Chemoenzymatic Site-Specific Labeling of DNA and RNA Oligonucleotides by Leveraging the Exclusive Reactivity of Glycine-Modified Nucleotide Analogs.

We report a systematic study on controlling the enzyme activity of a terminal uridylyl transferase (TUTase) called SpCID1, which provides methods to effect site-specific incorporation of a single modified nucleotide analog at the 3'-end of an RNA oligonucleotide (ON). Responsive heterocycle-modified fluorescent UTP probes that are useful in analyzing non-canonical nucleic acid structures and azide- and alkyne-modified UTP analogs that are compatible for chemoenzymatic functionalization were used as study systems. In the first strategy, we balanced the concentration of essential metal ion cofactors (Mg2+ and Mn2+ ions) to restrict the processivity of the enzyme, which gave a very good control on the incorporation of clickable nucleotide analogs. In the second approach, borate that complexes with 2' and 3' oxygen atoms of a ribose sugar was used as a reversibly binding chelator to block repeated addition of nucleotide analogs. Notably, in the presence of heterocycle-modified fluorescent UTPs, we obtained single-nucleotide incorporated RNA products in reasonable yields, while with clickable nucleotides yields were very good. Further, 3'-end azide- and alkyne-labeled RNA ONs were post-enzymatically functionalized by CuAAC and SPAAC reactions with fluorescent probes. These strategies broaden the scope of TUTase in site-specifically installing modifications of different types onto RNA for various applications.

DNA polymerases can process a wide variety of structurally diverse nucleotide substrates, but the molecular basis by which the analogs are processed is not completely understood. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of DNA polymerases in real time and at the atomic level. The nucleotide analogs containing a selenophene, benzofuran, or benzothiophene moiety at the C5 position of 2'-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. KlenTaq (A family DNA polymerase) is sensitive to the size of the modification as it incorporates only one heterobicycle-modified nucleotide into the growing polymer, whereas it efficiently incorporates the selenophene-modified nucleotide analog at multiple positions. Notably, in the single nucleotide incorporation assay, irrespective of the heterocycle size, it exclusively adds a single nucleotide at the 3'-end of a primer, which enabled devising a simple two-step site-specific ON labeling technique. KOD and Vent(exo-) DNA polymerases, belonging to the B family, tolerate all the three modified nucleotides and produce ONs with multiple labels. Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real-time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides.

Antiviral nucleoside analogues have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogues can be limited by drug toxicity because the 5'-triphosphates of these nucleoside analogues (nucleotide analogues) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogues are substrates for the recently discovered human X- and Y-family DNA polymerases. Using presteady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogues approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogues were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3'-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator, while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogues catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (∼10e7 M−1s−1), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. https://doi.org/10.7554/eLife.01775.001 eLife digest The body produces proteins by transcribing DNA (genes) to make messenger RNA, which is then translated to make a protein. Transcription begins when an enzyme called RNA polymerase binds to the DNA and catalyzes the process by which genetic information from the double helix is copied to a complementary RNA transcript, which subsequently becomes the messenger RNA. Because a living cell usually contains only one or a few copies (alleles) of a given gene, molecular fluctuations play a crucial role in cellular transcription. Therefore, studying transcription kinetics at the level of single molecules may provide critical insights into how cells deal with—or even take advantage of—molecular fluctuations. A number of different single-molecule techniques can be used to follow transcription, but these techniques are often relatively slow compared to transcription in living cells, or they suffer from other problems such as only being able to study one step in the transcription process. Now, Zhang, Revyakin et al. have systematically