Canonical Watson-Crick Base Pairs Research Articles

Harnessing noncanonical crRNA for highly efficient genome editing

The CRISPR-Cas12a system is more advantageous than the widely used CRISPR-Cas9 system in terms of specificity and multiplexibility. However, its on-target editing efficiency is typically much lower than that of the CRISPR-Cas9 system. Here we improved its on-target editing efficiency by simply incorporating 2-aminoadenine (base Z, which alters canonical Watson-Crick base pairing) into the crRNA to increase the binding affinity between crRNA and its complementary DNA target. The resulting CRISPR-Cas12a (named zCRISPR-Cas12a thereafter) shows an on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with much lower off-target effects than the CRISPR-Cas9 system in mammalian cells. In addition, zCRISPR-Cas12a can be used for precise gene knock-in and highly efficient multiplex genome editing. Overall, the zCRISPR-Cas12a system is superior to the CRISPR-Cas9 system, and our simple crRNA engineering strategy may be extended to other CRISPR-Cas family members as well as their derivatives.

Incorporation of noncanonical base Z yields modified mRNA with minimal immunogenicity and improved translational capacity in mammalian cells

Chemically modified mRNAs hold great potential for therapeutic applications invivo. Currently, the base modification scheme largely preserves the canonical Watson-Crick base pairing, thus missing one mode of mRNA modulation by altering its secondary structure. Here we report the incorporation of base Z (2-aminoadenine) into mRNA to create Z-mRNA with improved translational capacity, decreased cytotoxicity, and drastically reduced immunogenicity compared to the unmodified mRNA in mammalian cells. In particular, the A-to-Z substitution renders modified mRNAs less immunogenic than the state-of-the-art base modification N1-methylpseudouridine (m1ψ) in mouse embryonic fibroblast cells. As a proof of concept, we developed a Z-mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antigen-encoding Z-mRNA elicited substantial humoral and cellular immune responses invivo in mice, albeit with relatively lower efficacy than the state-of-the-art m1ψ-mRNA. Z-mRNA expands the scope of mRNA base modifications toward noncanonical bases and could offer an advantageous platform for mRNA-based therapeutics where minimal immunogenicity is desired.

Dynamics and conformational heterogeneity of the PIM1 G-quadruplex from polarizable molecular dynamics simulations.

Reproductive cancers, such as triple-negative breast cancer (TNBC), are often challenging to treat due to a lack of specific chemotherapeutic targets. Overproduction of PIM1, an anti-apoptotic protein, is common in TNBC. Thus, counteracting this overproduction could serve as a viable therapeutic strategy against TNBC. The promoter region of the PIM1 gene can adopt two G-quadruplexes (GQs) with distinct topologies. GQs are higher-order, noncanonical nucleic acid structures that are rich in guanine and have become intensely explored as potential novel drug targets due to their proposed involvement in transcriptional regulation and other critical cellular processes. Two GQ-duplex hybrid structures have been found to exist in equilibrium in the promoter region of PIM1. The dominant GQ structure, form 1, contains conventional guanine tetrads and a duplex loop region of three Watson-Crick base pairs, while form 2 adopts a fold containing a mixed guanine-cytosine (GCGC) tetrad via slippage of two canonical Watson-Crick base pairs, and also contains a duplex loop consisting of two additional Watson-Crick pairs. We performed unbiased simulations and Gaussian-accelerated molecular dynamics simulations of both GQs with the Drude-2017 polarizable force field to better understand the properties of these GQs and how they might interconvert. Given the role of ion binding and noncanonical base pairing in stabilizing GQ structures, we monitored ion coordination and electronic properties (base dipole moments and electric fields). We observed an increased dipole moment for cytosine bases in the GCGC mixed tetrad of form 2, suggesting electronic plasticity as a factor in structural rearrangement between forms 1 and 2. We also report on conformational sampling in each structure to provide atomistic details that will be essential in the future design of more specific chemotherapeutic agents that target PIM1 on the nucleic acid level.

