Bacteria; Base Pairing; Evolution, Molecular; RNA, Bacterial; RNA, Small Untranslated; Selection, Genetic; Species Specificity
PubMed: 25934120
DOI: 10.1093/femsre/fuv014