Consecutive Base Pairs Research Articles

CircularSTAR3D: a stack-based RNA 3D structural alignment tool for circular matching.

The functions of non-coding RNAs usually depend on their 3D structures. Therefore, comparing RNA 3D structures is critical in analyzing their functions. We noticed an interesting phenomenon that two non-coding RNAs may share similar substructures when rotating their sequence order. To the best of our knowledge, no existing RNA 3D structural alignment tools can detect this type of matching. In this article, we defined the RNA 3D structure circular matching problem and developed a software tool named CircularSTAR3D to solve this problem. CircularSTAR3D first uses the conserved stacks (consecutive base pairs with similar 3D structures) in the input RNAs to identify the circular matched internal loops and multiloops. Then it performs a local extension iteratively to obtain the whole circular matched substructures. The computational experiments conducted on a non-redundant RNA structure dataset show that circular matching is ubiquitous. Furthermore, we demonstrated the utility of CircularSTAR3D by detecting the conserved substructures missed by regular alignment tools, including structural motifs and conserved structures between riboswitches and ribozymes from different classes. We anticipate CircularSTAR3D to be a valuable supplement to the existing RNA 3D structural analysis techniques.

Systematic generation and imaging of tandem repeats reveal base-pairing properties that promote RNA aggregation

A common pathological feature of RNAs containing expanded repeat sequences is their propensity to aggregate in cells. While some repeat RNA aggregates have been shown to cause toxicity by sequestering RNA-binding proteins, the molecular mechanism of aggregation remains unclear. Here, we devised an efficient method to generate long tandem repeat DNAs de novo and applied it to systematically determine the sequence features underlying RNA aggregation. Live-cell imaging of repeat RNAs indicated that aggregation was promoted by multivalent RNA-RNA interactions via either canonical or noncanonical base pairs. While multiple runs of two consecutive base pairs were sufficient, longer runs of base pairs such as those formed by GGGGCC hexanucleotide repeats further enhanced aggregation. In summary, our study provides a unifying model for the molecular basis of repeat RNA aggregation and a generalizable approach for identifying the sequence and structural determinants underlying the distinct properties of repeat DNAs and RNAs.

Affinity and Correlation in DNA

A statistical analysis of important DNA sequences and related proteins has been performed to study the relationships between monomers, and some general considerations about these macromolecules can be provided from the results. First, the most important relationship between sites in all the DNA sequences examined is that between two consecutive base pairs. This is an indication of an energetic stabilization due to the stacking interaction of these couples of base pairs. Secondly, the difference between human chromosome sequences and their coding parts is relevant both in the relationships between sites and in some specific compositional rules, such as the second Chargaff rule. Third, the evidence of the relationship in two successive triplets of DNA coding sequences generates a relationship between two successive amino acids in the proteins. This is obviously impossible if all the relationships between the sites are statistical evidence and do not involve causes; therefore, in this article, due to stacking interactions and this relationship in coding sequences, we will divide the concept of the relationship between sites into two concepts: affinity and correlation, the first with physical causes and the second without. Finally, from the statistical analyses carried out, it will emerge that the human genome is uniform, with the only significant exception being the Y chromosome.

RNA folding using quantum computers.

The 3-dimensional fold of an RNA molecule is largely determined by patterns of intramolecular hydrogen bonds between bases. Predicting the base pairing network from the sequence, also referred to as RNA secondary structure prediction or RNA folding, is a nondeterministic polynomial-time (NP)-complete computational problem. The structure of the molecule is strongly predictive of its functions and biochemical properties, and therefore the ability to accurately predict the structure is a crucial tool for biochemists. Many methods have been proposed to efficiently sample possible secondary structure patterns. Classic approaches employ dynamic programming, and recent studies have explored approaches inspired by evolutionary and machine learning algorithms. This work demonstrates leveraging quantum computing hardware to predict the secondary structure of RNA. A Hamiltonian written in the form of a Binary Quadratic Model (BQM) is derived to drive the system toward maximizing the number of consecutive base pairs while jointly maximizing the average length of the stems. A Quantum Annealer (QA) is compared to a Replica Exchange Monte Carlo (REMC) algorithm programmed with the same objective function, with the QA being shown to be highly competitive at rapidly identifying low energy solutions. The method proposed in this study was compared to three algorithms from literature and, despite its simplicity, was found to be competitive on a test set containing known structures with pseudoknots.

