Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the α-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA^Asp by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.Microcins are small (<10-kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (17). Three microcins, B, C, and J, form a subgroup of posttranslationally modified microcins. Members of this subgroup have highly unusual structures and inhibit cellular enzymes that are validated targets for antibacterial drug development (25). Posttranslationally modified microcins are attractive as drug candidates because of their strong antibacterial action and because virtually limitless numbers of their derivatives can be generated by means of mutation, chemical synthesis, or both. Microcin B (McB), a 43-residue peptide with thiazole and indole rings (13), inhibits DNA gyrase (21). Microcin J, a 21-amino-acid peptide, assumes an unusual threaded lasso structure (2, 23, 27) and inhibits bacterial RNA polymerase (1, 18). The structure of the subject of this study, McC (compound 1) is shown in Fig. ​Fig.1a.1a. McC is a heptapeptide with a formylated N-terminal methionine and a C-terminal aspartate whose α-carboxyl group is covalently linked to adenosine through an N-acyl phosphoramide bond (10, 14). The phosphoramidate of McC is additionally modified by an O-propylamine group (9).Open in a separate windowFIG. 1.Structures and synthesis of McC analogs. (a) Structures of microcin C (compound 1) and its processing product (compound 2). (b) Structures of synthetic McC analogs 7 to 9 and their expected processing products, compounds 4 to 6, which are established inhibitors of AspRS, GluRS, and LeuRS, respectively. (c) Structure of Asp-AMP (compound 3), the natural reaction intermediate of AspRS. Compounds 2 and 4 are nonhydrolyzable analogs of this compound. (d) Synthesis of compounds 7 to 9, which starts from compounds 4 to 6. Hereto the hexapeptide was coupled to the sulfamoyl precursors 4-6 via the coupling agent DIC, followed by removal of the Fmoc protecting group: (i) Fmoc-MRTGNA-OH, HOBt, DIC, DIPEA; (ii) Et₃N/DMF (1:1 [vol/vol]).The passage of McC through the inner layer of the Escherichia coli cell wall is carried out by the YejABEF transporter (19). Once inside the cell, McC is specifically processed by one of the several broad-specificity E. coli cytoplasmic aminopeptidases (12). The product of processing, modified aspartyl-adenylate (compound 2) (15), closely resembles Asp-AMP (compound 3) (Fig. ​(Fig.1c),1c), the natural reaction intermediate of the tRNA^Asp aminoacylation reaction catalyzed by AspRS. However, because the bond between the α-carboxyl of C-terminal aspartate and the phosphoramidate nitrogen is nonhydrolyzable, compound 2 inhibits AspRS. Unprocessed McC has no effect on tRNA^Asp aminoacylation, while processed McC has no effect on McC-sensitive cells at concentrations at which intact McC strongly inhibits cell growth. Thus, McC is a Trojan horse inhibitor (22): the peptide part allows McC to enter sensitive cells, where it gets processed, liberating the inhibitory part of the drug.Aminoacyl-tRNA synthetases (aaRSs) carry out the condensation of genetically encoded amino acids with cognate tRNAs. When 1 of the 20 aaRSs present in the cell is inhibited, the corresponding tRNA is not charged. This leads to protein synthesis inhibition and cell growth arrest. In principle, variation of the last amino acid of the McC peptide, the product of the mccA gene, should allow investigators to obtain McC derivatives targeting aaRSs other than AspRS. Unfortunately, the results of systematic structure-activity analyses of the McC peptide revealed that substitutions in the seventh codon of mccA invariably prevented McC production, presumably by interfering with posttranslational modifications of the MccA peptide by the McC maturation enzymes (11). Indeed, in vitro analysis showed that the C-terminal asparagine of MccA is required for the addition of the adenosine moiety by the MccB protein (24).Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of their corresponding aaRSs (5, 20, 26). However, these compounds show low in vivo activities due to limited membrane permeability and the absence of a transporter for these compounds. Here, we show that through chemical attachment of aminoacyl-sulfamoyl adenosines to the first 6 amino acids of the MccA peptide, potent antibacterial agents can be generated. The new compounds share the Trojan horse mechanism of action with McC but target aaRSs specified by the last amino acid of the peptide moiety.     

On the Evolution of Structure in Aminoacyl-tRNA Synthetases

The aminoacyl-tRNA synthetases are one of the major protein components in the translation machinery. These essential proteins are found in all forms of life and are responsible for charging their cognate tRNAs with the correct amino acid. The evolution of the tRNA synthetases is of fundamental importance with respect to the nature of the biological cell and the transition from an RNA world to the modern world dominated by protein-enzymes. We present a structure-based phylogeny of the aminoacyl-tRNA synthetases. By using structural alignments of all of the aminoacyl-tRNA synthetases of known structure in combination with a new measure of structural homology, we have reconstructed the evolutionary history of these proteins. In order to derive unbiased statistics from the structural alignments, we introduce a multidimensional QR factorization which produces a nonredundant set of structures. Since protein structure is more highly conserved than protein sequence, this study has allowed us to glimpse the evolution of protein structure that predates the root of the universal phylogenetic tree. The extensive sequence-based phylogenetic analysis of the tRNA synthetases (Woese et al., Microbiol. Mol. Biol. Rev. 64:202-236, 2000) has further enabled us to reconstruct the complete evolutionary profile of these proteins and to make connections between major evolutionary events and the resulting changes in protein shape. We also discuss the effect of functional specificity on protein shape over the complex evolutionary course of the tRNA synthetases.     

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.     

Horizontal Gene Transfer in Aminoacyl-tRNA Synthetases Including Leucine-Specific Subtypes

Aminoacyl-tRNA synthetases catalyze a fundamental reaction for the flow of genetic information from RNA to protein. Their presence in all organisms known today highlights their important role in the early evolution of life. We investigated the evolutionary history of aminoacyl-tRNA synthetases on the basis of sequence data from more than 200 Archaea, Bacteria, and Eukaryota. Phylogenetic profiles are in agreement with previous observations that many genes for aminoacyl-tRNA synthetases were transferred horizontally between species from all domains of life. We extended these findings by a detailed analysis of the history of leucyl-tRNA synthetases. Thereby, we identified a previously undetected case of horizontal gene transfer from Bacteria to Archaea based on phylogenetic profiles, trees, and networks. This means that, finally, the last subfamily of aminoacyl-tRNA synthetases has lost its exceptional position as the sole subfamily that is devoid of horizontal gene transfer. Furthermore, the leucyl-tRNA synthetase phylogenetic tree suggests a dichotomy of the archaeal/eukaryotic-cytosolic and bacterial/eukaryotic-mitochondrial proteins. We argue that the traditional division of life into Prokaryota (non-chimeric) and Eukaryota (chimeric) is favorable compared to Woese’s trichotomy into Archaea/Bacteria/Eukaryota. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the “gemini group.” (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.     

Partitioning of tRNA-dependent Editing between Pre- and Post-transfer Pathways in Class I Aminoacyl-tRNA Synthetases

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pre-transfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre- and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.     

MIST,a Novel Approach to Reveal Hidden Substrate Specificity in Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (AARSs) constitute a family of RNA-binding proteins, that participate in the translation of the genetic code, by covalently linking amino acids to appropriate tRNAs. Due to their fundamental importance for cell life, AARSs are likely to be one of the most ancient families of enzymes and have therefore been characterized extensively. Paradoxically, little is known about their capacity to discriminate tRNAs mainly because of the practical challenges that represent precise and systematic tRNA identification. This work describes a new technical and conceptual approach named MIST (Microarray Identification of Shifted tRNAs) designed to study the formation of tRNA/AARS complexes independently from the aminoacylation reaction. MIST combines electrophoretic mobility shift assays with microarray analyses. Although MIST is a non-cellular assay, it fully integrates the notion of tRNA competition. In this study we focus on yeast cytoplasmic Arginyl-tRNA synthetase (yArgRS) and investigate in depth its ability to discriminate cellular tRNAs. We report that yArgRS in submicromolar concentrations binds cognate and non-cognate tRNAs with a wide range of apparent affinities. In particular, we demonstrate that yArgRS binds preferentially to type II tRNAs but does not support their misaminoacylation. Our results reveal important new trends in tRNA/AARS complex formation and potential deep physiological implications.     

Aminoacyl-tRNA Synthetases in Triticum aestivum L. During Seed Development and Germination

Changes in the level of total and individual aminoacyl-tRNAsynthetase activity in the various tissues of wheat (Triticumaestivum L.) were followed by the ATP-pyrophosphate (ATP-PP₁)exchange procedure throughout seed maturation and germination. During seed development the total synthetase activity in theendosperm increased up to the 5th week after fertilization andthereafter decreased rapidly. Over the same period, synthetaseactivity in the testa-pencarp decreased markedly, whilst theactivity in the developing embryo increased. Many of the individualsynthetases conformed with this general pattern although therewere several exceptions. The total synthetase activity of both the coleoptile and coleorhiza(root) increased rapidly during the first 2 d of germinationwhilst the total activity of these enzymes in the scutellumremained constant. After an initial increase on germination,aminoacyl-tRNA synthetase activity in the testa-aleurone layerremained almost constant until most of the endosperm had beendigested. With a few exceptions the relative levels of individualsynthetases in the various tissues did not change significantlyduring seed maturation or germination.     

