Synthesis of Fluorobenzene and Benzimidazole Nucleic‐Acid Analogues and Their Influence on Stability of RNA Duplexes

Six different ribonucleoside phosphoramidites with fluorobenzenes or fluorobenzimidazoles as base analogues, one abasic site, and inosine were synthesized and incorporated into oligoribonucleotides. The oligomers were investigated by means of UV and CD spectroscopy to assess the contribution of H‐bonding, base stacking, and solvation to the stability of the RNA duplex. CD Spectra show that the incorporation of modified nucleosides does not lead to changes in the structure of RNA. The T_m differences determined are based on changes in base stacking and solvation. Individual contributions of base stacking and solvation of the modified nucleosides could be determined. In fluorobenzene⋅fluorobenzimidazole‐modified base pairs, a duplex‐stabilizing force was found that points to a weak F⋅⋅⋅H H‐bond.     

Selective pairing of polyfluorinated DNA bases

Novel selective non-hydrogen-bonding DNA base pairs utilizing fluorinated nucleoside analogues have been investigated. Melting studies of DNA duplexes containing 2,3,4,5-tetrafluorobenzene and 4,5,6,7-tetrafluoroindole bases on opposite strands show greater stabilization of the duplex compared with nonfluorinated hydrocarbon controls. Overall, these hydrophobic analogues are destabilizing compared with natural base pairs but are stabilizing compared with natural base mismatches. Such selective pairing may be due to solvent avoidance of these hydrophobic structures, burying their surfaces within the duplex. Our findings suggest that polyfluoroaromatic bases might be employed as a new, selective base-pairing system orthogonal to the natural genetic system.     

Hydrophobic, Non-Hydrogen-Bonding Bases and Base Pairs in DNA

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.     

yDNA versus xDNA pyrimidine nucleobases: computational evidence for dependence of duplex stability on spacer location

The structural and binding properties of the natural and x- and y-pyrimidines were compared using computational methods. Our calculations show that although the x-pyrimidines favor different orientations about the glycosidic bond compared to the natural pyrimidines, which could have implications for the formation and resulting stability of xDNA duplexes and jeopardize the selectivity of expanded nucleobases, y-pyrimidines have rotational profiles more similar to the natural bases. Increasing the pyrimidine size using a benzene spacer leads to relatively minor changes in the hydrogen-bond strength of isolated Watson-Crick base pairs. However, differences in the anomeric carbon distances in pairs composed of x- or y-pyrimidines suggest yDNA may yield a more optimal expanded structure. By stacking two monomers via their centers of mass, we find that the expanded nucleobases stack much stronger than the natural bases. Additionally, although replacing xT by yT changes the stacking energy by less than 5 kJ mol (-1), replacing xC by yC significantly strengthens complexes with the natural nucleobases (by up to 30%). Calculations on larger duplex models composed of four nucleobases reveal that x- and y-pyrimidines can increase duplex stability of natural helices by strengthening both the intra and interstrand stacking interactions. Furthermore, when the total stability (sum of all hydrogen-bonding and (intrastrand and interstrand) stacking interactions) of the larger models is considered, y-pyrimidines lead to more stable complexes than x-pyrimidines for all but three duplex sequences. Thus, through analysis of a variety of properties, our calculations suggest that the location of the benzene spacer affects the properties of expanded nucleobases and the stability of expanded duplexes, and therefore should be carefully considered when designing future expanded analogues.     

How do size-expanded DNA nucleobases enhance duplex stability? Computational analysis of the hydrogen-bonding and stacking ability of xDNA bases