RNA Structural Requirements for Nucleocapsid Protein-Mediated Extended Dimer Formation.

Retroviruses package two copies of their genomic RNA (gRNA) as non-covalently linked dimers. Many studies suggest that the retroviral nucleocapsid protein (NC) plays an important role in gRNA dimerization. The upper part of the L3 RNA stem-loop in the 5′ leader of the avian leukosis virus (ALV) is converted to the extended dimer by ALV NC. The L3 hairpin contains three stems and two internal loops. To investigate the roles of internal loops and stems in the NC-mediated extended dimer formation, we performed site-directed mutagenesis, gel electrophoresis, and analysis of thermostability of dimeric RNAs. We showed that the internal loops are necessary for efficient extended dimer formation. Destabilization of the lower stem of L3 is necessary for RNA dimerization, although it is not involved in the linkage structure of the extended dimer. We found that NCs from ALV, human immunodeficiency virus type 1 (HIV-1), and Moloney murine leukemia virus (M-MuLV) cannot promote the formation of the extended dimer when the apical stem contains ten consecutive base pairs. Five base pairs correspond to the maximum length for efficient L3 dimerization induced by the three NCs. L3 dimerization was less efficient with M-MuLV NC than with ALV NC and HIV-1 NC.

A Predicted Stem Loop in Coat Protein-Coding Sequence of Tobacco Vein Banding Mosaic Virus Is Required for Efficient Replication.

Potyviral coat protein (CP) is involved in the replication and movement of potyviruses. However, little information is available on the roles of CP-coding sequence in potyviral infection. Here, we introduced synonymous substitutions to the codon C574G575C576 coding conserved residue arginine at position 192 (R192) of tobacco vein banding mosaic virus (TVBMV) CP. Substitution of the codon C574G575C576 to A574G575A576 or A574G575G576, but not C574G575A576, C574G575T576, or C574G575G576, reduced the replication, cell-to-cell movement, and accumulation of TVBMV in Nicotiana benthamiana plants, suggesting that C574 was critical for replication of TVBMV. Nucleotides 531 to 576 of the TVBMV CP-coding sequence were predicted to form a stem-loop structure, in which four consecutive C-G base pairs (C576-G531, C532-G575, C574-G533, and C534-G573) were located at the stem. Synonymous substitutions of R178-codon C532G533C534 to A532G533A534 and A532G533G534, but not C532G533A534, C532G533T534, or C532G533G534, reduced the replication levels, cell-to-cell, and systemic movement of TVBMV, suggesting that C532 was critical for TVBMV replication. Synonymous substitutions disrupting base pairs C576-G531 and C534-G573 did not affect viral accumulation. After three serial-passage inoculations, the accumulation of spontaneous mutant viruses was restored, and codons A532G533A534, A532G533G534, A574G575A576, or A574G575G576 of mutants were each separately changed to C532G533A534, C532G533G534, C574G575A576, or C574G575G576. Synonymous mutation of R178 and R192 also reduced viral accumulation in N. tabacum plants. Therefore, we concluded that the two consecutive C532-G575 and C574-G533 base pairs played critical roles in TVBMV replication via maintaining the stability of the stem-loop structures formed by nucleotides 531 to 576 of the CP-coding sequence.

