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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 -
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 -
Angewandte Chemie (International Ed. in... Oct 2015All biological information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two... (Review)
Review
All biological information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long-standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi-synthetic life that stores increased information.
Topics: Animals; Artificial Cells; Base Pairing; DNA; DNA Replication; Genetic Code; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Nucleotides; Synthetic Biology
PubMed: 26304162
DOI: 10.1002/anie.201502890 -
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 -
Chemical & Pharmaceutical Bulletin 2018In this review, we have summarized the research effort into the development of unnatural base pairs beyond standard Watson-Crick (WC) base pairs for synthetic biology.... (Review)
Review
In this review, we have summarized the research effort into the development of unnatural base pairs beyond standard Watson-Crick (WC) base pairs for synthetic biology. Prior to introducing our research results, we present investigations by four outstanding groups in the field. Their research results demonstrate the importance of shape complementarity and stacking ability as well as hydrogen-bonding (H-bonding) patterns for unnatural base pairs. On the basis of this research background, we developed unnatural base pairs consisting of imidazo[5',4':4.5]pyrido[2,3-d]pyrimidines and 1,8-naphthyridines, i.e., Im : Na pairs. Since Im bases are recognized as ring-expanded purines and Na bases are recognized as ring-expanded pyrimidines, Im : Na pairs are expected to satisfy the criteria of shape complementarity and enhanced stacking ability. In addition, these pairs have four non-canonical H-bonds. Because of these preferable properties, ImN : NaO, one of the Im : Na pairs, is recognized as a complementary base pair in not only single nucleotide insertion, but also the PCR.
Topics: Base Pairing; Hydrogen Bonding; Naphthyridines; Physical Phenomena; Purines; Pyrimidines; Synthetic Biology
PubMed: 29386463
DOI: 10.1248/cpb.c17-00685 -
Metallomics : Integrated Biometal... Apr 2021Artificial metal base pairs have become increasingly important in nucleic acids chemistry due to their high thermal stability, water solubility, orthogonality to natural... (Review)
Review
Artificial metal base pairs have become increasingly important in nucleic acids chemistry due to their high thermal stability, water solubility, orthogonality to natural base pairs, and low cost of production. These interesting properties combined with ease of chemical and enzymatic synthesis have prompted their use in several practical applications, including the construction of nanomolecular devices, ions sensors, and metal nanowires. Chemical synthesis of metal base pairs is highly efficient and enables the rapid screening of novel metal base pair candidates. However, chemical synthesis is limited to rather short oligonucleotides and requires rather important synthetic efforts. Herein, we discuss recent progress made for the enzymatic construction of metal base pairs that can alleviate some of these limitations. First, we highlight the possibility of generating metal base pairs using canonical nucleotides and then describe how modified nucleotides can be used in this context. We also provide a description of the main analytical techniques used for the analysis of the nature and the formation of metal base pairs together with relevant examples of their applications.
Topics: Base Pairing; Coordination Complexes; DNA-Directed DNA Polymerase; Metals; Nucleic Acids
PubMed: 33791776
DOI: 10.1093/mtomcs/mfab016 -
Analytical Sciences : the International... Mar 2021Anisotropic gold nanoparticles have attracted great interest due to their unique physicochemical properties derived from the shape anisotropy. Manipulation of their... (Review)
Review
Anisotropic gold nanoparticles have attracted great interest due to their unique physicochemical properties derived from the shape anisotropy. Manipulation of their interfacial interactions, and thereby the assembling behaviors are often requisite in their applications ranging from optical sensing and diagnosis to self-assembly. Recently, the control of interfacial force based on base pair stacking of DNA terminals have offered a new avenue to surface engineering of nanostructures. In this review, we focus on the DNA base stacking-induced assembly of anisotropic gold nanoparticles, such as nanorods and nanotriangles. The fundamental aspects of anisotropic gold nanoparticles are provided, including the mechanism of the anisotropic growth, the properties arising from the anisotropic shape, and the construction of DNA-grafted anisotropic gold nanoparticles. Then, the advanced applications of their functional assemblies in biosensing and ordered assembly are summarized, followed by a comparison with gold nanospheres. Finally, conclusions and the direction of outlooks are given including future challenges and opportunities in this field.
Topics: Anisotropy; Base Pairing; Biosensing Techniques; DNA; Gold; Metal Nanoparticles
PubMed: 33071270
DOI: 10.2116/analsci.20SCR02 -
Physical Review Letters Oct 2017We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence...
We present a method of detecting sequence defects by supercoiling DNA with magnetic tweezers. The method is sensitive to a single mismatched base pair in a DNA sequence of several thousand base pairs. We systematically compare DNA molecules with 0 to 16 adjacent mismatches at 1 M monovalent salt and 3.6 pN force and show that under these conditions, a single plectoneme forms and is stably pinned at the defect. We use these measurements to estimate the energy and degree of end-loop kinking at defects. From this, we calculate the relative probability of plectoneme pinning at the mismatch under physiologically relevant conditions. Based on this estimate, we propose that DNA supercoiling could contribute to mismatch and damage sensing in vivo.
Topics: Base Pair Mismatch; Base Pairing; Base Sequence; DNA; DNA, Superhelical; Nucleic Acid Conformation
PubMed: 29053317
DOI: 10.1103/PhysRevLett.119.147801 -
Current Protocols in Nucleic Acid... Dec 2014Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base-pair opening and closing kinetics of individual... (Review)
Review
Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base-pair opening and closing kinetics of individual double-stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state-of-the art techniques and NMR instrumentation, including cryoprobes, is discussed.
Topics: Base Pairing; DNA; Kinetics; Magnetic Resonance Spectroscopy
PubMed: 25501592
DOI: 10.1002/0471142700.nc0720s59 -
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