ssDNA Oligomers Research Articles

Interactions of the RepA Helicase Hexamer of Plasmid RSF1010 with the ssDNA. Quantitative Analysis of Stoichiometries, Intrinsic Affinities, Cooperativities, and Heterogeneity of the Total ssDNA-binding Site

Interactions between the replicative RepA helicase hexamer of plasmid RSF1010 with the single-stranded DNA (ssDNA) have been studied, using the quantitative fluorescence titration, analytical sedimentation velocity, and sedimentation equilibrium techniques. Experiments were performed with fluorescein-labeled ssDNA oligomers. Studies with unmodified ssDNA oligomers were accomplished using the macromolecular competition titration method. Analyses of RepA helicase interactions with a series of the ssDNA provide direct evidence that the total site-size of the RepA hexamer-ssDNA complex is 19 +/- 1 nucleotide residues. The total ssDNA-binding site of the hexamer has a heterogeneous structure. Part of the total binding site constitutes the proper ssDNA-binding site of the enzyme, an area that possesses strong ssDNA-binding capability and encompasses only 8 +/- 1 residues of the ssDNA. The statistical effect on the macroscopic binding constant for the proper ssDNA-binding site indicates that it is structurally separated from the remaining part of the total ssDNA-binding site. Engagement in interactions with the ssDNA is accompanied by net ion release. Moreover, the proper ssDNA-binding site shows little base specificity. On the other hand, with long ssDNA oligomers, the entire total ssDNA-binding site of the RepA hexamer engages in interactions with the ssDNA resulting in a dramatic change in the nature of interactions with the nucleic acid. The association includes an uptake of ions by the protein. Moreover, unlike the proper-ssDNA-binding site, the total binding site shows a significant preference for pyrimidine oligomers. In this aspect, the RepA helicase is different from the Escherichia coli DnaB hexamer that shows large preference for purine homo-oligomers. In similar solution conditions, the ssDNA intrinsic affinity of the RepA hexamer is similar to the intrinsic affinity of the DnaB helicase. The RepA helicase binds to ssDNA oligomers that can accept more than one RepA hexamer with significant positive cooperative interactions.

Independent and Coordinated Functions of Replication Protein A Tandem High Affinity Single-stranded DNA Binding Domains

The initial high affinity binding of single-stranded DNA (ssDNA) by replication protein A (RPA) is involved in the tandem domains in the central region of the RPA70 subunit (RPA70AB). However, it was not clear whether the two domains, RPA70A and RPA70B, bind DNA simultaneously or sequentially. Here, using primarily heteronuclear NMR complemented by fluorescence spectroscopy, we have analyzed the binding characteristics of the individual RPA70A and RPA70B domains and compared them with the intact RPA70AB. NMR chemical shift comparisons confirmed that RPA70A and RPA70B tumble independently in solution in the absence of ssDNA. NMR chemical shift perturbations showed that all ssDNA oligomers bind to the same sites as observed in the x-ray crystal structure of RPA70AB complexed to d(C)8. Titrations using a variety of 5'-mer ssDNA oligomers showed that RPA70A has a 5-10-fold higher affinity for ssDNA than RPA70B. Detailed analysis of ssDNA binding to RPA70A revealed that all DNA sequences interact in a similar mode. Fluorescence binding measurements with a variety of 8-10'-mer DNA sequences showed that RPA70AB interacts with DNA with approximately 100-fold higher affinity than the isolated domains. Calculation of the theoretical "linkage effect" from the structure of RPA70AB suggests that the high overall affinity for ssDNA is a byproduct of the covalent attachment of the two domains via a short flexible tether, which increases the effective local concentration. Taken together, our data are consistent with a sequential model of DNA binding by RPA according to which RPA70A binds the majority of DNA first and subsequent loading of RPA70B domain is facilitated by the linkage effect.

Solution structure of the ActD-5'-CCGTT3GTGG-3' complex: drug interaction with tandem G.T mismatches and hairpin loop backbone.

Binding of actinomycin D (ActD) to the seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3 GTGG-3' has been studied in solution using high-resolution nuclear magnetic resonance (NMR) techniques. A strong binding constant (8 x 10(6) M(-1)) and high quality NMR spectra have allowed us to determine the initial DNA structure using distance geometry as well as the final ActD-5'-CCGTT3 GTGG-3' complex structure using constrained molecular dynamics calculations. The DNA oligomer 5'-CCGTT3GTGG-3' in the complex forms a hairpin structure with tandem G.T mismatches at the stem region next to a loop of three stacked thymine bases pointing toward the major groove. Bipartite T2O-GH1 and T2O-G2NH2 hydrogen bonds were detected for the G.T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ActD intercalates nicely between the tandem G.T mismatches in essentially one major orientation. Additional hydrophobic interactions between the ActD quinoid amino acid residues with the loop T5-T6-T7 backbone protons were also observed. The hydrophobic G-phenoxazone-G interaction in the ActD-5'-CCGTT3GTGG-3' complex is more robust than that of the classical ActD- 5'-CCGCT3GCGG-3' complex, consistent with the roughly 2-fold stronger binding of ActD to the 5'-CCGTT3GTGG-3' sequence than to its 5'-CCG CT3GCGG-3' counterpart. Stabilization by ActD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ActD or other related compounds may serve as a sequence- specific ssDNA-binding agent that inhibits human immunodeficiency virus (HIV) and other retroviruses replicating through ssDNA intermediates.

Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution.

The free solution mobility of DNA molecules of different molecular weights, the sequence dependence of the mobility, and the diffusion coefficients of small single- and double-stranded DNA (ss- and dsDNA) molecules can be measured accurately by capillary zone electrophoresis, using coated capillaries to minimize the electroosmotic flow (EOF) of the solvent. Very small differences in mobility between various analytes can be quantified if a mobility marker is used to correct for small differences in EOF between successive experiments. Using mobility markers, the molecular weight at which the free solution mobility of dsDNA becomes independent of molecular weight is found to be approximately 170 bp in 40 mM Tris-acetate-EDTA buffer. A DNA fragment containing 170 bp has a contour length of approximately 58 nm, close to the persistence length of DNA under these buffer conditions. Hence, the approach of the free solution mobility of DNA to a plateau value may be associated with the transition from a rod-like to a coil-like conformation in solution. Markers have also been used to determine that the free solution mobilities of ss- and dsDNA oligomers are sequence-dependent. Double-stranded 20-bp oligomers containing runs of three or more adenine residues in a row (A-tracts) migrate somewhat more slowly than 20-mers without A-tracts, suggesting that somewhat larger numbers of counterions are condensed in the ion atmospheres of A-tract DNAs, decreasing their net effective charge. Single-stranded 20-mers with symmetric sequences migrate approximately 1% faster than their double-stranded counterparts, and faster than single-stranded 20-mers containing A(5)- or T(5)-tracts. Interestingly, the average mobility of two complementary single-stranded 20-mers is equal to the mobility of the double-stranded oligomer formed upon annealing. Finally, the stopped migration method has been used to measure the diffusion coefficients of single- and double-stranded oligomers. The diffusion coefficients of ssDNA oligomers containing 20 nucleotides are approximately 50% larger than those of double-stranded DNA oligomers of the same size, reflecting the greater flexibility of ssDNA molecules. The methods used to carry out these experiments are also described in detail.

Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA.

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.

Escherichia coli Replicative Helicase PriA Protein-Single-stranded DNA Complex: STOICHIOMETRIES, FREE ENERGY OF BINDING, AND COOPERATIVITIES

Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.

Interactions of Escherichia coli Replicative Helicase PriA Protein with Single-Stranded DNA

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.

Reduction and oxidation of peptide nucleic acid and DNA at mercury and carbon electrodes

Abstract Peptide nucleic acid (PNA) is a DNA mimic that binds strongly and specifically to complementary DNA or RNA oligomers, but in contrast to DNA its backbone does not carry any electric charge. We used voltammetry in cyclic and square-wave modes to study reduction and oxidation signals of single stranded (ss)PNA and DNA decamers and pentadecamers with the same base sequences at mercury and carbon electrodes. The signals produced by the ssDNA and ssPNA oligomers at the hanging mercury drop electrode (HMDE), i.e. the cathodic peak CA (due to reduction of cytosine and adenine) and the anodic peak G (due to oxidation of the guanine reduction product) corresponded roughly to those observed earlier with ssDNAs. ssPNA peak potentials were more negative compared to DNA. Differences in the signals of ssPNA and ssDNA were explained primarily by different adsorption properties of these compounds. At an accumulation time of 5 min the detection limit of ssPNA was below 5 ng ml −1 . Constant current derivative chronopotentiometric stripping analysis (CPSA) at a pyrolytic graphite electrode produced two well-separated oxidation peaks of guanine and adenine residues in ssDNA and ssPNA in contrast to the poorly developed signals obtained by linear sweep (LS) and square wave (SW) voltammetries. The voltammetric signals were improved greatly as a result of application of a suitable baseline correction method. Using the polynomic method for LSV and moving average baseline correction for SWV, the ssDNA detection limits were comparable to those of CPSA at carbon electrodes as well to those obtained with peak G measurements at the HMDE.

