Nucleotide Loop Research Articles

Optimizing the Kinetics and Thermodynamics of DNA i‐Motif Folding

Under slightly acidic conditions, single cytidine-rich DNA strands can form four-stranded structures called i-motifs. The stability of the i-motif structure is based on the intercalation of hemiprotonated C-C(+) base pairs. In addition, the stability of these structures is influenced by pH, temperature, salt concentration, number of cytidines per C-rich stretch, and length of sequence; it also depends on the nucleotides in the connecting loop regions. Here, we investigated the influence of the loop nucleotides on i-motif stability, structure, and kinetics of folding, in five structures with the same loop-size but different adenosine and thymidine residues within the loop. The stabilities of the i-motif structures were determined by CD melting, and structure and kinetics of folding were studied by static and time-resolved NMR experiments.

APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are ‘hotspots’ for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3′ and 5′ of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

Simple and highly sensitive molecular beacon probe based on target-induced structure-switching DNA for mercury(ii) detection

A simple and highly sensitive molecular beacon (MB) probe, based on coupling target-induced conformational switching of DNA hairpins with thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry, was designed for the determination of mercury(II) (Hg2+). In this MB, the DNA hairpins contain a stem of 6 base pairs enclosing a 27 nucleotide (nt) loop and an additional 12 nt sticky end at the 3′ end. The twelfth base from the 3′ end was labeled with a quencher of 4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL), and the 5′ end was labeled with a fluorophore fluorescein amidite (FAM). In the absence of Hg2+, the DNA hairpin is in a closed form resulting in fluorescence quenching. In the presence of the target analyte, Hg2+-mediated base pairs induce the conformational change from the sticky end to open the hairpins; the fluorophore and the quencher are moved away from each other, leading to the restoration of fluorescence. The dynamic concentration range was from 0.5 to 50 nM Hg2+ with a detection limit of 0.3 nM at the 3σ level. The assay strategy afforded high selectivity for Hg2+ against other environmentally related metal ions. The developed method is very simply, only requiring a one-step reaction to realize the assay of Hg2+.

Loop Nucleotide Polymorphism in a Putative miRNA Precursor Associated with Seed Length in Rice (Oryza sativa L.)

The terminal loop region of primary miRNA transcripts is an important determinant controlling miRNA function in human, animals and plants. However, the effects of polymorphisms in loop region of miRNA precusors on phenotypic variation have not been reported, especially on agronomic traits in rice. From rice japonica variety Koshihikari and indica Guichao2, we isolated a precursor of putative miRNA osa-MIR2923a. We detected a GG/AA polymorphism in the loop structure from japonica and indica rice varieties, which was corresponding to japonica/indica rice differentiation. By using high-resolution melting (HRM) analysis, we measured the polymorphisms in a RIL (recombinant inbred lines) population derived from japonica variety Koshihikari and indica Guichao2. We found that the GG/AA polymorphism in the osa-MIR2923a loop was correlated to grain length and length-width ratio. We further found the significant association between seed length and GG/AA polymorphism in a population consisting of 72 rice landraces. Three targets were predicted, whose expressions showed significant differences between the two varieties. Our results suggested that the putative miRNA precursor and the three target genes could play functional roles for indica/japonica seed differentiation.

New insights from molecular dynamic simulation studies of the multiple binding modes of a ligand with G-quadruplex DNA

G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences. These structures have recently emerged as a new class of potential molecular targets for anticancer drugs. An understanding of the three-dimensional interactions between small molecular ligands and their G-quadruplex targets in solution is crucial for rational drug design and the effective optimization of G-quadruplex ligands. Thus far, rational ligand design has been focused mainly on the G-quartet platform. It should be noted that small molecules can also bind to loop nucleotides, as observed in crystallography studies. Hence, it would be interesting to elucidate the mechanism underlying how ligands in distinct binding modes influence the flexibility of G-quadruplex. In the present study, based on a crystal structure analysis, the models of a tetra-substituted naphthalene diimide ligand bound to a telomeric G-quadruplex with different modes were built and simulated with a molecular dynamics simulation method. Based on a series of computational analyses, the structures, dynamics, and interactions of ligand-quadruplex complexes were studied. Our results suggest that the binding of the ligand to the loop is viable in aqueous solutions but dependent on the particular arrangement of the loop. The binding of the ligand to the loop enhances the flexibility of the G-quadruplex, while the binding of the ligand simultaneously to both the quartet and the loop diminishes its flexibility. These results add to our understanding of the effect of a ligand with different binding modes on G-quadruplex flexibility. Such an understanding will aid in the rational design of more selective and effective G-quadruplex binding ligands.

Abstract PL06-02: Inflammation, DNA mismatch repair, and colon cancer.

Abstract Colorectal neoplasia is driven by at least two pathogenic pathways for cancer development. Chromosomal instability, observed in ~80% of sporadic colorectal cancers, is named due to the rearrangement of chromosomes, resulting in aneuploid nuclei. The etiology for CIN is not known. A common finding is allelic mutation of a tumor suppressor gene, followed by loss of the remaining normal allele (termed loss of heterozygosity, LOH). Microsatellite instability (MSI), observed in up to 20% of sporadic colorectal cancers, is named for frameshifts (or instability) that occur at DNA microsatellite sequences. Cells remain diploid. The etiology for MSI is inactivation of DNA mismatch repair (MMR), an enzyme system functioning in the nucleus to repair interstrand nucleotide mispairs and loops of DNA that are formed on one strand at microsatellites. The specificity of mismatch recognition is directed by hMutSa and hMutSb; hMutSa (hMSH2-hMSH6 heterodimer) binds to single mispairs and single insertion-deletion loops, whereas hMutSb (hMSH2-hMSH3 heterodimer) binds larger insertion-deletion loops. MSI sporadic cancers are caused by hypermethylation of the MMR gene hMLH1's promoter, preventing its transcription and translation. With loss of hMLH1, MMR completely fails and mutations accumulate in the genome due to non-repair of DNA polymerase errors. Clinically, MSI tumors contain surrounding and intratumoral mononuclear inflammatory cells which are thought responsible in containing the tumor in place, leading to improved survival over same staged patients with non-MSI tumors. Although the types of inflammatory cells within MSI tumors have not been characterized, it is believed that neo-peptides from frameshifted proteins as a result of MMR failure are generated to attract mononuclear cells into the tumor, producing a protective immune response. Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colon cancers. Unlike MSI colorectal cancers in which hypermethylation of hMLH1 drives multiple target gene mutations, the cause of EMAST has been recently associated with reduced expression of hMSH3, which we have also observed in hamartomatous polyps and during the adenoma-to-carcinoma progression, and is hypothesized to be triggered as a consequence of inflammation with subsequent relaxation of MMR function. EMAST-positive human colorectal cancers are strongly associated with inflammation, particularly within tumor nests, but unlike MSI tumors, this inflammation is associated with advance staged colorectal cancer, implying reduced survival. Citation Format: John M. Carethers. Inflammation, DNA mismatch repair, and colon cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL06-02.

Electrochemiluminescence Resonance Energy Transfer Between CdS:Eu Nancrystals and Au Nanorods for Sensitive DNA Detection

This work provides a novel electrochemiluminescence resonance energy transfer (ECL-RET) system using CdS:Eu nanoscrytals (NCs) as an ECL donor and Au nanorods (Au NRs) as an ECL acceptor. CdS:Eu NC, prepared by doping 1.5% Eu3+ ions into CdS NCs, exhibits strong and stable cathodic ECL emissions in the presence of coreactant S2O82– ions with two ECL spectral bands at 450–550 nm from the host CdS and at 600–700 nm due to the energy transfer from host CdS to Eu3+ ions. Au nanorods (Au NRs) have two absorption peaks that are easily tuned to match well with the ECL emission spectrum of the CdS:Eu NCs film by adjusting the aspect ratio of the nanorods to get a highly effective ECL-RET. Here, we studied the spectrum, distance and shape dependence of the efficiency of ECL-RET between the NCs' ECL and different Au nanoparticles (Au NPs) on the basis of the stem-loop structure DNA with a 6-base-pair (bp) stem and a 12, 30, or 45 nucleotide (nct) loop. At the optimized conditions, the system could be used to ultras...

Target-induced structure-switching DNA hairpins for sensitive electrochemical monitoring of mercury (II)

A simple, sensitive and reusable electrochemical sensor was designed for determination of mercury (II) (Hg2+) by coupling target-induced conformational switch of DNA hairpins with thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry. The hairpin probe consisted of a stem of 6 base pairs enclosing a 14 nucleotide (nt) loop and an additional 12 nt sticky end at the 3′ end. Each hairpin was labeled with ferrocene (Fc) redox tag in the middle of the loop, which was immobilized on the electrode via self-assembly of the terminal thiol moiety at the 5′ end. In the presence of target analyte, Hg2+-mediated base pairs induced the conformational change from the sticky end to open the hairpins, resulting in the ferrocene tags close to the electrode for the increasing redox current. The strong coordination reaction of T–Hg2+–T resulted in a good repeatability and intermediate precision down to 10%. The dynamic concentration range spanned from 5.0nM to 1.0μM Hg2+ with a detection limit of 2.5nM at the 3sblank level. The strategy afforded exquisite selectivity for Hg2+ against other environmentally related metal ions. Inspiringly, the developed sensor could be reused by introduction of iodide (I−).

Thermodynamic Characterization of RNA 2 × 3 Nucleotide Internal Loops

To better elucidate RNA structure-function relationships and to improve the design of pharmaceutical agents that target specific RNA motifs, an understanding of RNA primary, secondary, and tertiary structure is necessary. The prediction of RNA secondary structure from sequence is an intermediate step in predicting RNA three-dimensional structure. RNA secondary structure is typically predicted using a nearest neighbor model based on free energy parameters. The current free energy parameters for 2 × 3 nucleotide loops are based on a 23-member data set of 2 × 3 loops and internal loops of other sizes. A database of representative RNA secondary structures was searched to identify 2 × 3 nucleotide loops that occur in nature. Seventeen of the most frequent 2 × 3 nucleotide loops in this database were studied by optical melting experiments. Fifteen of these loops melted in a two-state manner, and the associated experimental ΔG°(37,2×3) values are, on average, 0.6 and 0.7 kcal/mol different from the values predicted for these internal loops using the predictive models proposed by Lu, Turner, and Mathews [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Res. 34, 4912-4924] and Chen and Turner [Chen, G., and Turner, D. H. (2006) Biochemistry 45, 4025-4043], respectively. These new ΔG°(37,2×3) values can be used to update the current algorithms that predict secondary structure from sequence. To improve free energy calculations for duplexes containing 2 × 3 nucleotide loops that still do not have experimentally determined free energy contributions, an updated predictive model was derived. This new model resulted from a linear regression analysis of the data reported here combined with 31 previously studied 2 × 3 nucleotide internal loops. Most of the values for the parameters in this new predictive model are within experimental error of those of the previous models, suggesting that approximations and assumptions associated with the derivation of the previous nearest neighbor parameters were valid. The updated predictive model predicts free energies of 2 × 3 nucleotide internal loops within 0.4 kcal/mol, on average, of the experimental free energy values. Both the experimental values and the updated predictive model can be used to improve secondary structure prediction from sequence.

Conformation and dynamics of nucleotides in bulges and symmetric internal loops in duplex DNA studied by EPR and fluorescence spectroscopies

The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe Ç, which becomes fluorescent (Çf) upon reduction of the nitroxide functional group, along with EPR and fluorescence spectroscopies. A one-base bulge was in a conformational equilibrium between looped-out and stacked states, the former favored at higher temperature and the latter at lower temperature. Stacking of bulge bases was favored in two- and three-base bulges, independent of temperature, resulting in DNA bending as evidenced by increased fluorescence of Çf. EPR spectra of Ç-labeled three-, four- and five-base symmetrical interior DNA bulges at 20°C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37°C increased as the loops became larger. A considerable variation in fluorescence between different loops was observed, as well as a temperature-dependence within constructs. Fluorescence unexpectedly increased as the size of the loop decreased at 2°C. Fluorescence of the smallest loops, where a single T·T mismatch was located between the stem region and the probe, was even larger than for the single strand, indicating a considerable local structural deformation of these loops from regular B-DNA. These results show the value of combining EPR and fluorescence spectroscopy to study non-helical regions of nucleic acids.

Structural Basis for Profilin-Mediated Actin Nucleotide Exchange

Actin is a ubiquitous eukaryotic protein that is responsible for cellular scaffolding, motility, and division. The ability of actin to form a helical filament is the driving force behind these cellular activities. Formation of a filament depends on the successful exchange of actin's ADP for ATP. Mammalian profilin is a small actin binding protein that catalyzes the exchange of nucleotide and facilitates the addition of an actin monomer to a growing filament. Here, crystal structures of profilin–actin have been determined to show an actively exchanging ATP. Structural analysis shows how the binding of profilin to the barbed end of actin causes a rotation of the small domain relative to the large domain. This conformational change is propagated to the ATP site and causes a shift in nucleotide loops, which in turn causes a repositioning of Ca2+ to its canonical position as the cleft closes around ATP. Reversal of the solvent exposure of Trp356 is also involved in cleft closure. In addition, secondary calcium binding sites were identified.

Unique Structural Features of Interconverting Monomeric and Dimeric G-Quadruplexes Adopted by a Sequence from the Intron of the N-myc Gene

A multidimensional heteronuclear NMR study has demonstrated that a guanine-rich DNA oligonucleotide originating from the N-myc gene folds into G-quadruplex structures in the presence of K(+), NH(4)(+), and Na(+) ions. A monomeric G-quadruplex formed in K(+) ion containing solution exhibits three G-quartets and flexible propeller-type loops. The 3D structure with three single nucleotide loops represents a missing element in structures of parallel G-quadruplexes. The structural features together with the high temperature stability are suggestive of the specific biological role of G-quadruplex formation within the intron of the N-myc gene. An increase in K(+) ion and oligonucleotide concentrations resulted in transformation of the monomeric G-quadruplex into a dimeric form. The dimeric G-quadruplex exhibits six stacked G-quartets, parallel strand orientations, and propeller-type loops. A link between the third and the fourth G-quartets consists of two adenine residues that are flipped out to facilitate consecutive stacking of six G-quartets.

Combined Spectroscopic/Computational Studies of Vitamin B12 Precursors: Geometric and Electronic Structures of Cobinamides

Vitamin B(12) (cyanocobalamin) and its biologically active derivatives, methylcobalamin and adenosylcobalamin, are members of the family of corrinoids, which also includes cobinamides. As biological precursors to cobalamins, cobinamides possess the same structural core, consisting of a low-spin Co(3+) ion that is ligated equatorially by the four nitrogens of a highly substituted tetrapyrrole macrocycle (the corrin ring), but differ with respect to the lower axial ligation. Specifically, cobinamides possess a water molecule instead of the nucleotide loop that coordinates axially to Co(3+)cobalamins via its dimethylbenzimidazole (DMB) base. Compared to the cobalamin species, cobinamides have proven much more difficult to study experimentally, thus far eluding characterization by X-ray crystallography. In this study, we have utilized combined quantum mechanics/molecular mechanics (QM/MM) computations to generate complete structural models of a representative set of cobinamide species with varying upper axial ligands. To validate the use of this approach, analogous QM/MM geometry optimizations were carried out on entire models of the cobalamin counterparts for which high-resolution X-ray structural data are available. The accuracy of the cobinamide structures was assessed further by comparing electronic absorption spectra computed using time-dependent density functional theory to those obtained experimentally. Collectively, the results obtained in this study indicate that the DMB → H(2)O lower axial ligand switch primarily affects the energies of the Co 3d(z(2))-based molecular orbital (MO) and, to a lesser extent, the other Co 3d-based MOs as well as the corrin π-based highest energy MO. Thus, while the energy of the lowest-energy electronic transition of cobalamins changes considerably as a function of the upper axial ligand, it is nearly invariant for the cobinamides.

Interaction of Replication Protein-A with G-Quadruplex Structures

We performed systematic single molecule and bulk studies on the interactions of Replication Protein A (RPA) with four different G-Quadruplex (GQ) structures. RPA, the most abundant single strand DNA binding protein in eukaryotes, is known to interact and unfold GQ structures however; this unfolding activity has not been systematically studied for different GQ structures. As a first step to study this dependence, we studied RPA-GQ interactions for GQ structures in which we varied the number of tetrad layers while keeping the loop length and loop sequence fixed. We studied GQ forming sequences of the form TT (G_n TTT)_3 G_n TT where 1< n <6. These sequences correspond to 2-5 layer GQ constructs (L2-L5) with 3 nucleotide loops (TTT) and two nucleotide overhang sequences (TT). Our results showed that 100%, 80%, 50%, and 20% of the L2, L3, L4, and L5 GQ molecules were unfolded in equilibrium and physiological RPA concentration. The systematic, almost linear, increase in stability of GQ structure as the number of layers is increased is very interesting and will provide an important ingredient in understanding protein-GQ interactions. In addition, we measured the timescales of GQ folding as a result of K+ addition, and GQ unfolding by RPA using a single molecule stopped flow type measurement. We found that the folding of all GQ constructs takes a similar time of 0.3±0.1 sec, with the exception of L2. L2 has two distinct transitions with transition times of 0.3±0.1 sec and 0.5±0.1 sec, and the longer transition time demonstrates a clear intermediate folding conformation. Surprisingly, GQ unfolding times by RPA were also similar for all constructs, 0.2 - 0.3 sec, despite significant differences in their equilibrium stability.

Interaction of water with the G-quadruplex loop contributes to the binding energy of G-quadruplex to protein

Unlike DNA duplexes that release water upon interaction with protein, the binding of DNA G-quadruplex of the thrombin-binding aptamer (TBA) to thrombin takes up water. Here, to reveal the mechanism of water uptake, we designed four mutants of TBA (ΔT3, ΔT7, ΔT9, ΔT12), in which thymine residues (T3, T7, T9 and T12) were deleted from the loop regions of TBA G-quadruplex. For the mutants the thermodynamics and the osmolyte effects on the interactions with thrombin were investigated. The mutants ΔT3, ΔT9 and ΔT12 decreased the binding constants of the G-quadruplex to thrombin. Furthermore, an osmotic stress analysis indicated that the number of water molecules binding to the complex decreased in the mutants ΔT3 and ΔT9. The decrease in the binding affinity was related to loss of binding of the loop nucleotides to water molecules. Therefore, the interaction between loops of the G-quadruplex and water molecules contributed to the binding energy of G-quadruplex to protein. Our study suggests that water binding is essential for the binding of G-quadruplex to protein.

Comparison of solution conformations and stabilities of modified helix 69 rRNA analogs from bacteria and human

The helix 69 (H69) region of the large subunit (28S) ribosomal RNA (rRNA) of Homo sapiens contains five pseudouridine (Ψ) residues out of 19 total nucleotides, three of which are highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine in double-stranded (stem) regions. These results were compared with previous hairpin (stem plus single-stranded loop) studies to understand the contributions of the loop sequences to H69 structure and stability. The role of a loop nucleotide substitution from an A in bacteria (position 1918 in Escherichia coli 23S rRNA) to a G in eukaryotes (position 3734 in H. sapiens 28S rRNA) was examined. Thermodynamic parameters for the duplex RNAs were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by circular dichroism spectroscopy. The overall folded structure of human H69 appears to be similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved; however, our results reveal subtle differences in structure and stability between the bacterial and human H69 RNAs in both the stem and loop regions. These findings may be significant with respect to H69 as a potential drug target site.

A Single Nucleotide in Stem Loop II of 5′-Untranslated Region Contributes to Virulence of Enterovirus 71 in Mice

BackgroundEnterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored.Principal FindingsIn this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5′-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5′-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5′-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo.ConclusionsThese results presented the first reported virulence determinant in EV71 5′-UTR and first position discovered from unadapted isolates.

Loop nucleotides control primary and mature miRNA function in target recognition and repression

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5’ end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs’ sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation.

G Quadruplexes Stabilised by 8-Oxo-2′-deoxyguanosine

G quadruplexes (GQPs) are four-stranded secondary structures formed of guanosine-rich nucleic acids. They consist of at least two G tetrads composed of four guanosine nucleotides. DNA GQPs have been shown to fold into a variety of different topologies that can be divided into antiparallel and parallel conformations (the latter is depicted in Figure 1 A).[!] The topology of DNA GQPs depends on the sequence and the loop length. For example, sequences with single nucleotide loops favour all-parallel strand orientationPJ Furthermore, the loop length also determines the stability of GQPs: the shorter the loop, the more stable is the

Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

−1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in −1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.