Sarcin/Ricin Domain RNA Retains Its Structure Better Than A-RNA in Adaptively Biased Molecular Dynamics Simulations.

Less than one in thirty of the RNA sequences transcribed in humans are translated into protein. The noncoding RNA (ncRNA) functions in catalysis, structure, regulation, and more. However, for the most part, these functions are poorly characterized. RNA is modular and described by motifs that include helical A-RNA with canonical Watson-Crick base-pairing as well as structures with only noncanonical base pairs. Understanding the structure and dynamics of motifs will aid in deciphering functions of specific ncRNAs. We present computational studies on a standard sarcin/ricin domain (SRD), citrus bark cracking viroid SRD, as well as A-RNA. We have applied enhanced molecular dynamics techniques that construct an inverse free-energy surface (iFES) determined by collective variables that monitor base-pairing and backbone conformation. Each SRD RNA is flanked on each side by A-RNA, allowing comparison of the behavior of these motifs in the same molecule. The RNA iFESs have single peaks, indicating that the combined motifs should denature as a single cohesive unit, rather than by regional melting. Local root-mean-square deviation (RMSD) analysis and communication propensity (CProp, variance in distances between residue pairs) reveal distinct motif properties. Our analysis indicates that the standard SRD is more stable than the viroid SRD, which is more stable than A-RNA. Base pairs at SRD to A-RNA transitions have limited flexibility. Application of CProp reveals extraordinary stiffness of the SRD, allowing residues on opposite sides of the motif to sense each other's motions.

Reconfigurable Two‐Dimensional DNA Lattices: Static and Dynamic Angle Control

AbstractBranched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle‐enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson–Crick base pairing or non‐canonical nucleobase interactions (e.g., i‐motif and G‐quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely‐ordered three‐dimensional architectures.

Reconfigurable Two-Dimensional DNA Lattices: Static and Dynamic Angle Control.

Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle-enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson-Crick base pairing or non-canonical nucleobase interactions (e.g., i-motif and G-quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely-ordered three-dimensional architectures.

Developing an Updated Strategy for Estimating the Free-Energy Parameters in RNA Duplexes

For the last 20 years, it has been common lore that the free energy of RNA duplexes formed from canonical Watson–Crick base pairs (bps) can be largely approximated with dinucleotide bp parameters and a few simple corrective constants that are duplex independent. Additionally, the standard benchmark set of duplexes used to generate the parameters were GC-rich in the shorter duplexes and AU-rich in the longer duplexes, and the length of the majority of the duplexes ranged between 6 and 8 bps. We were curious if other models would generate similar results and whether adding longer duplexes of 17 bps would affect the conclusions. We developed a gradient-descent fitting program for obtaining free-energy parameters—the changes in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS), and the melting temperature (Tm)—directly from the experimental melting curves. Using gradient descent and a genetic algorithm, the duplex melting results were combined with the standard benchmark data to obtain bp parameters. Both the standard (Turner) model and a new model that includes length-dependent terms were tested. Both models could fit the standard benchmark data; however, the new model could handle longer sequences better. We developed an updated strategy for fitting the duplex melting data.

Role of pH in the stability of cytosine-cytosine mismatch and canonical AT and GC base pairs mediated with silver ion: a DFT study

Metallo-nucleic acids have been investigated for their applications in the field of nanodevices and genetic expansion. The cytosine-Ag+-cytosine mismatch base pair interactions and their stability in nucleic acids have attracted the attention of chemists. We report a systematic study of canonical, mismatch, Ag+ mediated system with CC, AT, and GC base pairs computationally. The stability of such mismatch base pairs is dependent on the pH range ~ 5 to 9 and the duplexes beyond this range are unstable. The DFT calculations performed with the model DNA duplexes comprising of such mismatch pairs reveal the stability trend while varying the pH conditions. The stability of canonical Watson–Crick ATGC base pairs was compared with the CCAT and CCGC mismatch base pairs and the calculated results at B3LYP-D3/6-31G(d) level of theory in the aqueous phase suggest that the base stacking and hydrogen bonding are well maintained in the former case, however, the larger perturbations in the geometry are observed with the mispair and relatively unstable. The calculated binding energy B3LYP-D3/6-31G(d) level of theory of ATGC is energetically more stable (~ 20 kcal/mol) than the mismatch base pairs. The Ag+ mediated mismatch base pairs i.e., C_CAT and C_CGC examined at the same level of theory suggest that the CC mismatch base pairs complexed with proper alignment to the Ag+ ion and the AT and GC bases maintained the hydrogen bonding interactions. The relative binding energies have been calculated with 6–311 + G(d,p) basis set using B3LYP-D3 and wb97xd DFT functionals in the study. The mismatched base pair duplex systems i.e., C_CAT and C_CGC are structurally similar to the canonical Watson–Crick base pairs and energetically stable by ~ 40 and ~ 50 kcal/mol compared to the canonical ATGC base pairs. The experimental report on the thermal transition profile in 5’-(A)10C(A)10–3’ and 5’ (T)10C(T)10–3’ duplexes showed remarkable stability and corroborate the calculated results (Ono et al. in Chem Commun 4825−4827, 2008). The stability of Ag+ mediated mismatch bases at the higher pH 9 was also examined and the nucleobases such as guanine and thymine would be deprotonated under this condition. The calculated results suggest that the CCA_T and CCG_C duplexes are largely distorted with the complexation of Ag+ with the AT and GC base pairs and would in turn denature the duplex. The AIM analysis performed at B3LYP-D3/6-31G(d) level of theory for all the studied Ag+ mediated complexes reveals that the Ag+ interaction with the corresponding nucleobases was electrostatic in nature. The role of pH in governing the stability of C–Ag+-C complex formation in mismatch base nucleic acids is crucial for their application of genetic expansion and nucleic acid-based nanodevices.Graphical abstract

A Self‐Assembled Rhombohedral DNA Crystal Scaffold with Tunable Cavity Sizes and High‐Resolution Structural Detail

AbstractDNA is an ideal molecule for the construction of 3D crystals with tunable properties owing to its high programmability based on canonical Watson–Crick base pairing, with crystal assembly in all three dimensions facilitated by immobile Holliday junctions and sticky end cohesion. Despite the promise of these systems, only a handful of unique crystal scaffolds have been reported. Herein, we describe a new crystal system with a repeating sequence that mediates the assembly of a 3D scaffold via a series of Holliday junctions linked together with complementary sticky ends. By using an optimized junction sequence, we could determine a high‐resolution (2.7 Å) structure containing R3 crystal symmetry, with a slight subsequent improvement (2.6 Å) using a modified sticky‐end sequence. The immobile Holliday junction sequence allowed us to produce crystals that provided unprecedented atomic detail. In addition, we expanded the crystal cavities by 50 % by adding an additional helical turn between junctions, and we solved the structure to 4.5 Å resolution by molecular replacement.

A Self-Assembled Rhombohedral DNA Crystal Scaffold with Tunable Cavity Sizes and High-Resolution Structural Detail.

DNA is an ideal molecule for the construction of 3D crystals with tunable properties owing to its high programmability based on canonical Watson-Crick base pairing, with crystal assembly in all three dimensions facilitated by immobile Holliday junctions and sticky end cohesion. Despite the promise of these systems, only a handful of unique crystal scaffolds have been reported. Herein, we describe a new crystal system with a repeating sequence that mediates the assembly of a 3D scaffold via a series of Holliday junctions linked together with complementary sticky ends. By using an optimized junction sequence, we could determine a high-resolution (2.7 Å) structure containing R3 crystal symmetry, with a slight subsequent improvement (2.6 Å) using a modified sticky-end sequence. The immobile Holliday junction sequence allowed us to produce crystals that provided unprecedented atomic detail. In addition, we expanded the crystal cavities by 50 % by adding an additional helical turn between junctions, and we solved the structure to 4.5 Å resolution by molecular replacement.

Functional analysis reveals G/U pairs critical for replication and trafficking of an infectious non-coding viroid RNA.

While G/U pairs are present in many RNAs, the lack of molecular studies to characterize the roles of multiple G/U pairs within a single RNA limits our understanding of their biological significance. From known RNA 3D structures, we observed that the probability a G/U will form a Watson–Crick (WC) base pair depends on sequence context. We analyzed 17 G/U pairs in the 359-nucleotide genome of Potato spindle tuber viroid (PSTVd), a circular non-coding RNA that replicates and spreads systemically in host plants. Most putative G/U base pairs were experimentally supported by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). Deep sequencing PSTVd genomes from plants inoculated with a cloned master sequence revealed naturally occurring variants, and showed that G/U pairs are maintained to the same extent as canonical WC base pairs. Comprehensive mutational analysis demonstrated that nearly all G/U pairs are critical for replication and/or systemic spread. Two selected G/U pairs were found to be required for PSTVd entry into, but not for exit from, the host vascular system. This study identifies critical roles for G/U pairs in the survival of an infectious RNA, and increases understanding of structure-based regulation of replication and trafficking of pathogen and cellular RNAs.

Bypass of the Major Alkylative DNA Lesion by Human DNA Polymerase η.

A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2′-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson–Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson–Crick base pair with dCTP through their keto tautomers.

DNA Surface Technology: From Gene Sensors to Integrated Systems for Life and Materials Sciences.

The evolution of DNA microarray technology has led to sophisticated DNA chips that are being used as routine tools for fundamental and applied genome research such as genotyping and expression profiling. Owing to their capability for highly parallel, site-directed immobilization of complementary nucleic acids through canonical Watson-Crick base-pairing, however, DNA-modified surfaces can also be used for the assembly of complex surface architectures comprised of non-nucleic acid compounds, such as proteins or colloidal materials. Furthermore, implementation of functional DNA devices and structural DNA nanotechnology can unlock the full potential of DNA surfaces. Based on case studies from diagnostics, sensing, proteome research, cell adhesion, and cell signaling, we show how classical gene sensors have developed into modern integrated systems and platforms for various applications in life sciences and materials research.

Selective prebiotic conversion of pyrimidine and purine anhydronucleosides into Watson-Crick base-pairing arabino-furanosyl nucleosides in water

Prebiotic nucleotide synthesis is crucial to understanding the origins of life on Earth. There are numerous candidates for life’s first nucleic acid, however, currently no prebiotic method to selectively and concurrently synthesise the canonical Watson–Crick base-pairing pyrimidine (C, U) and purine (A, G) nucleosides exists for any genetic polymer. Here, we demonstrate the divergent prebiotic synthesis of arabinonucleic acid (ANA) nucleosides. The complete set of canonical nucleosides is delivered from one reaction sequence, with regiospecific glycosidation and complete furanosyl selectivity. We observe photochemical 8-mercaptopurine reduction is efficient for the canonical purines (A, G), but not the non-canonical purine inosine (I). Our results demonstrate that synthesis of ANA may have been facile under conditions that comply with plausible geochemical environments on early Earth and, given that ANA is capable of encoding RNA/DNA compatible information and evolving to yield catalytic ANA-zymes, ANA may have played a critical role during the origins of life.

Molecular mechanism of substrate recognition and specificity of tRNAHis guanylyltransferase during nucleotide addition in the 3'-5' direction.

The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3′–5′ direction onto the 5′-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the −1 position (G−1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis. Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G−1 addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A73 in tRNAHis revealed that the G−1 addition reaction was more efficient onto the template containing the opposite A73 than onto the template with cytidine (C73) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A73, which plays a role in the prevention of continuous templated 3′–5′ polymerization. This study provides important insights into the mechanism of accurate tRNAHis maturation.

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

AbstractMetallo‐base pairs have been extensively studied for applications in nucleic acid‐based nanodevices and genetic code expansion. Metallo‐base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo‐base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing THgIIT base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases CAgIC formed in an RNA duplex. One AgI occupies the center between two cytosines and forms a CAgIC base pair through N3AgIN3 linear coordination. The CAgIC base pair formation does not disturb the standard A‐form conformation of RNA. Since the CAgIC base pair is structurally similar to the canonical Watson–Crick base pairs, it can be a useful building block for structure‐based design and fabrication of nucleic acid‐based nanodevices.

High-Resolution Crystal Structure of a Silver(I)-RNA Hybrid Duplex Containing Watson-Crick-like C-Silver(I)-C Metallo-Base Pairs.

Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T-Hg(II)-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-Ag(I)-C formed in an RNA duplex. One Ag(I) occupies the center between two cytosines and forms a C-Ag(I)-C base pair through N3-Ag(I)-N3 linear coordination. The C-Ag(I)-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-Ag(I)-C base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices.

Stacking geometry for non-canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion-corrected DFT-D study.

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson-Crick base pairs, different orientations of the bases to form hydrogen-bonded non-canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non-canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non-canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non-canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll-slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences.

Enhanced Dynamics of Mismatched Base Pairs Associated with Msh2-Msh6 Recognition

The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner. In eukaryotic cells, the initiation of MMR is achieved by recognition by MutS homologs (Msh). The Msh2-Msh6 heterodimer plays this important initial role in recognizing single base mismatches and small insertion/deletion loops (IDL) in MMR. Although Msh2-Msh6 recognizes mismatched DNA with high affinity, the exact mechanism by which Msh2-Msh6 distinguishes different types of mismatched base pairs from a large excess of canonical Watson-Crick base pairs is still unknown. In this study, we use the intrinsic fluorescent probe 6-methylisoxanthopterin (6-MI, guanosine analog) in the context of the ATFAA (F = 6-MI) pentamer sequence where it exhibits enhanced fluorescence, to measure the binding affinity of S. cerevisiae Msh2-Msh6 to different single base pair mismatches. Fluorescence anisotropy measurements reveal the following order for Msh2-Msh6 mismatch bp binding affinity: A:A ≈ A:G > A:C ≈ G:T ≈ +T > T:T ≈ G:G > T:C ≈ G:C. Fluorescence intensity measurements suggest a greater degree of DNA distortion accompanies binding to well-recognized mismatches. We employ Forster resonance energy transfer to measure the bending angle and compare with that observed in MutS/Msh2-Msh6 co-crystal structures. We have also investigated DNA dynamics upon Msh2-Msh6 binding using time-resolved fluorescence spectroscopy. Specific placement of the probe at the mismatch site or adjacent to it reveals significant local motion prior to protein binding. We observe that high affinity binding is associated with those mismatches that exhibit the greatest amount of motion. Protein binding stabilizes mismatch local motion, which is consistent with Phe intercalation at the site, as observed in Msh2-Msh6-DNA co-crystal structures.

Simple molecular engineering of glycol nucleic acid: Progression from self-pairing to cross-pairing with cDNA and RNA

The acyclic chiral nucleic acid analogue, Glycol Nucleic Acid (GNA), displayed exceptional structural simplicity and atom economy while forming self-paired duplexes, using canonical Watson–Crick base pairing. We disclose here that the replacement of phosphodiester linker in GNA with somewhat rigid and shorter carbamate linker in Glycol Carbamate Nucleic Acid (GCNA) backbone allows unprecedented stability to the antiparallel self-paired duplexes. The R-GCNA oligomers were further found to form cross-paired antiparallel duplexes with cDNA and RNA following Watson–Crick base pairing. The stability of cross-paired GCNA:DNA and GCNA:RNA duplexes was higher than the corresponding DNA:DNA and DNA:RNA duplexes. The chiral (R) and (S) precursors were easily accessible from naturally occurring l-serine.