DNA computing

Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science.     

DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology.     

Dynamics and consequences of DNA looping by the FokI restriction endonuclease

Genetic events often require proteins to be activated by interacting with two DNA sites, trapping the intervening DNA in a loop. While much is known about looping equilibria, only a few studies have examined DNA-looping dynamics experimentally. The restriction enzymes that cut DNA after interacting with two recognition sites, such as FokI, can be used to exemplify looping reactions. The reaction pathway for FokI on a supercoiled DNA with two sites was dissected by fast kinetics to reveal, in turn: the initial binding of a protein monomer to each site; the protein–protein association to form the dimer, trapping the loop; the subsequent phosphodiester hydrolysis step. The DNA motion that juxtaposes the sites ought on the basis of Brownian dynamics to take ~2 ms, but loop capture by FokI took 230 ms. Hence, DNA looping by FokI is rate limited by protein association rather than DNA dynamics. The FokI endonuclease also illustrated activation by looping: it cut looped DNA 400 times faster than unlooped DNA.     

Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting ‘top’ and ‘bottom’ strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.     

Subunit assembly for DNA cleavage by restriction endonuclease SgrAI

The SgrAI endonuclease usually cleaves DNA with two recognition sites more rapidly than DNA with one site, often converting the former directly to the products cut at both sites. In this respect, SgrAI acts like the tetrameric restriction enzymes that bind two copies of their target sites before cleaving both sites concertedly. However, by analytical ultracentrifugation, SgrAI is a dimer in solution though it aggregates to high molecular mass species when bound to its specific DNA sequence. Its reaction kinetics indicate that it uses different mechanisms to cleave DNA with one and with two SgrAI sites. It cleaves the one-site DNA in the style of a dimeric restriction enzyme acting at an individual site, mediating neither interactions in trans, as seen with the tetrameric enzymes, nor subunit associations, as seen with the monomeric enzymes. In contrast, its optimal reaction on DNA with two sites involves an association of protein subunits: two dimers bound to sites in cis may associate to form a tetramer that has enhanced activity, which then cleaves both sites concurrently. The mode of action of SgrAI differs from all restriction enzymes characterised previously, so this study extends the range of mechanisms known for restriction endonucleases.     

Linear DNA must have free ends to transform rat cells efficiently

Summary We have observed that failure to remove certain restriction enzymes after digestion reduced the transforming ability of DNA from 10- to 50-fold. The DNA found integrated in the transformed cells isolated under these conditions had lost little or no sequences. We interpret these results as indicating that certain restriction enzymes remain bound to the DNA ends after digestion, thus generating a substrate unfavorable both for integration and exonucleolytic degradation. As expected from this interpretation, removal of the restriction enzymes before transfection restored the full transforming ability of linear DNA, but also resulted in the integrated sequences being significantly shorter than the transfected DNA. These findings strongly argue for the hypothesis that integration of linear DNA by illegitimate recombination requires free ends and further suggest that exonucleolytic degradation of such ends may generate a preferred substrate for integration. Finally, a comparison of the sequences found integrated after transfection with circular or linear molecules, led us to conclude that circular molecules need not be linearized to become integrated.     

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.     

An MboII/FokI trimming plasmid allowing consecutive cycles of precise 1- to 12-base-pair deletions in cloned DNA

A novel trimming plasmid has been designed which allows, in a preprogrammed fashion, the precise deletion of up to 12 bp per cleavage cycle, from one end of a cloned fragment. The plasmid, which carries the dhfr gene, contains unique recognition sites for two class-IIS restriction enzymes, MboII and FokI, which are arranged in the form of a cassette, so that consecutive cleavages with these endonucleases, followed by blunting with mung bean nuclease (MB), will precisely delete 12 bp of adjacent cloned DNA. When either MboII or FokI is used alone (followed by MB), 1 or 4 bp are removed, respectively. The final step in the trimming cycle is religation of the plasmid with T4 ligase. After required number of cycles, plasmids were transformed into Escherichia coli C600, and transformants selected by resistance to trimethoprim. Since the MboII/FokI cassette remains intact during these operations, one can repeat the cycle, consisting of cleaving, MB blunting and religation, several times, each time removing up to 12 bp from the cloned target DNA. Examples are provided of one-, two- and three-cycle trimmings.     

Unusual base sequence arrangement in phage phi 29 DNA.

Susceptibility of Bacillus subtilis phage phi 29 DNA to 34 different restriction endoculceases was determined. Three enzymes, BglI, XbaI and BstEII, were found to cleave phi 29 DNA only once at specific sites. The sites of these single cleavages have been mapped. Thirteen enzymes did not cut phi 29 DNA. phi 29 HindIII DNA fragments inserted into pBR313 plasmid and propagated in Escherichia coli, were resistant to these restriction endonucleases. This result suggests that the insusceptibility is due to the absence of the nucleotide sequences on phi 29 recognized by the enzymes, and not to the presence of modified nucleotides.     

Specificity of DNA binding and methylation by the M.FokI DNA methyltransferase

The M.FokI adenine-N(6) DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine-N(6) DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M.FokI interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2-3-fold. In addition, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C(m)ATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein-protein interaction assays show that both domains of M.FokI are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding of both domains is mutually exclusive in full-length M.FokI and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification systems are discussed.     

Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region

Plant genomes contain a heavily methylated region in which cytosines are methylated in both the symmetrical and asymmetrical sequences. The physical mapping of such a hypermethylated region is difficult because many restriction enzymes are sensitive to methylated cytosine residues in their recognition sites. The Phi29 DNA polymerase provides an efficient and representative amplification of the genomic DNA that is methylation-free. Using this amplified genomic DNA, we were able to show that a heavily methylated genomic DNA region becomes amenable to physical mapping with any restriction enzymes. This protocol will be especially useful for analysis of the heavily methylated region of plant genomes.     

DNA计算机的分子生物学研究进展

DNA(脱氧核糖核酸)计算机研究是一个新领域。从字面上看,它既包含DNA研究也包含计算机的研究,因而也包含DNA技术与计算机技术如何交融的研究。1994年,Adleman在Science上报道了首例DNA计算的研究结果;2001年,Benenson等在Nature报道了一种由DNA分子和相应的酶分子构成的、有图灵机功能的可程序试管型DNA计算机,标志着DNA计算机研究的重大进展。DNA计算机最大的特点是超大规模的并行运算能力和潜在的巨大的数据储存能力。目前DNA计算机研究已涉及许多领域,包括生物学、数学、物理、化学、计算机科学和自动化工程等具体应用,是计算概念上的一次革命。DNA计算机的研究大大促进了DNA分子操作技术尤其是在纳米尺度下操作DNA分子的研究速度。从DNA计算机的基本原理、应用形式、与基因组学研究的重要关系等方面总结和评述了相关研究进展。     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

FokI requires two specific DNA sites for cleavage

FokI is a bipartite restriction endonuclease that recognizes a non-palindromic DNA sequence, and then makes double-stranded cuts outside of that sequence to leave a 5' overhang. Earlier kinetic and crystallographic studies suggested that FokI might function as a dimer. Here, we show, using dynamic light-scattering, gel-filtration and analytical ultracentrifugation, that FokI dimerizes only in the presence of divalent metal ions. Furthermore, analysis of the DNA-bound complex reveals that two copies of the recognition sequence are incorporated into the dimeric complex and that formation of this complex is essential for full activation of cleavage. These results have broad implications for the mechanism by which monomeric type II endonucleases achieve high fidelity.     

Bromodeoxyuridine substitution in mammalian DNA can both stimulate and inhibit restriction cleavage

Total replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA causes a 5-fold stimulation in the rate of DNA cleavage by Mbo I. This is the first report of the stimulation of restriction endonuclease activity by 5-bromodeoxyuridine and is in contrast to the inhibition found with other restriction enzymes. We propose a hypothesis to rationalize these results.     

Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligodeoxynucleotide and enzyme moieties

Class IIS restriction endonucleases cleave double-stranded (ds) DNA at precise distances from their recognition sequences. A method is proposed which utilizes this separation between the recognition site and the cut site to allow a class IIS enzyme, e.g., FokI, to cleave practically any predetermined sequence by combining the enzyme with a properly designed oligodeoxynucleotide adapter. Such an adapter is constructed from the constant recognition site domain (a hairpin containing the ds sequence, e.g., GGATG CCTAC for FokI) and a variable, single-stranded (ss) domain complementary to the ss sequence to be cleaved (at 9 and 13 nucleotides on the paired strands from the recognition sequence in the example of FokI). The ss sequence designated to be cleaved could be provided by ss phage DNA (e.g., M13), gapped ds plasmids, or supercoiled ds plasmids that were alkali denatured and rapidly neutralized. Combination of all three components, namely the class IIS enzyme, the ss DNA target sequence, and the complementing adapter, would result in target DNA cleavage at the specific predetermined site. The target ss DNA could be converted to the precisely cleaved ds DNA by DNA polymerase, utilizing the adapter oligodeoxynucleotide as primer. This novel procedure represents the first example of changing enzyme specificity by synthetic design. A practically unlimited assortment of new restriction specificities could be produced. The method should have many specific and general applications when its numerous ramifications are exploited.     

一种新的DNA分子克隆方法

DNA分子克隆是基本的分子生物学实验技术,传统的分子克隆方法大多需经过酶切链接过程,但在某些情况下,没有合适的酶切位点往往会成为阻碍克隆进行的障碍.本文描述了一种新的分子克隆方法,称为不依赖酶切和链接的分子克隆（RLIC）.利用RLIC,将3种不同大小的DNA片段克隆到3种不同载体,证明了这种方法的有效性和可靠性.由于该方法不受限制性酶切序列限制,省去了酶切连接步骤,因此具有很大的灵活性和简便性,在分子生物学研究方面有广泛应用前景.     

Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Zinc-finger–FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP–scFokI). DNA cleavage assays revealed that the AZP–scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP–FokI dimer. The DNA cleavage pattern of AZP–scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP–scFokI. Furthermore, we demonstrated that AZP–scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP–scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.     

The methylation status of DNA derived from potato plants recovered from slow growth

The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.