Transition between different binding modes in rat DNA polymerase β-ssDNA complexes

Interactions of rat DNA polymerase β with a single-stranded (ss) DNA have been studied using the quantitative fluorescence titration technique. Examination of the fluorescence changes accompanying the binding, as a function of the thermodynamically rigorous binding density of rat pol β-ssDNA complexes, reveals the existence of two binding phases. In the first high affinity phase, rat pol β forms a complex with the ssDNA in which 16 nucleotides are occluded by the enzyme. In the second low affinity phase, a transition to a complex where the polymerase occludes only five nucleotides occurs. Thus, the data show that rat pol β binds the ssDNA in two binding modes which differ in the number of occluded nucleotides. We designate the first complex as the (pol β) 16 binding mode and the second as the (pol β) 5 binding mode. The formation of the (pol β) 16 and (pol β) 5 modes has been fully confirmed in experiments with short ssDNA oligomers, a 16mer which can form either the (pol β) 16 or the (pol β) 5 mode, and a 10mer which can form only the (pol β) 5 mode. Binding of rat pol β to the ssDNA has been analyzed using a statistical thermodynamic model which accounts for the existence of the two binding modes, cooperative interactions, and the overlap of potential binding sites. The results indicate that the 8 kDa domain of the enzyme is involved in ssDNA binding in both modes. Binding studies show that an isolated 8 kDa domain has the same intrinsic affinity for the ssDNA as the entire intact enzyme in its (pol β) 5 mode. However, the site size of the 8 kDa domain-ssDNA complex is ten nucleotides, suggesting that the formation of the (pol β) 5 mode is accompanied by a significant conformational transition of the intact protein. A higher intrinsic affinity, a higher net number of ions released, and a lower fluorescence change accompanying the formation of the (pol β) 16 than the (pol β) 5 mode indicate that the 31 kDa catalytic domain of the enzyme interacts with the ssDNA only in the (pol β) 16 mode. The significance of these results for understanding the functioning of rat pol β in the DNA metabolism is discussed.

Does Single-stranded DNA Pass through the Inner Channel of the Protein Hexamer in the Complex with the Escherichia coli DnaB Helicase?: FLUORESCENCE ENERGY TRANSFER STUDIES

The structure of the complex of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method. In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis. The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates. Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer. This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution. In the stationary complex with the 5' arm of the replication fork, without ATP hydrolysis, the distance between the 5' end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork. These data indicate that both ssDNA and the 5' arm of the fork bind in the same manner to the DNA binding site. Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation. In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2-3 base pairs. This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork. Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.

CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants.

Foreign DNA has been shown to impinge on immune cell function by an as yet unidentified mechanism. We and others have demonstrated that single-stranded (ss) DNA containing the motif CpG flanked by two 5' purines and two 3' pyrimidines are mitogenic for B cells and activate macrophages to release tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-6 or IL-12. Because of these pro-inflammatory responses we investigated if ssDNA would serve as a potential vaccine adjuvant. Here we show that CpG-containing oligonucleotides represent a powerful adjuvant for both humoral and cellular immune responses. When ssDNA was incorporated into inocula, specific antibody titers of the IgG2 isotype were enhanced by greater than 100-fold. Primary cytotoxic T lymphocyte responses generated to either unprocessed protein antigen or major histocompatibility complex class I-restricted peptide were exceedingly strong. Evidence is also provided that oligomers directly influenced T cell receptor-triggered T cell proliferation. Thus ssDNA oligomers may serve as inexpensive and safe vaccine adjuvants and, in addition, differential effects due to sequence may allow for directed responses.

Structural and thermodynamical analysis of drug binding to single-stranded DNA oligomers Self-association of non-self-complementary deoxytetranucleotides of different base sequence and their complexation with ethidium bromide in aqueous solution