devised a technique called ‘fastFISH’ that is fast enough to track the production of single RNA molecules directly and instantaneously. FastFISH builds on an existing technique called FISH—short for fluorescence in situ hybridization—in which fluorescent molecules are attached to single strands of DNA or RNA. These single strands pair with specific regions of complementary DNA or RNA molecules, and they can be visualized with a fluorescence microscope. However, conventional FISH is a ‘snap-shot’ technique that is not suitable for making real-time observations under physiological conditions. FastFISH relies on single strands of fluorescently labeled DNA and RNA that bind to complementary strands of DNA or RNA extremely quickly, even under physiological conditions, because they contain only three of the four ‘regular’ nucleotides that make up DNA or RNA. As a proof of principle, Zhang, Revyakin et al. used fastFISH to study the kinetics of transcription by the bacteriophage T7 RNA polymerase and were able to measure multiple stages of the transcription cycle in a single-molecule experimental setup. By allowing each stage of transcription to be tracked in real-time at the level of single-molecules, fastFISH will permit a more in-depth analysis of the factors that regulate how genes are expressed as proteins in our cells. Moreover, the ability to design single-strand probes that bind rapidly to DNA and RNA targets could have many additional applications, including new strategies for more efficient gene silencing. https://doi.org/10.7554/eLife.01775.002 Introduction Transcription is the first, and frequently the most regulated step in the flow of genetic information from DNA to protein. Transcription is a dynamic, multi-step process in which RNA polymerase (RNAP) (i) binds to the promoter to form the closed complex (RPc); (ii) melts the promoter to form the open complex (RPo); (iii) performs several abortive cycles of synthesis and release of short 2–12 nt RNA products as the initial transcribing complex (RPitc); (iv) escapes the promoter; (v) undergoes some promoter-proximal pausing; (vi) forms an elongating complex (RDe) whose processivity can either be interrupted by more pauses or stimulated by trailing RNAPs and elongation factors; and, finally (vii) terminates at the end of the transcription unit (reviewed in DeHaseth et al., 1998; Murakami and Darst, 2003; Cheung and Cramer, 2012). Due to the low copy number of genes in a cell (usually one in prokaryotes and two in eukaryotes), molecular fluctuations at any of the above steps may cause large cell-to-cell variability in the amount of the final RNA transcript produced in populations of otherwise genetically identical cells grown under identical conditions, and thus can affect gene expression, and cell physiology (Ogawa, 1993; Raj and Van Oudenaarden, 2009; Yamanaka, 2009; Gupta et al., 2011; Lionnet and Singer, 2012). Therefore, understanding how molecular fluctuations at different steps of the transcription cycle alter transcriptional outcomes is required to dissect the mechanism of gene regulation. Single-molecule techniques are ideally suited to directly monitor molecular fluctuations in multi-step reactions in real-time, without averaging out their inherent stochasticity (Weiss, 1999), and have provided important insights into the dynamics of transcription, unattainable by conventional ensemble methods (Bai et al., 2006). For instance, single-molecule assays based on optical nanomanipulation have revealed pausing and backtracking of RDe (Neuman et al., 2003; Shaevitz et al., 2003; Galburt et al., 2007) and measured the force and the torque generated by RDe (Wang et al., 1998; Ma et al., 2013). Methods based on magnetic nanomanipulation and single-molecule fluorescence resonance energy transfer have probed conformational changes in DNA and RNAP within RPo, and RPitc (Kapanidis et al., 2006; Revyakin et al., 2006; Tang et al., 2009; Chakraborty et al., 2012; Robb et al., 2013). Finally, single-molecule localization studies have probed the dynamics of the initial promoter search by RNAP (Wang et al., 2012; Friedman et al., 2013). However, most current single-molecule methods focus on only one step of transcription, and are not well suited to relate protein-DNA complex assembly and dynamics during multiple stages of the transcription cycle to RNA production. In addition, most methods do not measure RNA production directly, but rather infer it from changes in DNA conformation or movement of RNAP along the DNA. Most recently, single-molecule tracking of key protein-DNA interactions coupled with detection of the RNA production has been demonstrated