Simple molecular engineering of glycol nucleic acid: Progression from self-pairing to cross-pairing with cDNA and RNA

The acyclic chiral nucleic acid analogue, Glycol Nucleic Acid (GNA), displayed exceptional structural simplicity and atom economy while forming self-paired duplexes, using canonical Watson–Crick base pairing. We disclose here that the replacement of phosphodiester linker in GNA with somewhat rigid and shorter carbamate linker in Glycol Carbamate Nucleic Acid (GCNA) backbone allows unprecedented stability to the antiparallel self-paired duplexes. The R-GCNA oligomers were further found to form cross-paired antiparallel duplexes with cDNA and RNA following Watson–Crick base pairing. The stability of cross-paired GCNA:DNA and GCNA:RNA duplexes was higher than the corresponding DNA:DNA and DNA:RNA duplexes. The chiral (R) and (S) precursors were easily accessible from naturally occurring l-serine.     

Solution spectroscopy of dipicolinic acid interaction with nucleic acids: Role in spore heat resistance

The effect of dipicolinic acid (DPA) or its calcium chelate (CaDPA) on the spectral characteristics of nucleic acids was examined. Dipicolinic acid was found to displace ethidium gromide from DNA; this indicates that it may bind by intercalation. On interaction with DNA, the ultraviolet absorption spectrum revealed downfield shifts and caused progressive diminution in both DNA and dipicolinate chromophores. The strength and type of interaction may be ion-specific but not discriminatory to any type of base pairing. Spectral analysis also indicated that both dipicolinate and calcium dipicolinate bound to different RNA species, although the mechanism of binding was not elucidated. We conclude that the interaction of dipicolinate/ calcium dipicolinate with nucleic acids is a mechanism whereby water can be removed from spore polynucleotides, increasing their stability to denaturation.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

Unnatural base pairs between 2-amino-6-(2-thienyl)purine and the complementary bases.

The unnatural base, 2-amino-6-(2-thienyl)purine (designated as s), instead of 2-amino-6-(N,N-dimethylamino)purine (designated as x), was designed in order to improve the specificity and efficiency of the base pairing with pyridin-2-one (designated as y). DNA fragments containing s were chemically synthesized, and the thermal stability and the enzymatic reactions involving the s-y pairing were examined. Thermal denaturation experiments showed that the DNA duplex (12-mer) containing the s-y pair was more stable than that containing the x-y pair. The incorporation of dyTP was also more advantageous to the s-y pairing than the x-y pairing in single-nucleotide insertion experiments using the Klenow fragment of Escherichia coli DNA polymerase I.     

Local base dynamics and local structural features in RNA and DNA duplexes

Local base motion and local structural base information are derived with a simple motional model from site specifically spin-labeled polyribo- and polydeoxyribonucleotides. The model was developed earlier for some nucleic acids and has now been applied to analyze 22 different nucleic acid systems. We conclude that the base motion of the spin-labeled nucleotide in single-stranded RNA, DNA, or non-base-paired bases in duplexes is of the order of 1 ns and that its base mobility decreases by about a factor of 4 upon base pairing. Also, the tether motion of the probe is slower in an RNA than in a DNA duplex.     

Reconstitution of biologically active 50S ribosomal subunits with artificial 5S RNA molecules carrying disturbances in the base pairing within the molecular stalk.

Bacillus stearothermophilus 50S ribosomal subunits were reconstituted in vitro using artificial 5S RNA molecules constructed by combining parts of major and minor type (Raué et al. (1976) Europ. J. Biochem. 68, 169-176) B. licheniformis 5S RNA. The artificial 5S RNA molecules carry defined disturbances (A.C juxtapositions and extra G.U pairs) in the base pairing between the 5'- and 3'-terminal sequences of the molecule (the molecular stalk region). The biological activity of the reconstituted subunits was determined in an E. coli cell-free system programmed with poly-U. The results show that conservation of the base pairing within the molecular stalk is not required for biological activity of 5S RNA. Disturbances of the base pairing within this region do reduce the rate of reconstitution, however. Normal base pairing in the molecular stalk is thus required to ensure efficient ribosome assembly.     

Accurate energies of hydrogen bonded nucleic acid base pairs and triplets in tRNA tertiary interactions

Tertiary interactions are crucial in maintaining the tRNA structure and functionality. We used a combined sequence analysis and quantum mechanics approach to calculate accurate energies of the most frequent tRNA tertiary base pairing interactions. Our analysis indicates that six out of the nine classical tertiary interactions are held in place mainly by H-bonds between the bases. In the remaining three cases other effects have to be considered. Tertiary base pairing interaction energies range from -8 to -38 kcal/mol in yeast tRNA(Phe) and are estimated to contribute roughly 25% of the overall tRNA base pairing interaction energy. Six analyzed posttranslational chemical modifications were shown to have minor effect on the geometry of the tertiary interactions. Modifications that introduce a positive charge strongly stabilize the corresponding tertiary interactions. Non-additive effects contribute to the stability of base triplets.     