Systematic evolution of initiation factor 3 and the ribosomal protein uS12 optimizes Escherichia coli growth with an unconventional initiator tRNA

The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNAcg/GC/cg ) having cg/GC/cg sequence instead of the conventional GC/GC/GC. Both suppressors have the common mutation of V93A in initiation factor 3 (IF3), and additional mutations of either V32L (Sup-1) or H76L (Sup-2) in small subunit ribosomal protein 12 (uS12). The V93A mutation in IF3 was necessary for relaxed fidelity of i-tRNA selection to sustain on i-tRNAcg/GC/cg though with a retarded growth. Subsequent mutations in uS12 salvaged the retarded growth by enhancing the fidelity of translation. The H76L mutation in uS12 showed better fidelity of i-tRNA selection. However, the V32L mutation compensated for the deficient fidelity of i-tRNA selection by ensuring an efficient fidelity check by ribosome recycling factor (RRF). We reveal unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, elongation factor-G (EF-G), RRF, and Pth (peptidyl-tRNA hydrolase) which, taken together, govern the fidelity of translation in bacteria.

Computational analysis of sense-antisense chimeric transcripts reveals their potential regulatory features and the landscape of expression in human cells.

Many human genes are transcribed from both strands and produce sense-antisense gene pairs. Sense-antisense (SAS) chimeric transcripts are produced upon the coalescing of exons/introns from both sense and antisense transcripts of the same gene. SAS chimera was first reported in prostate cancer cells. Subsequently, numerous SAS chimeras have been reported in the ChiTaRS-2.1 database. However, the landscape of their expression in human cells and functional aspects are still unknown. We found that longer palindromic sequences are a unique feature of SAS chimeras. Structural analysis indicates that a long hairpin-like structure formed by many consecutive Watson-Crick base pairs appears because of these long palindromic sequences, which possibly play a similar role as double-stranded RNA (dsRNA), interfering with gene expression. RNA–RNA interaction analysis suggested that SAS chimeras could significantly interact with their parental mRNAs, indicating their potential regulatory features. Here, 267 SAS chimeras were mapped in RNA-seq data from 16 healthy human tissues, revealing their expression in normal cells. Evolutionary analysis suggested the positive selection favoring sense-antisense fusions that significantly impacted the evolution of their function and structure. Overall, our study provides detailed insight into the expression landscape of SAS chimeras in human cells and identifies potential regulatory features.

Base-Pairs' Correlated Oscillation Effects on the Charge Transfer in Double-Helix B-DNA Molecules.

By introducing a suitable renormalization process, the charge carrier and phonon dynamics of a double-stranded helical DNA molecule are expressed in terms of an effective Hamiltonian describing a linear chain, where the renormalized transfer integrals explicitly depend on the relative orientations of the Watson–Crick base pairs, and the renormalized on-site energies are related to the electronic parameters of consecutive base pairs along the helix axis, as well as to the low-frequency phonons’ dispersion relation. The existence of synchronized collective oscillations enhancing the - orbital overlapping among different base pairs is disclosed from the study of the obtained analytical dynamical equations. The role of these phonon-correlated, long-range oscillation effects on the charge transfer properties of double-stranded DNA homopolymers is discussed in terms of the resulting band structure.

Ultrafast excited state dynamics of silver ion-mediated cytosine-cytosine base pairs in metallo-DNA.

To better understand the nexus between structure and photophysics in metallo-DNA assemblies, the parallel-stranded duplex formed by the all-cytosine oligonucleotide, dC20, and silver nitrate was studied by circular dichroism (CD), femtosecond transient absorption spectroscopy, and time-dependent-density functional theory calculations. Silver(I) ions mediate Cytosine-Cytosine (CC) base pairs by coordinating to the N3 atoms of two cytosines. Although these silver(I) mediated CC base pairs resemble the proton-mediated CC base pairs found in i-motif DNA at first glance, a comparison of experimental and calculated CD spectra reveals that silver ion-mediated i-motif structures do not form. Instead, the parallel-stranded duplex formed between dC20 and silver ions is proposed to contain consecutive silver-mediated base pairs with high propeller twist-like ones seen in a recent crystal structure of an emissive, DNA-templated silver cluster. Femtosecond transient absorption measurements with broadband probing from the near UV to the near IR reveal an unusually long-lived (>10 ns) excited state in the dC20 silver ion complex that is not seen in dC20 in single-stranded or i-motif forms. This state is also absent in a concentrated solution of cytosine-silver ion complexes that are thought to assemble into planar ribbons or sheets that lack stacked silver(I) mediated CC base pairs. The large propeller twist angle present in metal-mediated base pairs may promote the formation of long-lived charged separated or triplet states in this metallo-DNA.