Methods developed for analysing the concentration and temperature dependences of NMR experimental parameters of drug–nucleic acid complexation in solution have been used to study the binding of the drug ethidium bromide (EB) with single-stranded (ss) DNA oligomers. Non-self-complementary (nsc) deoxytetranucleotide triphosphates of different base sequence, 5′-d(CpGpApA), 5′-d(ApApGpC), 5′-d(CpTpGpA) and 5′-d(GpApApG) have been used as model systems of ss nucleotide sequences. 1D and 2D 1 H NMR spectroscopy (500 and 600 MHz) have been used to investigate self-association of the deoxytetranucleotides, and their complexation with the drug in aqueous solution. 2D homonuclear 1 H NMR spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignment of the proton signals of the deoxytetranucleotides and to determine qualitatively the binding sites of the dye with tetramers. Experimental results for self-association of the tetranucleotides have been analysed using the dimer model. It has been shown that there is a relatively low probability of dimer formation for nsc compared with self-complementary (sc) tetranucleotides, so that complexation of the drug with ss tetranucleotides is expected to dominate the complex equilibrium in solution. The results show that dimerisation constants for nsc deoxytetranucleotides depend on the base sequences, being higher when there is the possibility of base-pairing in the tetranucleotide sequence. Thermodynamic parameters ΔG, ΔH and ΔS for the dimerisation reactions of nsc tetranucleotides have also been determined and confirm the role of base sequences in dimer formation. NMR data for EB complexation with nsc deoxytetranucleotides of different base sequence have been interpreted in terms of equilibrium reaction constants and limiting proton chemical shifts of different complexes (1:1, 1:2 and 2:1) in aqueous solution. Analysis of the relative content of the different complexes has been made and specific features of the dynamic equilibrium have been revealed as a function of the ratio of the drug and tetranucleotide concentrations. The results show that there is a sequence-specific binding of EB with ss DNA and that the pyrimidine–purine sequence is preferred, especially the d(CG)-site. However, it is found that the differences in binding affinities of EB to different sites containing alternating base sequence in the chain are not as great as for drug intercalation to the duplex. A much lower probability of binding is observed for formation of EB complexes with sites of ss sequence containing identical types of bases in the chain. The experimentally determined induced chemical shifts have been analysed in terms of the structures of the complexes. The most favourable structures of the 1:1 drug–tetranucleotide complexes have been calculated taking into account that two different orientations of the drug chromophore with respect to its longitudinal axis occur with equal probability in the 1:1 EB–tetranucleotide complexes. The results confirm that complexes of the dye with ss sequences have substantially higher conformational freedom compared to complexes with sc tetranucleotide duplexes. The enthalpies and entropies of complex formation between EB and nsc deoxytetranucleotides have been determined from the temperature dependence of the 500 MHz 1 H NMR chemical shifts. The contributions have been determined for formation of the different types of complexes (1:1, 2:1 and 1:2) in solution. The nature of the intermolecular interactions involved in EB complexation with single strands and dimers of nsc tetramers of different base sequence is discussed.

Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA. The nucleic acid does not wrap around the protein hexamer.

The interactions of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA have been studied using the thermodynamically rigorous fluorescence titration technique, which allowed us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal change and the degree of binding. Binding of the DnaB protein to the ssDNA fluorescent derivative poly(d epsilon A) is accompanied by a strong increase of the nucleic acid fluorescence. We show that, in the presence of the ATP nonhydrolyzable analog AMP-PNP, the DnaB helicase binds polymer ssDNA with the site-size of 20 +/- 3 nucleotides per protein hexamer. This stoichiometry has been fully confirmed in the binding experiments with ssDNA oligomers of 40 and 20 residues in length. Two DnaB hexamers bind to 40-mer, and one DnaB hexamer binds to 20-mer. Thermodynamic studies of the 20-mer binding to the DnaB hexamer show that the hexamer has a single, strong binding site for ssDNA. Moreover, photo-cross-linking experiments indicate that only a single subunit is primarily in contact with ssDNA. This surprisingly very low site-size of the large hexameric helicase--ssDNA complex, the existence of only a single, strong ssDNA binding site on the hexamer, and the results of photo-cross-linking experiments preclude the possibility of extensive wrapping of the ssDNA around the hexamer and formation of the complex in which all six protomers are simultaneously bound to ss nucleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-of-flight mass spectrometry of underivatized single-stranded DNA oligomers by matrix-assisted laser desorption.

Matrix-assisted laser desorption with concomitant ionization (MALDI) in conjunction with time-of-flight mass spectrometry (TOF-MS) has been used to analyze underivatized random-base single-stranded DNA (ssDNA) oligomers ranging from 10 to 89 nucleotides in length by embedding them in a solid matrix of 3-hydroxypicolinic acid. At 355-nm desorption wavelength, mass spectra of positive and negative ions measured by reflecting and linear time-of-flight mass spectrometers are compared. Results from the linear system show the ionization yield is approximately equal for each polarity. Metastable ion decay is significant for the larger ssDNA oligomer ions, which results in a decrease in signal intensity and the broadening of mass peaks. In order to obtain an acceptable signal-to-noise ratio on a reflecting TOF system, a higher laser irradiance is needed, which consequently causes further degradation of mass resolution. With the apparent advantages of better sensitivity and mass resolution, it is concluded that a linear TOF system is better suited for the mass spectrometric analysis of ssDNA oligomers larger than about a 25-mer. The current system permits one-base resolution up to about a 40-mer. Mass accuracy for a 20-mer or smaller is within +/- 0.05%. Comparison of mass spectra from 5-ns and 35-ps pulse widths at the same energy density shows no significant differences. Mechanisms for oligonucleotide ion production in these experiments are discussed.

Use of Single-stranded DNA oligonucleotides in programming ribosomes for translation

Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation. These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin. While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG. However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent. Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA. At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF. We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination. Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis.