Consecutive Base Pairs Research Articles

A novel form of RNA double helix based on G·U and C·A+ wobble base pairing.

Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double-helical structures of nucleic acids. We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs (W helix) with two G·U and four rare C·A+ wobble pairs. Four adenines within the W helix are N1-protonated and wobble-base-paired with the opposing cytosine through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A+ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson–Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A+ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA nanotechnology and pH biosensors.

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas

ABSTRACTInitiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the Escherichia coli model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.

Reaction Path Averaging: Characterizing the Structural Response of the DNA Double Helix to Electron Transfer.

A polarizable environment, prominently the solvent, responds to electronic changes in biomolecules rapidly. The knowledge of conformational relaxation of the biomolecule itself, however, may be scarce or missing. In this work, we describe in detail the structural changes in DNA undergoing electron transfer between two adjacent nucleobases. We employ an approach based on averaging of tens to hundreds of thousands of nonequilibrium trajectories generated with molecular dynamics simulation, and a reduction of dimensionality suitable for DNA. We show that the conformational response of the DNA proceeds along a single collective coordinate that represents the relative orientation of two consecutive base pairs, namely, a combination of helical parameters shift and tilt. The structure of DNA relaxes on time scales reaching nanoseconds, contributing marginally to the relaxation of energies, which is dominated by the modes of motion of the aqueous solvent. The concept of reaction path averaging (RPA), conveniently exploited in this context, makes it possible to filter out any undesirable noise from the nonequilibrium data, and is applicable to any chemical process in general.

Thermodynamics of DNA with “hump” Morse potential

The thermal denaturation of DNA, i.e. the separation of the two strands is a phenomenon caused by the amplitude of the vibrations of the bases, therefore it is necessary to know how such separation is generated in order to implement alternative model of the melting behavior as a function of nucleotide sequence and therapies to combat the cancer. We propose to use the extended nonlinear Peyrard-Bishop(PB) model of DNA to include an anharmonic potential representing the aromatic stacking interaction between n- and (n-1)-th consecutive base pairs to treat the problem. We use Finite-difference methods for determine the mean value of the displacement for the “hump Morse” potential of the Peyrard-Bishop model of DNA. We show how the extended “pseudo-Schrödinger” combined with finite difference method can be used to obtain the mean value displacements for the thermal denaturation of DNA with “hump” Morse potential.

Noncovalent Binding to DNA: Still a Target in Developing Anticancer Agents.

DNA-binding compounds are of extraordinary importance in medicine, accounting for a substantial portion of antitumor drugs in clinical usage. However, their mechanisms of action remain sometimes incompletely understood. This review critically examines two broad classes of molecules that bind noncovalently to DNA: intercalators and groove binders. Intercalators bind to DNA by inserting their chromophore moiety between two consecutive base pairs, whereas groove binders fit into the grooves of DNA. Noncovalent DNAinteractive drugs can recognize certain supramolecular DNA structures such as the Gquadruplexes found in telomeres and in numerous gene promoters, and they can act as topoisomerase I and II poisons. We discuss how DNA-binding compounds affect transcription and compete with protein factors for binding to consensus binding sites in gene promoters both in vitro and in cultured cancer cells. Moreover, we comment on the design of molecules that can tightly and specifically bind to any desired target DNA, such as various hairpin polyamides which efficacy as chemotherapeutic agents is being evaluated. At present, genome-wide studies, which provide details of events that may influence both cancer progression and therapeutic outcome, are a common way used to analyze the effects of DNA-binding compounds. A conclusive feature that emerges from reviewing the information on DNA-binding compounds is that both natural sources and chemical approaches can be productively used to obtain drugs to manipulate gene expression in cancer cells.

An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3' and 5' ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.

Site-Specific Isotope-Labeling of Inosine Phosphoramidites and NMR Analysis of an Inosine-Containing RNA Duplex.

Structural features and internal dynamics of inosine-containing RNAs are poorly understood. NMR studies of such RNAs require 13 C,15 N-labeling, which cannot be achieved using in vitro transcription as inosine and guanosine are not distinguished by RNA polymerase. Herein, we report the synthesis of an inosine phosphoramidite with selective 13 C8 and 15 N7-isotope incorporation in the base and uniform 13 C-labeling of the ribose. Chemical synthesis of an RNA duplex containing four consecutive IU base pairs with this optimized isotope-labeling scheme greatly simplifies NMR spectra and resolves signal overlap. The absence of detectable NMR signals of imino protons and unusual inter-residue NOE correlations in this RNA indicate deviations from standard A-form geometry, consistent with reduced stability of this duplex seen in UV melting studies compared to its nonedited RNA counterparts. These studies indicate that the introduction of IU base pairs distorts and destabilizes RNA helices significantly compared to the also noncanonical GU base-pairs. Our optimized isotope-labeling scheme enables high-resolution NMR studies of inosine-edited RNAs.

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

A label-free fluorescence turn-on sensor for rapid detection of cysteine

A Hg2+-mediated fluorescence turn-on sensor for cysteine (Cys) detection was developed using the nucleic acid minor groove binding dye DAPI. In this work, two fully complementary DNA sequences, a T-rich single-stranded molecule (ssDNA) and an A-rich single-stranded molecule, were employed to constitute consecutive “AT/TA” base pairs, which could strongly enhance the fluorescence of DAPI. In the absence of cysteine, Hg2+ reacted with T-rich single-stranded DNA and “T–Hg2+–T” base pairs formed, this seriously disrupted consecutive AT base pairs. As a result, the fluorescence of DAPI was not increased efficiently. However, considering that cysteine binds strongly to Hg2+, the structure of the “T–Hg2+–T” complexes was destroyed in the presence of cysteine, resulting in the re-formation of consecutive AT base pairs and increased DAPI fluorescence. Obviously, the amount of cysteine could be easily measured based on the enhancement of DAPI fluorescence, and it took only 20min to complete the whole cysteine-sensing process. Therefore, a label-free fluorescent “turn-on” sensor for the rapid detection of cysteine was designed, and the detection limit of this sensor was as low as 2.4nM, which was much lower than those of the most of the previously reported cysteine sensors.

Structure of the complete bacterial SRP Alu domain.

The Alu domain of the signal recognition particle (SRP) arrests protein biosynthesis by competition with elongation factor binding on the ribosome. The mammalian Alu domain is a protein–RNA complex, while prokaryotic Alu domains are protein-free with significant extensions of the RNA. Here we report the crystal structure of the complete Alu domain of Bacillus subtilis SRP RNA at 2.5 Å resolution. The bacterial Alu RNA reveals a compact fold, which is stabilized by prokaryote-specific extensions and interactions. In this ‘closed’ conformation, the 5′ and 3′ regions are clamped together by the additional helix 1, the connecting 3-way junction and a novel minor groove interaction, which we term the ‘minor-saddle motif’ (MSM). The 5′ region includes an extended loop–loop pseudoknot made of five consecutive Watson–Crick base pairs. Homology modeling with the human Alu domain in context of the ribosome shows that an additional lobe in the pseudoknot approaches the large subunit, while the absence of protein results in the detachment from the small subunit. Our findings provide the structural basis for purely RNA-driven elongation arrest in prokaryotes, and give insights into the structural adaption of SRP RNA during evolution.

Analysis of stacking overlap in nucleic acid structures: algorithm and application.

RNA contains different secondary structural motifs like pseudo-helices, hairpin loops, internal loops, etc. in addition to anti-parallel double helices and random coils. The secondary structures are mainly stabilized by base-pairing and stacking interactions between the planar aromatic bases. The hydrogen bonding strength and geometries of base pairs are characterized by six intra-base pair parameters. Similarly, stacking can be represented by six local doublet parameters. These dinucleotide step parameters can describe the quality of stacking between Watson-Crick base pairs very effectively. However, it is quite difficult to understand the stacking pattern for dinucleotides consisting of non canonical base pairs from these parameters. Stacking interaction is a manifestation of the interaction between two aromatic bases or base pairs and thus can be estimated best by the overlap area between the planar aromatic moieties. We have calculated base pair overlap between two consecutive base pairs as the buried van der Waals surface between them. In general, overlap values show normal distribution for the Watson-Crick base pairs in most double helices within a range from 45 to 50Å(2) irrespective of base sequence. The dinucleotide steps with non-canonical base pairs also are seen to have high overlap value, although their twist and few other parameters are rather unusual. We have analyzed hairpin loops of different length, bulges within double helical structures and pseudo-continuous helices using our algorithm. The overlap area analyses indicate good stacking between few looped out bases especially in GNRA tetraloop, which was difficult to quantitatively characterise from analysis of the base pair or dinucleotide step parameters. This parameter is also seen to be capable to distinguish pseudo-continuous helices from kinked helix junctions.

Fulfilling the dream of a perfect genome editing tool.

Adaptive nucleic acid-based immune systems of bacteria and archaea provided new tools for genome editing applications and transcription silencing. Despite the rapid success in developing targetable nucleases based on the clustered regularly interspersed short palindromic repeats (CRISPR)-associated (Cas) enzymes, our understanding of how these ribonucleoprotein complexes recognize and process their DNA targets remains rudimentary. A report in PNAS (1) delves into the mechanism of R-loop formation by two very different CRISPR/Cas systems, Cas9 and Cascade (CRISPR-associated complex for antiviral defense). Our ability to tailor the genomes of various organisms has been revolutionized by the development of targetable nucleases with flexible specificity (2). These powerful reagents generate double-strand breaks (DSBs) in the chromosomal DNA with exquisite specificity. Once generated, the DSB is repaired through nonhomologous end-joining or through homologous recombination. The former often leads to gene inactivation, whereas the latter incorporates the desired genetic alterations by using the information from an exogenously supplied template to introduce, correct, or disrupt specific genes. Three types of targetable nucleases are currently in use: zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and the RNA-guided nucleases. Zinc-finger nucleases consist of multiple Zn2+-finger DNA-binding modules selected or engineered to confer specificity for a particular triplet of consecutive base pairs. DNA recognition modules of TALENs are derived from transcription factors found in plant-pathogenic bacteria. Each DNA-binding module of a TALEN specifically interacts with a single base pair, yielding a straightforward recognition code. Two sets of zinc-finger arrays or transcription activator-like effector arrays are engineered to bind the two adjacent DNA sites in a head-to-head orientation, and are fused to the nuclease domains of FokI restriction enzyme. Dimerization activates FokI, allowing it to generate the desired DSB. Requirement for FokI dimerization is an important contributor to cutting specificity, as DSB can only occur when both DNA sequence … [↵][1]1Email: maria-spies{at}uiowa.edu. [1]: #xref-corresp-1-1

A general pairwise interaction model provides an accurate description of in vivo transcription factor binding sites.

The identification of transcription factor binding sites (TFBSs) on genomic DNA is of crucial importance for understanding and predicting regulatory elements in gene networks. TFBS motifs are commonly described by Position Weight Matrices (PWMs), in which each DNA base pair contributes independently to the transcription factor (TF) binding. However, this description ignores correlations between nucleotides at different positions, and is generally inaccurate: analysing fly and mouse in vivo ChIPseq data, we show that in most cases the PWM model fails to reproduce the observed statistics of TFBSs. To overcome this issue, we introduce the pairwise interaction model (PIM), a generalization of the PWM model. The model is based on the principle of maximum entropy and explicitly describes pairwise correlations between nucleotides at different positions, while being otherwise as unconstrained as possible. It is mathematically equivalent to considering a TF-DNA binding energy that depends additively on each nucleotide identity at all positions in the TFBS, like the PWM model, but also additively on pairs of nucleotides. We find that the PIM significantly improves over the PWM model, and even provides an optimal description of TFBS statistics within statistical noise. The PIM generalizes previous approaches to interdependent positions: it accounts for co-variation of two or more base pairs, and predicts secondary motifs, while outperforming multiple-motif models consisting of mixtures of PWMs. We analyse the structure of pairwise interactions between nucleotides, and find that they are sparse and dominantly located between consecutive base pairs in the flanking region of TFBS. Nonetheless, interactions between pairs of non-consecutive nucleotides are found to play a significant role in the obtained accurate description of TFBS statistics. The PIM is computationally tractable, and provides a general framework that should be useful for describing and predicting TFBSs beyond PWMs.

A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats

Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the “CA motif.” The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides. Several active variants of the CA motif aptamer lack the ability to form consecutive Watson-Crick base pairs in any register, while others consist of repeats containing only cytidine and adenosine residues, indicating that noncanonical interactions play important roles in its structure. The circular dichroism spectrum of the CA motif aptamer is distinct from that of A-form RNA and other major classes of nucleic acid structures. Bioinformatic searches indicate that the CA motif is absent from most archaeal and bacterial genomes, but occurs in at least 70 percent of approximately 400 eukaryotic genomes examined. These searches also uncovered several phylogenetically conserved examples of the CA motif in rodent (mouse and rat) genomes. Together, these results reveal the existence of a second class of naturally occurring GTP aptamer whose sequence requirements, like that of the G motif, are not consistent with those of a canonical secondary structure. They also indicate a new and unexpected potential biochemical activity of certain naturally occurring tandem repeats.

Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides

Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2′-O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine–purine–pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2′-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA.

Su1991 Reactivation of Reprimo by an Artificial Transcription Factor in Gastric Cancer Cell Lines

Introduction: Gastric cancer (GC) is the second cause of cancer-related death worldwide and the leading one in Latin America. It has been proposed that the silencing of Reprimo (RPRM), by epigenetic mechanisms, is a putative biomarker of GC. RPRM is a p53 dependent G2 arrest mediator and is considered to be tumor suppressor gene involved in the pathogenesis of numerous cancers such as gastric, colon, pituitary, among others. We propose that the reactivation of RPRM expression, by means of an activator Artificial Transcription Factor (ATF), modifies the proliferation rate of GC cell lines in vitro. Methods: An activator ATF for RPRM was designed using the method proposed by zincfingertools.org. Thus, an ATF with a unique domain of six zinc-finger capable of binding specifically to 18 consecutive base pairs, located in the promoter region of RPRM, was engineered. This ATF was then inserted into a modified pcDNA3.1+ vector coupled with a potent transcription activator domain (VP64). A luciferase reporter assay was performed using a pGL3 vector (Promega), linked to a 634 base pairs region of the promoter region of RPRM, in order to evaluate the efficiency and specificity of the synthetized ATF. Finally, the ATF was transiently transfected with lipofectamine2000 (Invitrogen). Subsequently, RPRM mRNA expression was evaluated by a RT-qPCR and proliferation rate was evaluated by a MTS assay. The experiments were conducted on AGS and MKN28 GC cell lines. Results: The ATF designed was able to significantly increase the luciferase activity vs controls, both in MKN28 and AGS cell lines. Accordingly, RPRM mRNA was upregulated in the presence of the ATF. These findings were associated with a lower proliferation rate in both cell lines. Since the AGS cell line is highly methylated in the promoter region of RPRM, it might explain a significantly lower response to the presence of ATF, in comparison with that of MKN28. Discussion: Our work suggests that a RPRM specific ATF could be used as a novel tool to reactivate this epigenetically silenced tumor suppressor gene. Moreover, the upregulation of RPRM modifies one of the hallmarks of cancer pathways by reducing the proliferation rate of cancerous cell lines in vitro. Grant Support: Fondecyt #1111014 and Fondef #D09I1137 from the Government of Chile to AHC.

Hx-amides: DNA sequence recognition by the fluorescent Hx (p-anisylbenzimidazole)•pyrrole and Hx•imidazole pairings

Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx–I/P–I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔTM studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T–A/T; Hx/IP prefers C–A/T; Hx/PI prefers A/T–C; and Hx/II prefers C–C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.

Simulations of A-RNA Duplexes. The Effect of Sequence, Solute Force Field, Water Model, and Salt Concentration

We have carried out an extended reference set of explicit solvent molecular dynamics simulations (63 simulations with 8.4 μs of simulation data) of canonical A-RNA duplexes. Most of the simulations were done using the latest variant of the Cornell et al. AMBER RNA force field bsc0χ(OL3), while several other RNA force fields have been tested. The calculations show that the A-RNA helix compactness, described mainly by geometrical parameters inclination, base pair roll, and helical rise, is sequence-dependent. In the calculated set of structures, the inclination varies from 10° to 24°. On the basis of simulations with modified bases (inosine and 2,6-diaminopurine), we suggest that the sequence-dependence of purely canonical A-RNA double helix is caused by the steric shape of the base pairs, i.e., the van der Waals interactions. The electrostatic part of stacking does not appear to affect the A-RNA shape. Especially visible is the role of the minor groove amino group of purines. This resembles the so-called Dickerson-Calladine mechanical rules suggested three decades ago for the DNA double helices. We did not identify any long-living backbone substate in A-RNA double helices that would resemble, for example, the B-DNA BI/BII dynamics. The variability of the A-RNA compactness is due to mutual movements of the consecutive base pairs coupled with modest change of the glycosidic χ torsion. The simulations further show that the A-RNA compactness is modestly affected by the water model used, while the effect of ionic conditions, investigated in the range from net-neutral condition to ~0.8 M monovalent ion excess salt, is smaller.

Quantitative experimental determination of primer–dimer formation risk by free‐solution conjugate electrophoresis

DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.

Encoding Phenotype in Bacteria with an Alternative Genetic Set

An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but homologated by the width of a benzene ring, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands and were transformed into Escherichia coli. Although they yielded fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) was used, the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, and xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.