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i-motif (iM) folds occur under slightly acidic conditions when sequences rich in 2'-deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT ) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly-dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH-induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly-dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly-dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values.

LocalSTAR3D: a local stack-based RNA 3D structural alignment tool.

A fast-growing number of non-coding RNA structures have been resolved and deposited in Protein Data Bank (PDB). In contrast to the wide range of global alignment and motif search tools, there is still a lack of local alignment tools. Among all the global alignment tools for RNA 3D structures, STAR3D has become a valuable tool for its unprecedented speed and accuracy. STAR3D compares the 3D structures of RNA molecules using consecutive base-pairs (stacks) as anchors and generates an optimal global alignment. In this article, we developed a local RNA 3D structural alignment tool, named LocalSTAR3D, which was extended from STAR3D and designed to report multiple local alignments between two RNAs. The benchmarking results show that LocalSTAR3D has better accuracy and coverage than other local alignment tools. Furthermore, the utility of this tool has been demonstrated by rediscovering kink-turn motif instances, conserved domains in group II intron RNAs, and the tRNA mimicry of IRES RNAs.

Investigations on crystal structure of a novel 3-((4,6-dimethylpyrimidin-2-yl)amino)isobenzofuran-1(3H)-one, and related theoretical studies

In this report, 3-((4,6-dimethylpyrimidin-2-yl)amino)isobenzofuran-1(3H)-one have been synthesized via reaction between phthalaldehydic acid and 2-amino-4,6-dimethylpyrimidine in 90% yields and characterized by Infrared (IR), Nuclear Magnetic Resonance (NMR), Ultraviolet–visible (UV–Vis), X-ray single crystal diffraction techniques. The single-crystal X-ray analysis shows that the title compound crystallizes in the triclinic space group P-1 with unit-cell parameters a = 7.9351(4) Å, b = 11.1687(6) Å, c = 16.1281(9) Å, α = 73.713(5)°, β = 80.362(5)°, γ = 72.882(4)° and Z = 4. A theoretical study with hybrid functional B3LYP 6-311G (d, p) basis set have been used in calculations. The structural and electronic properties have been detailed. The title compound was screened for its antioxidant activity by (1,1-diphenyl-2-picryl hydrazyl) free radical scavenging (DPPH), Ferric ion reducing antioxidant power (FRAP), total phenolic contents (TP) assays and its ferrous ions chelating property. Electronic absorption titration, thermal denaturation measurement and viscosity techniques were used to determine the interaction between double stranded DNA (dsDNA) and compound 1. In three techniques, the mode of binding of compound 1 to dsDNA is minor groove. The UV–Vis measurement results allowed the calculation of the binding constant showing the binding strength of compound 1 to dsDNA was calculated as 8.13 × 104 ± 0.07 L mol−1. Moreover, the molecular docking calculations have been performed to investigate the compound–DNA interactions, computationally. In molecular docking calculations, it was observed that for the title compound, the lowest energy docking pose takes place in the minor groove of DNA and in addition to minor groove binding, interactions between the compound and the consecutive base pairs of DNA which may cause a partial intercalation were also observed. Results showed that title compound – DNA complex is stabilized by several hydrogen bonds, and Pi-alkyl interactions also take part in the stabilization of the complex. Binding affinities of the lowest energy docking pose of the title compound was found to be −8.3 kcal/mol.