Intra-protein Compensatory Mutations Analysis Highlights the tRNA Recognition Regions in Aminoacyl-tRNA Synthetases

Abstract

The aminoacyl-tRNA synthetases (aaRSs) covalently attach amino acids to their corresponding nucleic acid adapter molecules, tRNAs. The interactions in the tRNA-aaRSs complexes are mostly non-specific, and largely electrostatic. Tracing a way of aaRS-tRNA mutual adaptation throughout evolution offers a clearer view of understanding how aaRS-tRNA systems preserve patterns of tRNA recognition and binding. In this study, we used the compensatory mutations analysis to explore adaptation of aaRSs in respond to random mutations that can occur in the tRNA-recognition area. We showed that the frequency of compensatory mutations among residues that belong to the recognition region is 1.75-fold higher than that of the exposed residues. The highest frequencies of compensatory mutations are observed for pairs of charged residues, wherein one residue is located within the tRNA-recognition area, while the second is placed outside of the area, and contributes to the formation of the aaRS electrostatic landscape. Given charged residues are compensated by buried charge residues in more than 60% of the analyzed mutations. The cytoplasmatic and mitochondrial aaRSs preserve similar patterns of compensatory mutations in the tRNA recognition areas. Moreover, we found that mitochondrial aaRSs demonstrate a significant increase in the frequency of compensatory mutations in the area. Our findings shed light on the physical nature of compensatory mutations in aaRSs, thereby keeping unchanged tRNA-recognition patterns.     

On the Classes of Aminoacyl-tRNA Synthetases,Amino Acids and the Genetic Code

The division of the aminoacyl-tRNA synthetases in two classes is compared with a division of the amino acids in two classes, obtained from the AAIndex databank by a principal component analysis. The division of the enzymes in Classes I and II follows to a great extent a division in the chemical and biological properties of their cognate amino acids. Furthermore, the phylogenetic trees of Classes I and II enzymes are highly correlated with dendrograms obtained for their cognate amino acids by using the indices in the AAIndex database. We argue that the evolution of aminoacyl-tRNA synthetases was determined by the characteristics of their corresponding amino acids. We interpret these results considering models for the origin and evolution of the genetic code in which an initial version, containing fewer amino acids, was modified by the incorporation of new amino acids following duplication and divergence of previous synthetases and tRNA molecules.     

Association of Aminoacyl-tRNA Synthetases Gene Polymorphisms with the Risk of Congenital Heart Disease in the Chinese Han Population

Aminoacyl-tRNA synthetases (ARSs) are in charge of cellular protein synthesis and have additional domains that function in a versatile manner beyond translation. Eight core ARSs (EPRS, MRS, QRS, RRS, IRS, LRS, KRS, DRS) combined with three nonenzymatic components form a complex known as multisynthetase complex (MSC).We hypothesize that the single-nucleotide polymorphisms (SNPs) of the eight core ARS coding genes might influence the susceptibility of sporadic congenital heart disease (CHD). Thus, we conducted a case-control study of 984 CHD cases and 2953 non-CHD controls in the Chinese Han population to evaluate the associations of 16 potentially functional SNPs within the eight ARS coding genes with the risk of CHD. We observed significant associations with the risk of CHD for rs1061248 [G/A; odds ratio (OR) = 0.90, 95% confidence interval (CI) = 0.81–0.99; P = 3.81×10⁻²], rs2230301 [A/C; OR = 0.73, 95%CI = 0.60–0.90, P = 3.81×10⁻²], rs1061160 [G/A; OR = 1.18, 95%CI = 1.06–1.31; P = 3.53×10⁻³] and rs5030754 [G/A; OR = 1.39, 95%CI = 1.11–1.75; P = 4.47×10⁻³] of EPRS gene. After multiple comparisons, rs1061248 conferred no predisposition to CHD. Additionally, a combined analysis showed a significant dosage-response effect of CHD risk among individuals carrying the different number of risk alleles (P _trend = 5.00×10⁻⁴). Compared with individuals with “0–2” risk allele, those carrying “3”, “4” or “5 or more” risk alleles had a 0.97-, 1.25- or 1.38-fold increased risk of CHD, respectively. These findings indicate that genetic variants of the EPRS gene may influence the individual susceptibility to CHD in the Chinese Han population.     

On the Relative Content of G,C Bases in Codons of Amino Acids Corresponding to Class I and II Aminoacyl-tRNA Synthetases

We have analyzed the relative G,C content from protein coding regions of 530 organisms and found that the ratio of the G,C content of the codons of the amino acids correspondingto Class II and Class I aminoacyl-tRNA synthetases decreases in a statistically significant way from prokaryotes to animals.This can be interpreted assuming that an initial asymmetry between the G,C content of codons of Class I and II amino acids existed and has decreased in the course of evolution.     

Aminoacyl-tRNA synthetases

Crystallographic studies of a number of aminoacyl-tRNA synthetases and their complexes with ATP, amino acid and cognate tRNA are leading to an increasingly detailed picture of how these sophisticated enzymes function. Within the two distinct structural classes of ten synthetases, many common features are apparent, although evolution has led to many interesting idiosyncrasies in certain enzymes. Recent advances, specially concerning class II enzymes, have increased out knowledge of both the role of electrophiles in the mechanism of amino acid activation and cross-subunit tRNA recognition and help solve the evolutionary puzzles that have emerged from the extension of the aminoacyl-tRNA synthetase database to include Archae     

Aminoacyl-tRNA synthetases database

Aminoacyl-tRNA synthetases (AARSs) are at the center of the question of the origin of life. They constitute a family of enzymes integrating the two levels of cellular organization: nucleic acids and proteins. AARSs arose early in evolution and are believed to be a group of ancient proteins. They are responsible for attaching amino acid residues to their cognate tRNA molecules, which is the first step in the protein synthesis. The role they play in a living cell is essential for the precise deciphering of the genetic code. The analysis of AARSs evolutionary history was not possible for a long time due to a lack of a sufficiently large number of their amino acid sequences. The emerging picture of synthetases' evolution is a result of recent achievements in genomics [Woese,C., Olsen,G.J., Ibba,M. and S?ll,D. (2000) Microbiol. Mol. Biol. Rev., 64, 202-236]. In this paper we present a short introduction to the AARSs database. The updated database contains 1047 AARS primary structures from archaebacteria, eubacteria, mitochondria, chloroplasts and eukaryotic cells. It is the compilation of amino acid sequences of all AARSs known to date, which are available as separate entries via the WWW at http://biobases.ibch.poznan.pl/aars/.     

hKCNQ1/hKCNQ2嵌合体的构建,表达及电生理学特性

目的:构建hKCNQ1/hKCNQ2嵌合体,研究嵌合体通道的电生理学特征,为分析PIP2对hKcnq1通道的调节机制打下基础.方法:利甩over-lapPCR方法构建hKCNQ1/hKCNQ2嵌合体质粒(Q1ctQ2-pCI、Q1ctQ2-pcDNA3.1),转染HEK293细胞,采用全细胞膜片钳技术记录HEK293细胞上嵌合体电流.结果:在全细胞膜片钳记录模式下,转染了嵌合体Q1ctQ2-pCI的HEK293细胞电流密度160为5.2±0.87 pA/pf,其半数激活电压V0.5为23.3±8.9 mV(n=6);转染了嵌合体Q2ctQ1-pcDNA3.1的HEK293细胞电流密度160为21.7±4.2 pA/pf,其半数激活电压V0.5为-37.5±3.6 mV(n=9).结论:成功构建了hKCNQ1/hKCNQ2嵌合体并完成了嵌合体重组质粒的异源性表达和膜片钳记录.     

Specificity in the Formation of Aminoacyl-tRNA

Abstract

It had been previously observed that at temparatures above 70°C alterations can occur in the specificity of aminoacyl-tRNA synthetases. The effect of several agents on this specificity has now been tested. Anomalous aminoacylation has been observed in the presence of 3M urea and 1095 ethanol. The realationship between the loss of secondary structure of tRNA in the presence of these agents and anomalous aminoacylation is briefly discussed.     

Generation of Viable Plant-Vertebrate Chimeras

The extreme dependence on external oxygen supply observed in animals causes major clinical problems and several diseases are related to low oxygen tension in tissues. The vast majority of the animals do not produce oxygen but a few exceptions have shown that photosynthetic capacity is physiologically compatible with animal life. Such symbiotic photosynthetic relationships are restricted to a few aquatic invertebrates. In this work we aimed to explore if we could create a chimerical organism by incorporating photosynthetic eukaryotic cells into a vertebrate animal model. Here, the microalgae Chlamydomonas reinhardtii was injected into zebrafish eggs and the interaction and viability of both organisms were studied. Results show that microalgae were distributed into different tissues, forming a fish-alga chimera organism for a prolonged period of time. In addition, microscopic observation of injected algae, in vivo expression of their mRNA and re-growth of the algae ex vivo suggests that they survived to the developmental process, living for several days after injection. Moreover microalgae did not trigger a significant inflammatory response in the fish. This work provides additional evidence to support the possibility that photosynthetic vertebrates can be engineered.     

PNA-DNA Chimeras Forming Quadruplex Structures

Abstract

¹H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized ^5′TGGG^3′-t (1) and ^5′TGG^3′-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]₄. ¹H-NMR spectrum for ^5′TGG^3′-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.