in the bacterial (Friedman and Gelles, 2012) and human transcription systems (Revyakin et al., 2012). The former study achieved time resolution for RNA detection on the order of ∼10 s, and thus provided a dynamic, quantitative view of the full transcription cycle of bacterial RNAP. However, time scales of many events in transcription are on the order of ∼1 s, particularly under physiological conditions (for instance, the residence time of transcription factors on DNA (reviewed in Hager et al., 2009), the rate of initiation and promoter clearance by RNAPs (Revyakin et al., 2006; Tang et al., 2009), and the expected time delay between cooperatively elongating RNAPs molecules (Epshtein and Nudler, 2003)). Thus, a real-time method for nascent transcript detection at ∼1 s time scales would significantly enhance our ability to dissect the dynamics of the transcription process, and to correlate stochastic fluctuations at different steps with transcriptional outcomes. Currently, the most sensitive and specific methods for detecting RNA transcripts continue to rely on complementary nucleic acid hybridization. However, oligonucleotide probes typically hybridize several orders of magnitude slower than diffusion under physiological conditions (effective rate constant kon less than 105 M−1 s−1, [Chan et al., 1995 and references therein]) and, as a result, do not permit real-time nascent RNA detection with common single-molecule setups, such as total internal reflection fluorescence (TIRF) microscopy. Here we present a fast fluorescence in situ hybridization method (fastFISH) to detect synthesis of nascent RNA transcripts at sub-second time resolution, at the single-molecule level. The method takes advantage of our finding that single-stranded nucleic acid probes with sequences comprised of just three of the four bases (A, U and C for RNA probes, and A, T, and G for the complementary DNA targets) are intrinsically unstructured and, as a result, hybridize at exceptionally fast rates (∼107 M−1s−1) without compromising sequence specificity. As a proof of concept, we applied fastFISH to probe the production of nascent RNA transcripts by the speedy bacteriophage T7 RNA Polymerase (T7 RNAP) in vitro. Furthermore, by using fastFISH in combination with fluorescently labeled RNAP, we dissected the full T7 RNAP transcription cycle (promoter binding, promoter escape, elongation, and termination). FastFISH can be used to study transcription by multi-subunit prokaryotic and eukaryotic RNA polymerases at the level of stochastic molecular interactions. The rules for generating fastFISH probe-target pairs can also be used for gene silencing, gene profiling and bottom-up assembly of nucleic acid nanostructures (Rothemund, 2006). Results Design and characterization of fastFISH hybridization probes To achieve real-time nascent RNA detection, we set out to find a general rule for designing RNA-probe pairs that hybridize at the fastest possible rates. Hybridization of two single-stranded nucleic acid fragments requires unfolding of the fragments into unstructured coils, which then anneal to form an intermolecular base paired helix (Lima et al., 1992 and references therein). In support of this notion, nucleic acid probes with less stable secondary structures show faster hybridization rates to complementary nucleic acid targets (Lima et al., 1992; Kushon et al., 2001; Wang and Drlica, 2004; Gao et al., 2006; Yilmaz et al., 2006). Likewise, decreasing the length of hairpins in conventional molecular beacons increases their hybridization rates (Tsourkas et al., 2003). Thus, we reasoned that intrinsically unstructured nucleic acid oligonucleotide pairs of optimal lengths should have the fastest hybridization kinetics under physiological conditions without compromising specificity. To systematically examine the propensity of RNA sequences to form base-paired secondary structures, we used the nucleic acid structure prediction tool Mfold (Zuker, 2003) to calculate the free energy of self-folding, ΔG37°C, of randomly selected short RNA sequences (N = 338,417). We chose RNA sequence with length of 19 nt and GC content between 0.4 and 0.6 as representative of a typical oligonucleotide primer. We found that ΔG37°C values were mostly negative and widely distributed (−1.7 ± 1.7 kCal mol−1), indicating that an average random 19-mer RNA sequence is mostly structured (Figure 1A). Next, we closely examined a subset of 19-mer RNA sequences that had positive ΔG37°C (>+2 kCal mol−1, N = 2,768, <1% of the total pool of random sequences), and found that these mostly unstructured sequences were composed predominantly of only three bases A, U, and C (∼84% had only 2 G residues or less, significantly less than the 4.75 G residues expected on average, Figure 1—figure supplement 1). The bias towards the lower G-content in the unstructured sequences was not surprising, because guanine is the most potent in base-paring interactions: it forms a triple hydrogen bond with cytosine and a wobble pair with uracil. We then calculated ΔG37°C for randomly picked 19-mers composed of only A, U, and C (N = 99,777, GC content between 0.4 and 0.6, Figure 1A), and found that these AUC-sequences had mostly positive ΔG37°C, with a much narrower distribution (+2.5 ± 0.6 kCal mol−1, Figure 1A). Analysis of other three-base-derived RNA sequences (AUG, CAG, CUG) indicated that AUC-sequences were unique in their largely positive ΔG37°C (Figure 1A). Calculation of ΔG37°C for random DNA 19-mers (that could be used as complementary probes for RNA targets) indicated that ATG-based and ATC-based DNA 19-mers were also significantly less structured than their four-base counterparts (Figure 1A). Therefore, we hypothesized that the use of the AUC alphabet for RNA targets, and ATG alphabet for DNA probes should allow the fastest annealing reactions under physiological temperature of 37°C. Figure 1 with 3 supplements see all Download asset Open asset Design of fastFISH probe-target pairs. (A) Probability distribution of Mfold-calculated free energies of self-folding of randomly selected, single-stranded 19-mer RNA (left) and DNA (right) oligonucleotides, composed of three or four bases. Results of analysis of three independent sets are shown as ‘+’, ‘x’, and ‘○’. About 100,000 three-letter sequences, and about 300,000 four-letter sequences were analyzed in each set. (B) Lempel–Ziv complexity analysis of three-letter 19-mer oligonucleotides (one set of ∼100,000 AUC sequences), four-letter 19-mer oligonucleotides (one set of ∼300,000 AUGC sequences), and all tiling 19-mers from the exome of the human chromosome 22. (C) Single-molecule measurements of hybridization rates of fastFISH probe-target pairs, and the effect of G-residues in the targets on the rates. Left: schematic of experiment. Cy3-labeled 90-base RNA oligonucleotides containing a single target sequence were immobilized on a glass surface through a biotin moiety at the 3′ end. Atto633-labeled DNA probes were injected into the imaging flow cell, and their hybridization was detected using TIRF/CoSMoS to obtain the probe arrival time Twait. Right: table of RNA target sequences, Mfold-calculated free energies of self-folding of RNA targets (ΔGtarget), DNA probes (ΔGprobe), combined energies of targets and probes, and on-rates calculated based on probe Twait and concentrations. (D) Self-quenching approach to reduce fluorescence background from unbound DNA probes in TIRF imaging of probe-target hybridization. Left: schematic of experiment. A quencher (e.g., Iowa Black FQ) is placed on one end of a DNA probe, and a fluorophore (e.g., Cy3) is placed on the opposite end of the DNA probe. The short persistence length of single-stranded DNA (lo ∼0.8 nm, Smith et al., 1996; Dessinges et al., 2002) enables quenching of Cy3. Upon hybridization to the target, the distance between Cy3 and Iowa Black FQ increases due to the larger lo ∼50 nm of the DNA-RNA duplex, leading to an increase of Cy3 fluorescence. Middle: representative TIRF image and a corresponding three-dimensional plot of target-hybridized F1 probes acquired in the presence of unbound, self-quenched F1 probe at 100 nM. Right: same set of molecules imaged in the presence of unbound, unquenched F1 probe at 100 nM. https://doi.org/10.7554/eLife.01775.003 To ensure that the use of the three-base alphabet did not compromise the specificity of probe-target hybridization, we calculated the textual complexity of randomly picked three-letter 19-mers using the algorithm of Lempel and Ziv (Ziv and Lempel, 1976; Kaspar and Schuster, 1987; Orlov and Potapov, 2004). This algorithm, commonly used in lossless data compression programs, calculates the minimal number of operations required to reconstruct a sequence of symbols by copying and inserting segments of an existing sub-sequence. Thus, nucleic acid sequences that contain repetitive elements would have a lower LZ complexity (and would be less specific in hybridization) (Wright and Church, 2002). We found that three-letter 19-mer sequences had complexity indices of 8.1 ± 0.8 (N = 99,777), while four-letter 19-mers had complexity indices of 9.3 ± 0.8 (N = 338,417, Figure 1B). As a comparative reference, we calculated complexity indices for tiling 19-mers in all exons of human chromosome 22 to be 8.9 ± 1.0 (Figure 1B). Importantly, a significant fraction of three-letter 19-mers (∼31%) had complexity indices of 9 and higher, which matched or exceeded the average complexity of human exons. These calculations indicate that a significant fraction of the random probe sequences composed of only three bases nevertheless retain sequence complexity and specificity that match their physiological, four-base derived counterparts. We applied the AUC rule and the complexity filter to generate two candidate probe-target pairs (referred to as F1 and F2). The calculated ΔG37°C for the F1 and F2 pairs were +1.5 and +2.2 kCal mol−1, respectively, for the AUC-based RNA targets, and +1.0 and +0.9 kcal mol−1 for their ATG-based complementary DNA probes (Figure 1C), suggesting that the pairs are mostly unstructured and likely to be fast-hybridizing. Indeed, the F1 and F2 probes annealed to their surface-immobilized RNA targets at exceptionally fast rates—6 × 106 M−1s−1 and 4 × 106 M−1s−1, respectively, as measured by TIRF-based Colocalization Single-Molecule Spectroscopy (CoSMoS, Friedman et al., 2006) (Figure 1C, Figure 1—figure supplement 2). These on-rates were at least 100-fold faster than typical four-base nucleic acid probes of similar lengths reported in literature (Lima et al., 1992; Kushon et al., 2001; Wang and Drlica, 2004; Friedman et al., 2006; Gao et al., 2006; Yilmaz et al., 2006). Consistent with the AUC rule, introducing back one or more G residues into the F2 RNA target sequence decreased the rate of its hybridization to the complementary DNA probe (10-fold reduction for one G residue and 300-fold reduction for four G residues), which correlated with the progressively lower free energies of self-folding (Figure 1C, Figure 1—figure supplement 2D). We conclude that the target sequences designed using the AUC rule, combined with the complexity filter, achieved our goal of generating superior hybridization kinetics for fast transcript detection. Although the F1 and F2 probes were exceptionally fast, detection of RNA on the sub-second time scales would require the use of fluorescent probe concentrations above 100 nM (Figure 1—figure supplement 2D). Such concentrations are generally incompatible with common single-molecule detection setups such as TIRF microscopy, due to high fluorescence background from the freely diffusing, unbound probe molecules. We overcame this problem by attaching a quencher to the single-stranded probe at the end opposite of the fluorophore (Marras et al., 2002). This self-quenching strategy enabled the use of free probes at concentrations up to 1 μM (Figure 1D, Figure 1—figure supplement 3). We believe that, in the absence of the secondary structure, the self-quenching effect was likely mediated by random polymer motion and/or contact quenching (Johansson et al., 2002; Marras et al., 2002). We refer to our method for fast nucleic acid detection using unstructured, sequence specific, self-quenched fluorescent probes as ‘fastFISH’. Real-time single-molecule detection of transcription with fastFISH To demonstrate that fastFISH can detect nascent transcripts in real time at single-molecule resolution, we used the bacteriophage T7 RNAP. This single-subunit RNAP is an excellent test case for fastFISH: at physiological temperature (37°C) it initiates transcripts at an effective rate of at least 1 s−1 (Martin and Coleman, 1987), and elongates transcripts at ∼240 nt s−1 (Golomb and Chamberlin, 1974; Bonner et al., 1994). As a model template, we used a fluorescently labeled linear DNA fragment containing the consensus promoter for T7 RNAP (Milligan et al., 1987) and the F1 target sequence between and from the transcription and at We surface-immobilized the Cy3-labeled DNA a biotin moiety attached to the end of the (Figure In this RNAP molecules were expected to transcription at the the nascent RNA in the from the and the end at with their nascent RNA Figure 2 Download asset Open asset Real-time single-molecule detection of transcription by T7 RNAP using (A) of experiment. DNA containing a single consensus promoter for T7 RNAP or a single promoter were immobilized on a with the promoter transcription towards the free end The the F1 fastFISH target sequence from the promoter to which was expected to for hybridization in the nascent RNA the RNAP (B) analysis of F1 interactions in a representative experiment. Left: Right: (C) and (D) as (A) and but for the F2 target and probe. The the F2 fastFISH target sequence to which was expected to for hybridization in the nascent RNA the RNAP We RNAP, and the self-quenched F1 probe to the imaging and the interactions of the F1 probe with the DNA by TIRF/CoSMoS (Friedman et al., 2006; Revyakin et al., 2012). We reasoned that the F1 target sequence in the nascent RNA would for hybridization the of elongating RNAP (RDe) into the bases of nascent RNA by RNAP and The RDe complex would then on the DNA it at the expected average RNAP elongation rate of nt s−1, the F1 probe would have a ∼1 s of to the nascent transcript at the DNA of hybridization to the RNA would then be as a fluorescent with the DNA Figure the analysis of interactions in a typical single-molecule fastFISH experiment. This analysis all DNA molecules that with a probe for more than 0.4 during the and the between the probe and DNA molecules as a (Revyakin et al., 2012). interactions were as indicated by a at of the In a typical of DNA in a of view with a probe. This was not due to probe hybridization Figure 4 and was likely by the of the DNA due to surface interactions were in a with a DNA containing a promoter et al., indicating that the was due to transcription. We also real-time single-molecule transcription using the other unstructured probe, and identical (Figure these that fastFISH can detect production of nascent transcripts in Single-molecule dynamics of T7 interactions To measure the of real-time detection of nascent transcripts by we a to the and end of each single-molecule transcription We reasoned that the interactions between RNAP molecules and the DNA during promoter and can this Therefore, we fluorescently labeled RNAP with using (Figure supplement and to an imaging flow cell containing immobilized DNA and interactions with the DNA molecules (Figure 3). We RNAP events that between and s, and were (Figure The events were comprised of two events whose well to a single distribution time and a towards events time The of interactions were not by the of the (Figure supplement 2). We the stochastic interactions to be but and RPo, because interactions were with containing a promoter sequence rates of Figure and similar interactions were in the absence of s, Figure Figure 3 with 2 supplements see all Download asset Open asset Single-molecule dynamics of T7 interactions. (A) of experiment. (B) analysis of interactions. Left: promoter DNA in the presence of promoter DNA in the presence of Right: promoter DNA in the absence of (C) of RNAP interactions with containing consensus promoter for a × of at a DNA = nm, imaged at fluorescence time corresponding to the shown on of Left: out in the presence of Right: out in the absence of indicate the of RNAP (D) time of Left: out in the presence of to a of single and is shown in Right: out in the absence of to a single is shown in of the time of interactions on the length of the DNA schematic of experiment. DNA containing segments from to or were and interactions of labeled RNAP were at time of interactions for the three DNA shown in N = for the N = for the and N = for the DNA The were calculated by the to a of single and of the time of interactions the length of the DNA of of RNAP fluorescence during interactions as an of elongation by RNAP. interactions than 0.8 s in were by RNAP = and by RNAP and of RNAP and were for DNA segments of different lengths between RNAP and were set at for the DNA measured in We the of events to be transcription because (i) the events were not in the absence of (Figure (ii) the time of the with the length of the DNA s, s, and s for DNA segments with = and respectively, Figure and (iii) the RNAP fluorescence on average, towards the end of events (Figure with RNAP transcription at the promoter to the surface or and then elongating towards the DNA the of the TIRF We conclude that the time of interactions transcription events which can be used as a to the of nascent RNA detection by and to dissect the full transcription cycle by RNAP. We that the of the plot of RNAP DNA ± × s (Figure an of RNAP elongation rate of nt The of the plot with the time = an of the time that RNAP on the DNA without = ± s, which the of promoter and abortive and and, the time RNAP at the free DNA end Single-molecule of the T7 RNAP transcription cycle with fastFISH To the of nascent RNA detection by and to demonstrate the use of fastFISH in the kinetics of the full transcription we interactions and the production

All four possible stereoisomers of dNTP with regard to deoxyribofuranose C-1' and C-4' carbon atoms were studied as substrates for several template-dependent DNA polymerases and template-independent terminal deoxynucleotidyl transferase. It was shown that DNA polymerases alpha, beta, and epsilon from human placenta and reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus incorporate into the DNA chain only natural beta-D-dNTPs, whereas calf thymus terminal deoxynucleotidyl transferase incorporates two nucleotide residues of alpha-D-dNTP and extends the resulting oligonucleotide in the presence of beta-D-dNTPs. The latter enzyme also extended alpha-anomeric D-oligodeoxynucleotide primers in the presence of beta-D-dNTPs. None of the studied enzymes utilized L-dNTPs. These data indicate that template-dependent DNA polymerases are highly stereospecific with regard to dNTPs, whereas template-independent terminal deoxynucleotidyl transferase shows less stereodifferentiation. It is likely that the active center of the latter enzyme forms no specific contacts with the nucleic bases of both nucleotide substrate and oligonucleotide primer.

The kinetics of double-strand formation were measured by using temperature-jump kinetic techniques for the DNA oligonucleotides dCA5G + dCT5G, the analogous RNA oligonucleotides rCA5G + rCU5G, and the hybrid rCA5G + dCT5G. The DNA oligonucleotides have a faster rate of recombination and a slower rate of dissociation at 12.0 degrees C than the RNA oligonucleotides; the hybrid has about the same recombination rate and a slightly faster dissociation rate than the RNA oligonucleotides. The activation energies for recombination for the DNA and RNA oligonucleotides are both near 0 kcal/mol. The difference in dissociation and recombination activation energies is consistent with the thermodynamic results obtained earlier. The relaxation process is composed of two exponential components for the RNA and hybrid oligonucleotides at temperatures of 12.0 degrees C and lower. One exponential component is observed for these oligonucleotides above 12.0 degrees C and for the DNA oligonucleotides at all temperatures.

During transcription elongation the nascent RNA remains base-paired to the template strand of the DNA before it is displaced and the two strands of the DNA reanneal, resulting in the formation of a transcription "bubble" of approximately 10 bp. To examine how the length of the RNA-DNA hybrid is maintained, we assembled transcription elongation complexes on synthetic nucleic acid scaffolds that mimic the situation in which transcript displacement is compromised and the polymerase synthesizes an extended hybrid. We found that in such complexes bacterial RNA polymerase exhibit an intrinsic endonucleolytic cleavage activity that restores the hybrid to its normal length. Mutations in the region of the RNA polymerase near the site of RNA-DNA separation result in altered RNA displacement and translocation functions and as a consequence in different patterns of proofreading activities. Our data corroborate structural findings concerning the elements involved in the maintenance of the length of the RNA-DNA hybrid and suggest interplay between polymerase translocation, DNA strand separation, and intrinsic endonucleolytic activity.

As interest in oligonucleotide (ON) therapeutics is increasing, there is a need to develop suitable analytical methods able to properly analyze those molecules. However, an issue exists in the adsorption of ONs on different parts of the instrumentation during their analysis. The goal of the present paper was to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IP-RPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA and RNA oligonucleotides. The whole sample flow path was considered under both conditions, including chromatographic columns, ultra-high-performance liquid chromatography (UHPLC) system, and ultraviolet (UV) flow cell. It was found that a negligible amount of non-specific adsorption might be attributable to the chromatographic instrumentation. However, the flow cell of a detector should be carefully subjected to sample-based conditioning, as the material used in the UV flow cell was found to significantly impact the peak shapes of the largest ONs (60- to 100-mer). Most importantly, we found that the choice of column hardware had the most significant impact on the extent of non-specific adsorption. Depending on the material used for the column walls and frits, adsorption can be more or less pronounced. It was proved that any type of bioinert RPLC/HILIC column hardware offered some clear benefits in terms of adsorption in comparison to their stainless-steel counterparts. Finally, the evaluation of a large set of ONs was performed, including a DNA duplex and DNA or RNA ONs having different base composition, furanose sugar, and modifications occurring at the phosphate linkage or at the sugar moiety. This work represents an important advance in understanding the overall ON adsorption, and it helps to define the best combination of materials when analyzing a wide range of unmodified and modified 20-mer DNA and RNA ONs.

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2'-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2'-methylseleno cytidine phosphoramidite and improved the accessibility of the 2'-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2'-selenium derivatization had no effect on the stability of the duplexes with the 3'-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3'-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2'-Se-C and 2'-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2'-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.

Base excision repair (BER) enzymes are attractive targets for antiviral and anticancer agents. A number of nucleotides and nucleotide analogues are potent competitive inhibitors of BER glycosylases when they are incorporated into synthetic oligonucleotides. However, these molecules often are not substrates for DNA polymerases, which limits their utility in cells and as potential therapeutic agents. 1'-Cyano-2'-deoxyuridine (CNdU) is a nanomolar competitive inhibitor of uracil DNA glycosylase. In addition, the respective nucleotide triphosphate is accepted as a substrate by the Klenow fragment (exo(-)) of DNA polymerase I from E. coli. This is the first competitive inhibitor of UDG that is incorporated into DNA by Klenow exo(-), a model replicative polymerase. 1'-Cyano-2'-deoxyuridine (CNdU) and related molecules may prove useful as a new family of therapeutic or experimental agents that target DNA repair by using the cells' polymerase(s) to incorporate them into DNA. A potential benefit of such a mechanism is that multiple incorporations can occur for longer DNA molecules leading to amplification of the inhibitory effect beyond that seen here with short DNA duplexes.

Nucleoside boranephosphonates are nucleotide analogues in which one of the non-bridging oxygen atom of the phosphate part has been replaced by a borane group (-BH3). This modification imparts a wide spectrum of biological activity, e.g., activation of ribonuclease H, resistance to endo- and exonucleases, and their respective triphosphates are good substrates for DNA and RNA polymerases. Nucleoside boranephosphonate derivatives are used in antisense therapy, silencing gene expression using siRNA strategies, and as potential antiviral and anti-cancer prodrugs. Boranephosphonates find also applications as aptamers and as substrates in a new method of DNA sequencing. This review briefly presents potential biological applications of nucleoside boranephosphonates.

Modified 2′-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

We are interested in creating artificial gene repressors based on duplex DNA recognition by nucleic acids rather than polypeptides. An in vitro model system involving repression of bacteriophage T7 RNA polymerase initiation has been employed to demonstrate that certain DNA oligonucleotides can repress transcription by site-specific triple-helix formation at two kinds of homopurine operator sequences [Maher, L. J., III, (1992) Biochemistry 31, 7587-7594]. Recognition in the purine motif is based on antiparallel oligonucleotide binding (G.G.C and T.A.T triplets). Recognition in the pyrimidine motif is based on parallel oligonucleotide binding (C+.G.C and T.A.T base triplets). Using this system, we report that the concentration-dependence of repression by DNA oligonucleotides provides triple-helix inhibition constant (Ki) estimates of approximately 2 x 10(-7) M for both purine motif and pyrimidine motif DNA complexes. RNA oligonucleotides are shown to repress promoters overlapping pyrimidine motif operators (Ki = 6 x 10(-7) M), but not purine motif operators. Although competent to hybridize to complementary single strands, RNA oligonucleotides fail to bind the purine motif operator. Partial substitution of deoxyribose residues tends to rescue repressor activity by RNA oligonucleotides in the purine motif. These results suggest prospects for, and constraints on, natural and artificial RNA-based repressors.

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.

LNA guanine and 2,6-diaminopurine. Synthesis, characterization and hybridization properties of LNA 2,6-diaminopurine containing oligonucleotides