An efficient simulated annealing algorithm for the RNA secondary structure prediction with Pseudoknots

BackgroundRNA pseudoknot structures play an important role in biological processes. However, existing RNA secondary structure prediction algorithms cannot predict the pseudoknot structure efficiently. Although random matching can improve the number of base pairs, these non-consecutive base pairs cannot make contributions to reduce the free energy.ResultIn order to improve the efficiency of searching procedure, our algorithm take consecutive base pairs as the basic components. Firstly, our algorithm calculates and archive all the consecutive base pairs in triplet data structure, if the number of consecutive base pairs is greater than given minimum stem length. Secondly, the annealing schedule is adapted to select the optimal solution that has minimum free energy. Finally, the proposed algorithm is evaluated with the real instances in PseudoBase.ConclusionThe experimental results have been demonstrated to provide a competitive and oftentimes better performance when compared against some chosen state-of-the-art RNA structure prediction algorithms.

Structural Pattern Differences in Unbranched Rod-like RNA of Hepatitis Delta Virus affect RNA Editing.

Hepatitis delta virus (HDV) RNA forms an unbranched rod-like structure and complexes with the delta antigen (HDAg). Host ADAR1-catalyzed RNA editing at the amber/W site of the small HDAg leads to the production of the large HDAg, which inhibits replication and is required for virion assembly. For HDV genotype 1, amber/W editing is controlled by HDAg and the RNA structure immediate vicinity and downstream of the editing site. Here, the effects of 20 mutants carrying an increased length of consecutive base-pairing at various sites in HDV RNA on amber/W site editing were examined. All nine mutants carrying genomic regions that formed up to 15 consecutive base pairs, which is also the maximum length observed in 41 naturally occurring HDV genomes, showed normal editing rate. However, mutants carrying a 16 or 17 consecutive base-paired antigenomic segment located as far as 114 nt upstream could increase editing efficiency, possibly by interfering with HDAg binding. These data show for the first time that extended base-pairing upstream of the amber/W site could increase HDV RNA editing efficiency. Furthermore, it appears that the naturally occurring HDV RNA structures have been selected for suboptimal amber/W RNA editing, which favors the HDV replication cycle.

IntaRNAhelix-composing RNA-RNA interactions from stable inter-molecular helices boosts bacterial sRNA target prediction.

Efficient computational tools for the identification of putative target RNAs regulated by prokaryotic sRNAs rely on thermodynamic models of RNA secondary structures. While they typically predict RNA-RNA interaction complexes accurately, they yield many highly-ranked false positives in target screens. One obvious source of this low specificity appears to be the disability of current secondary-structure-based models to reflect steric constraints, which nevertheless govern the kinetic formation of RNA-RNA interactions. For example, often- even thermodynamically favorable- extensions of short initial kissing hairpin interactions are kinetically prohibited, since this would require unwinding of intra-molecular helices as well as sterically impossible bending of the interaction helix. Another source is the consideration of instable and thus unlikely subinteractions that enable better scoring of longer interactions. In consequence, the efficient prediction methods that do not consider such effects show a high false positive rate. To increase the prediction accuracy we devise IntaRNAhelix, a dynamic programming algorithm that length-restricts the runs of consecutive inter-molecular base pairs (perfect canonical stackings), which we hypothesize to implicitly model the steric and kinetic effects. The novel method is implemented by extending the state-of-the-art tool IntaRNA. Our comprehensive bacterial sRNA target prediction benchmark demonstrates significant improvements of the prediction accuracy and enables more than 40-times faster computations. These results indicate- supporting our hypothesis- that stable helix composition increases the accuracy of interaction prediction models compared to the current state-of-the-art approach.

Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

Nonfilament-forming RecA dimer catalyzes homologous joint formation.

