TNA synthesis by DNA polymerases

Threose nucleic acid (TNA), which has a repeat unit one atom shorter than that of DNA, is capable of Watson-Crick base pairing with DNA, RNA, and TNA. Because of its chemical simplicity, TNA is considered to be a possible progenitor of RNA. As an initial step toward developing the molecular tools necessary to investigate the functional capabilities of TNA by in vitro selection, we have screened a variety of DNA polymerases for TNA synthesis activity on a DNA template. We wish to report that several polymerases show surprisingly good ability to synthesize TNA using alpha-l-threofuranosyl thymidine-3'-triphosphate as a substrate.     

Crystal structure of a B-form DNA duplex containing (L)-alpha-threofuranosyl (3'-->2') nucleosides: a four-carbon sugar is easily accommodated into the backbone of DNA

(L)-alpha-Threofuranosyl-(3'-->2')-oligonucleotides (TNA) containing vicinally connected phosphodiester linkages undergo informational base pairing in an antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. TNA is derived from a sugar containing only four carbon atoms and is one of the simplest potentially natural nucleic acid alternatives investigated thus far in the context of a chemical etiology of nucleic acid structure. Compared to DNA and RNA that contain six covalent bonds per repeating nucleotide unit, TNA contains only five. We have determined the atomic-resolution crystal structure of the B-form DNA duplex [d(CGCGAA)Td(TCGCG)](2) containing a single (L)-alpha-threofuranosyl thymine (T) per strand. In the modified duplex base stacking interactions are practically unchanged relative to the reference DNA structure. The orientations of the backbone at the TNA incorporation sites are slightly altered in order to accommodate fewer atoms and covalent bonds. The conformation of the threose is C4'-exo with the 2'- and 3'-substituents assuming quasi-diaxial orientation.     

An in vitro selection system for TNA

(3'-2')-alpha-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in vitro selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of selection.     

The structure of a TNA-TNA complex in solution: NMR study of the octamer duplex derived from alpha-(L)-threofuranosyl-(3'-2')-CGAATTCG

TNA (alpha-( l)-threofuranosyl-(3'-2') nucleic acid) is a nucleic acid in which the ribofuranose building block of the natural nucleic acid RNA is replaced by the tetrofuranose alpha-( l)-threose. This shortens the repetitive unit of the backbone by one bond as compared to the natural systems. Among the alternative nucleic acid structures studied so far in our laboratories in the etiological context, TNA is the only one that exhibits Watson-Crick pairing not only with itself but also with DNA and, even more strongly, with RNA. Using NMR spectroscopy, we have determined the structure of a duplex consisting entirely of TNA nucleotides. The TNA octamer (3'-2')-CGAATTCG forms a right-handed double helix with antiparallel strands paired according to the Watson-Crick mode. The dominant conformation of the sugar units has the 2'- and 3'-phosphodiester substituents in quasi-diaxial position and corresponds to a 4'-exo puckering. With 5.85 A, the average sequential P i -P i+1 distances of TNA are shorter than for A-type DNA (6.2 A). The helix parameters, in particular the slide and x-displacement, as well as the shallow and wide minor groove, place the TNA duplex in the structural vicinity of A-type DNA and RNA.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

Sequence‐Specific Incorporation of Enzyme–Nucleotide Chimera by DNA Polymerases

DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High‐fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high‐fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template‐dependent DNA synthesis, despite this “cargo” being more than 100‐fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked‐eye detection of DNA and RNA at single nucleotide resolution.     

2,6-diaminopurine in TNA: effect on duplex stabilities and on the efficiency of template-controlled ligations

Replacement of adenine by 2,6-diaminopurine-two nucleobases to be considered equivalent from an etiological point of view-strongly enhances the stability of TNA/TNA, TNA/RNA, or TNA/DNA duplexes and efficiently accelerates template-directed ligation of TNA ligands. [reaction: see text]     

Base-pairing systems related to TNA: alpha-threofuranosyl oligonucleotides containing phosphoramidate linkages

(3'NH)- and (2'NH)-TNA, two isomeric phosphoramidate analogues of TNA (alpha-threofuranosyl-(3'-->2') oligonucleotides), are shown to be efficient Watson-Crick base-pairing systems and to undergo intersystem cross-pairing with TNA, RNA, and DNA. [reaction: see text]     

Kinetic analysis of an efficient DNA-dependent TNA polymerase

alpha-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day beta-d-2'-deoxyribofuranosyl nucleoside triphosphates (dNTPs). Therminator DNA polymerase, a variant of the 9 degrees N DNA polymerase, is an efficient DNA-directed threosyl nucleic acid (TNA) polymerase. Here we report a detailed kinetic comparison of Therminator-catalyzed TNA and DNA syntheses. We examined the rate of single-nucleotide incorporation for all four tNTPs and dNTPs from a DNA primer-template complex and carried out parallel experiments with a chimeric DNA-TNA primer-DNA template containing five TNA residues at the primer 3'-terminus. Remarkably, no drop in the rate of TNA incorporation was observed in comparing the DNA-TNA primer to the all-DNA primer, suggesting that few primer-enzyme contacts are lost with a TNA primer. Moreover, comparison of the catalytic efficiency of TNA synthesis relative to DNA synthesis at the downstream positions reveals a difference of no greater than 5-fold in favor of the natural DNA substrate. This disparity becomes negligible when the TNA synthesis reaction mixture is supplemented with 1.25 mM MnCl(2). These results indicate that Therminator DNA polymerase can recognize both a TNA primer and tNTP substrates and is an effective catalyst of TNA polymerization despite changes in the geometry of the reactants.     

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarchaeota, two DNA polymerases of B family(polB1 and polB3), and one DNA polymerase of Y family(polIV) were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polB1(Saci_1537) and polB3(Saci_0074) possessed DNA polymerase and 3' to 5' exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV(Saci_0554) was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn²⁺ and Mg²⁺. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KCl with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

Implications of active site constraints on varied DNA polymerase selectivity

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.     

Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.     

The α‐L‐Threofuranosyl‐(3′→2′)‐oligonucleotide System (‘TNA'): Synthesis and Pairing Properties

Our studies of α‐L ‐Threofuranosyl‐(3′→2′)‐oligonucleotides (‘TNA') are part of a systematic experimental inquiry into the base‐pairing properties of potentially natural nucleic acid alternatives taken from RNA's close structural neighborhood. TNA is an efficient Watson‐Crick base‐pairing system and has the capability of informational cross‐pairing with both RNA and DNA. This property, together with the system's constitutional and (presumed) generational simplicity, warrants special scrutiny of TNA in the context of the search for chemical clues to RNA's origin.     

The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been developed whereby alternative hydrogen-bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base-pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.     

Unnatural substrate repertoire of A, B, and X family DNA polymerases

As part of an effort to develop unnatural base pairs that are stable and replicable in DNA, we examined the ability of five different polymerases to replicate DNA containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs. The unnatural pairs were developed based on intensive studies using the Klenow fragment of DNA polymerase I from E. coli (Kf) and found to be recognized to varying degrees. The five additional polymerases characterized here include family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9(o)N-7, and the family X polymerase, human polymerase beta. While we find that some aspects of unnatural base pair recognition are conserved among the polymerases, for example, the pair formed between two d3FB nucleotides is typically well recognized, the detailed recognition of most of the unnatural base pairs is generally polymerase dependent. In contrast, we find that the pair formed between d5SICS and dMMO2 is generally well recognized by all of the polymerases examined, suggesting that the determinants of efficient and general recognition are contained within the geometric and electronic structure of these unnatural nucleobases themselves. The data suggest that while the d3FB:d3FB pair is sufficiently well recognized by several of the polymerases for in vitro applications, the d5SICS:dMMO2 heteropair is likely uniquely promising for in vivo use. T7-mediated replication is especially noteworthy due to strong mispair discrimination.