Role of the heat capacity change in understanding and modeling melting thermodynamics of complementary duplexes containing standard and nucleobase-modified LNA

Melting thermodynamic data obtained by differential scanning calorimetry (DSC) are reported for 43 duplexed oligonucleotides containing one or more locked nucleic acid (LNA) substitutions. The measured heat capacity change (ΔC(p)) for the helix-to-coil transition is used to compute the changes in enthalpy and entropy for melting of an LNA-bearing duplex at the T(m) of its corresponding isosequential unmodified DNA duplex to allow rigorous thermodynamic analysis of the stability enhancements provided by LNA substitutions. Contrary to previous studies, our analysis shows that the origin of the improved stability is almost exclusively a net reduction (ΔΔS° < 0) in the entropy gain accompanying the helix-to-coil transition, with the magnitude of the reduction dependent on the type of nucleobase and its base pairing properties. This knowledge and our average measured value for ΔC(p) of 42 ± 11 cal mol(-1) K(-1) bp(-1) are then used to derive a new model that accurately predicts melting thermodynamics and the increased melting temperature (ΔT(m)) of heteroduplexes formed between an unmodified DNA strand and a complementary strand containing any number and configuration of standard LNA nucleotides A, T, C, and G. This single-base thermodynamic (SBT) model requires only four entropy-related parameters in addition to ΔC(p). Finally, DSC data for 20 duplexes containing the nucleobase-modified LNAs 2-aminoadenine (D) and 2-thiothymine (H) are reported and used to determine SBT model parameters for D and H. The data and model suggest that along with the greater stability enhancement provided by D and H bases relative to their corresponding A and T analogues, the unique pseudocomplementary properties of D-H base pairs may make their use appealing for in vitro and in vivo applications.     

Analysis of the role of the pseudoknot component in the SRV-1 gag-pro ribosomal frameshift signal: loop lengths and stability of the stem regions.

The simian retrovirus-1 (SRV-1) gag-pro frameshift signal was identified in previous work, and the overall structure of the pseudoknot involved was confirmed (ten Dam E, Brierley I, Inglis S, Pleij C, 1994, Nucleic Acids Res 22:2304-2310). Here we report on the importance of specific elements within the pseudoknot. Some mutations in stem S1 that maintain base pairing have reduced frameshift efficiencies. This indicates that base pairing in itself is not sufficient. In contrast, frameshifting correlates qualitatively with the calculated stability of mutations in S2. The stems thus play different roles in the frameshift event. The nature of the base in L1 has little influence on frameshift efficiency. It is however required to bridge S2; deleting it lowers frameshifting from 23 to 9%. In L2, frameshift efficiency was not affected in a mutant that changed 10 to 12 bases. This makes it unlikely that the primary sequence of L2 plays a role in -1 frameshifting, in contrast to readthrough in Moloney murine leukemia virus (Wills N, Gesteland R, Atkins J, 1994, EMBO J 13:4137-4144). Deletions of 2 and 3 bases gave more frameshifting than the wild type, probably reflecting the increased stability of the pseudoknot due to a shorter loop L2. Deleting even more bases reduces frameshifting compared to wild-type levels. At this point, stress will build up in L2, and this will reduce overall pseudoknot stability.     

An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo[(4,5)-b]pyridine

To develop unnatural base pairs that function in replication, we designed 4-propynylpyrrole-2-carbaldehyde (designated as Pa′) and synthesized the nucleoside derivatives of Pa′. The base pairing of Pa′ with the partner, 9-methylimidazo[(4,5)-b]pyridine (Q), was compared to that of pyrrole-2-carbaldehyde (Pa), which was previously developed as a specific pairing partner of Q. The thermal stability of a DNA duplex containing the Q–Pa′ pair and the incorporation efficiency of the Pa′ substrate (dPa′TP) into DNA opposite Q by the Klenow fragment of Escherichia coli DNA polymerase I were improved, in comparison with those of the Q–Pa pair. These improvements result from the increased hydrophobicity and stacking stability of Pa′ by the introduction of the propynyl group to Pa, providing valuable information for the further development of unnatural base pairs toward the expansion of the genetic alphabet.     

Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.     

Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins

Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins. J. Bacteriol. 92:739-745. 1966.-The ribonucleic acid (RNA) molecules and coat proteins of two RNA coliphages, MS-2 and Qbeta, have been characterized. MS-2 RNA shows an S(20,w) of 25.8 and a molecular weight by light scattering of 10(6). The corresponding parameters for Qbeta-RNA were 28.9 and 0.9 x 10(6). A difference in base composition was reflected in the adenine-uracil ratio, which was 0.95 for MS-2 and 0.75 for Qbeta. The two RNA preparations are readily separated by chromatography on columns of methylated albumin. Both gave identical bouyant densities in cesium sulfate of 1.64 g/ml. The coat protein subunits were of similar molecular weights: 15,500 (Qbeta) and 14,000 (MS-2). They differed, however, in that the Qbeta-protein lacked tryptophan and histidine, whereas the MS-2 protein lacked only histidine.     

On the Deletion of Inverted Repeated DNA in Escherichia Coli: Effects of Length, Thermal Stability, and Cruciform Formation in Vivo

We have studied the deletion of inverted repeats cloned into the EcoRI site within the CAT gene of plasmid pBR325. A cloned inverted repeat constitutes a palindrome that includes both EcoRI sites flanking the insert. In addition, the two EcoRI sites represent direct repeats flanking a region of palindromic symmetry. A current model for deletion between direct repeats involves the formation of DNA secondary structure which may stabilize the misalignment between the direct repeats during DNA replication. Our results are consistent with this model. We have analyzed deletion frequencies for several series of inverted repeats, ranging from 42 to 106 bp, that were designed to form cruciforms at low temperatures and at low superhelical densities. We demonstrate that length, thermal stability of base pairing in the hairpin stem, and ease of cruciform formation affect the frequency of deletion. In general, longer palindromes are less stable than shorter ones. The deletion frequency may be dependent on the thermal stability of base pairing involving approximately 16-20 bp from the base of the hairpin stem. The formation of cruciforms in vivo leads to a significant increase in the deletion frequency. A kinetic model is presented to describe the relationship between the physical-chemical properties of DNA structure and the deletion of inverted repeats in living cells.     

A tolerance of DNA heterology in the mammalian targeted gene repair reaction

Targeted gene repair consists of at least two major steps, the pairing of an oligonucleotide to a site bearing DNA sequence complementarity followed by a nucleotide exchange reaction directed by the oligonucleotide. In this study, oligonucleotides with different structures were designed to target a stably integrated (mutant) enhanced green fluorescent protein (EGFP) gene and used to direct the repair of a single base mutation. We show that the efficiency of correction is influenced by the degree of DNA sequence homology existing between the oligonucleotide and target gene. Correction is reduced when a heterologous stretch of DNA sequence is placed in the center of the oligonucleotide and the mismatched base pair is then formed near the terminus. The negative impact of heterology is dependent on the type of DNA sequence inserted and on the size of the heterologous region. If the heterologous sequence is palindromic and adopts a secondary structure, the negative impact on the correction frequency is removed, and wild-type levels of repair are restored. Although differences in the efficiency of correction are observed in various cell types, the effect of structural changes on gene repair is consistent. These results reveal the existence of a directional-specific repair pathway that relies on the pairing stability of a bilateral complex and emphasize the importance of sequence homology between pairing partners for efficient catalysis of gene repair.     

Rapid binding and release of Hfq from ternary complexes during RNA annealing

The Sm protein Hfq binds small non-coding RNA (sRNAs) in bacteria and facilitates their base pairing with mRNA targets. Molecular beacons and a 16 nt RNA derived from the Hfq binding site in DsrA sRNA were used to investigate how Hfq accelerates base pairing between complementary strands of RNA. Stopped-flow fluorescence experiments showed that annealing became faster with Hfq concentration but was impaired by mutations in RNA binding sites on either face of the Hfq ring or by competition with excess RNA substrate. A fast bimolecular Hfq binding step (∼10⁸ M⁻¹s⁻¹) observed with Cy3-Hfq was followed by a slow transition (0.5 s⁻¹) to a stable Hfq–RNA complex that exchanges RNA ligands more slowly. Release of Hfq upon addition of complementary RNA was faster than duplex formation, suggesting that the nucleic acid strands dissociate from Hfq before base pairing is complete. A working model is presented in which rapid co-binding and release of two RNA strands from the Hfq ternary complex accelerates helix initiation 10 000 times above the Hfq-independent rate. Thus, Hfq acts to overcome barriers to helix initiation, but the net reaction flux depends on how tightly Hfq binds the reactants and products and the potential for unproductive binding interactions.     

A second-generation copper(II)-mediated metallo-DNA-base pair

Metal-dependent pairing of nucleobases represents an alternative DNA base pairing scheme. Our first-generation copper(II)-mediated pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) metallo-base pair has a stability comparable to the natural base pairs dA:dT and dC:dG but does not have the selectivity of the Watson Crick base pairs. In order to increase the selectivity of base pair formation, a second-generation metallo-base pair was generated consisting of a pyridine-2,6-dicarboxamide (Dipam) and a pyridine (Py) nucleobase. This new metallo-base pair is more stable than the natural base pairs dA:dT and dC:dG and highly selective against mispairing. In addition, incorporation of multiple metallo-base pairs into DNA results in the formation of stable duplexes demonstrating that hydrogen bonding base pairs can efficiently be replaced by metal-dependent base pairs at multiple sites in DNA.     

Sugar modified oligonucleotides: synthesis, nuclease resistance and base pairing of oligodeoxynucleotides containing 1-(4'-thio-beta-D-ribofuranosyl)-thymine.

XdT12 and dT5XdT6, where X is 1-(4'-thio-beta-d-ribofuranosyl)-thymine, were synthesized. The first oligonucleotide presents a better stability against calf spleen phosphodiesterase than natural dT12. The second one hybridizes with complementary sequence. However the X:A base pairing stability is lower than natural T:A one.     

Fluorinated peptide nucleic acid

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.     

Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: implications for antisense design and targeting a group I intron

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.     

Optimizing the design of oligonucleotides for homology directed gene targeting

Background

Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit.

Methodology/Principal Findings

In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex.

Conclusion and Significance

A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.