Homologous recombination is essential to genome maintenance, and also to genome diversification. In virtually all organisms, homologous recombination depends on the RecA/Rad51-family recombinases, which catalyze ATP-dependent formation of homologous joints—critical intermediates in homologous recombination. RecA/Rad51 binds first to single-stranded (ss) DNA at a damaged site to form a spiral nucleoprotein filament, after which double-stranded (ds) DNA interacts with the filament to search for sequence homology and to form consecutive base pairs with ssDNA (‘pairing’). How sequence homology is recognized and what exact role filament formation plays remain unknown. We addressed the question of whether filament formation is a prerequisite for homologous joint formation. To this end we constructed a nonpolymerizing (np) head-to-tail-fused RecA dimer (npRecA dimer) and an npRecA monomer. The npRecA dimer bound to ssDNA, but did not form continuous filaments upon binding to DNA; it formed beads-on-string structures exclusively. Although its efficiency was lower, the npRecA dimer catalyzed the formation of D-loops (a type of homologous joint), whereas the npRecA monomer was completely defective. Thus, filament formation contributes to efficiency, but is not essential to sequence-homology recognition and pairing, for which a head-to-tail dimer form of RecA protomer is required and sufficient.

Immune lipoprotein nanostructures inspired relay drug delivery for amplifying antitumor efficiency

Chemo-immunotherapy represents an appealing approach to improving cancer treatment. Simultaneously administrating chemotherapeutics with immunoadjuvants can elicit potent tumor death and immune responses. Herein, high density lipoprotein (HDL) inspired immune lipoprotein was proposed for relay drug delivery and amplifying antitumor therapy. Lipophilic AS1411 aptamer-immunoadjuvant CpG fused sequences (Apt-CpG-DSPE) were conjugated to facilitate decoration onto HDLs; and doxorubicin (Dox) was successively intercalated into the consecutive base pairs of Apt-CpG to complete immune HDL nanodrug imHDL/Apt-CpG-Dox. For relay drug delivery, imHDL/Apt-CpG-Dox underwent site-specific structure collapse in tumor intercellular substances inspired from HDL biofunctions (sequential module I); subsequently, dissociated Apt-CpG-Dox was endocytosed into tumor cells mediated by the recognition of AS1411 and nucleolin (sequential module II), translocating Dox to nucleus and enabling tumor ablation and antigens release. The liberated CpG motif further evoked antigen recognition, induced vast secretion of pro-inflammatory cytokines and potentiated host antitumor immunity. Our studies demonstrated that HDL biomimetic platform based relay drug delivery strategy outperformed the monotherapy counterparts in malignant tumor models, eventually generating an augmented antitumor efficacy.

The sequence preference of DNA cleavage by T4 endonuclease VII

The enzyme T4 endonuclease VII is a resolvase that acts on branched DNA intermediates during genetic recombination, by cleaving DNA with staggered cuts approximately 3–6 bp apart. In this paper, we investigated the sequence preference of this cleavage reaction utilising two different DNA sequences. For the first time, the DNA sequence preference of T4 endonuclease VII cleavage sites has been examined without the presence of a known DNA substrate to mask any inherent nucleotide preference. The use of the ABI3730 platform enables the cleavage site to be determined at nucleotide resolution. We found that T4 endonuclease VII cleaves DNA with a sequence preference. We calculated the frequency of nucleotides surrounding the cleavage sites and found that following nucleotides had the highest incidence: AWTAN*STC, where N* indicates the cleavage site between positions 0 and 1, N is any base, W is A or T, and S is G or C. An A at position −1 and T at position +2 were the most predominant nucleotides at the cleavage site. Using a Sequence Logo method, the sequence TATTAN*CT was derived at the cleavage site. Note that A and T nucleotides were highly preferred 5′ to the cleavage sites in both methods of analysis. It was proposed that the enzyme recognises the narrower minor groove of these consecutive AT base pairs and cleaves DNA 3′ to this feature.