Computational chemistry (B3LYP, MP2) is used to study the properties of size-expanded DNA nucleobases generated by inserting a benzene spacer into the natural nucleobases. Although the addition of the spacer does not significantly affect the hydrogen-bonding properties of natural nucleobases, the orientation of the base about the glycosidic bond necessary for Watson-Crick binding is destabilized, which could have implications for the selectivity of expanded bases, as well as the stability of expanded duplexes. Consideration of the (stacked) binding energies in the preferred relative orientation of natural and expanded nucleobases aligned according to their centers of mass reveals that the stacking within natural dimers can be increased by up to 50% upon expansion of one nucleobase and up to 90% upon expansion of two nucleobases. The implications of these findings to the stability of expanded duplexes were revealed by considering simplified models of natural and mixed duplexes composed of four nucleobases. Although intra- and interstrand interactions within double helices are typically less than those predicted when nucleobases are stacked according to their centers of mass, some nucleobases utilize their full stacking potential within double helices, where both intra- and interstrand interactions can be significant. Most importantly, increasing the size of nucleobases within the duplex significantly increases both intra- and interstrand stacking interactions. Specifically, some interactions are double the magnitude of the corresponding intrastrand interactions in natural helices, and even greater increases in interstrand interactions are sometimes found. Thus, our work suggests that mixed duplexes composed of natural bases hydrogen bound to expanded bases may exploit the increase in the inherent stacking ability of the expanded bases in more than one way and thereby afford duplexes with greater stability than natural DNA.     

Understanding the role of base stacking in nucleic acids. MD and QM analysis of tandem GA base pairs in RNA duplexes

Preceding NMR experiments show that the conformation of tandem GA base pairs, an important recurrent non-canonical building block in RNA duplexes, is context dependent. The GA base pairs adopt "sheared" N3(G)-N6(A), N2(G)-N7(A) geometry in the r(CGAG)(2) and r(iGGAiC)(2) contexts while switching to "imino" N1(G)-N1(A), O6(G)-N6(A) geometry in the r(GGAC)(2) and r(iCGAiG)(2) contexts (iC and iG stand for isocytosine and isoguanine, respectively). As base stacking is likely to be one of the key sources of the context dependence of the conformation of GA base pairs, we calculated base stacking energies in duplexes containing such base pairs, to see if this dependence can be predicted by stacking energy calculations. When investigating the context dependence of the GA geometry two different conformations of the same duplex were compared (imino vs. sheared). The geometries were generated via explicit solvent MD simulations of the respective RNA duplexes, while the subsequent QM energy calculations focused on base stacking interactions of the four internal base pairs. Geometrical relaxation of nucleobase atoms prior to the stacking energy computations has a non-negligible effect on the results. The stacking energies were derived at the DFT-D/6-311++G(3df,3pd) level. We show a rather good correspondence between the intrinsic gas-phase stacking energies and the NMR-determined GA geometries. The conformation with more favorable gas-phase stacking is in most cases the one observed in experiments. This correlation is not improved when including solvent effects via the COSMO method. On the other side, the stacking calculations do not predict the relative thermodynamic stability of duplex formation for different sequences.     

Synthesis and pairing properties of alpha-tricyclo-DNA

We describe the synthesis of the phosphoramidite building blocks of alpha-tricyclo-DNA (alpha-tc-DNA) covering all four natural bases, starting from the already known corresponding alpha-tc-nucleosides. These building blocks were used for the preparation of three alpha-tc-oligonucleotide 10-mers representing a homopurine, a homopyrimidine, and a mixed purine/pyrimidine base sequence. The base-pairing properties with complementary parallel and antiparallel oriented DNA and RNA were studied by UV-melting analysis and CD spectroscopy. We found that alpha-tc-DNA binds preferentially to parallel nucleic acid complements through Watson-Crick duplex formation, with a preference for RNA over DNA. In comparison with natural DNA, alpha-tc-DNA shows equal to enhanced affinity to RNA and also pairs to antiparallel DNA or RNA complements, although with much lower affinity. In the mixed-base sequence these antiparallel duplexes are of the reversed Watson-Crick type, while in the homopurine/homopyrimidine sequences Hoogsteen and/or reversed Hoogsteen pairing is observed. Antiparallel duplex formation of two alpha-tc-oligonucleotides was also observed, although the thermal stability of this duplex was surprisingly low. The base-pairing properties of alpha-tc-DNA are discussed in the context of alpha-DNA, alpha-RNA, and alpha-LNA.     

pH-independent triple-helix formation with 6-oxocytidine as cytidine analogue

The syntheses of six different phosphoramidite building blocks of 6-oxocytosine and 5-allyl-6-oxocytosine as analogues of N(3)-protonated cytosine are described. These compounds have been incorporated into oligonucleotides by standard solid-phase synthesis. Hybridization of 15-mer Hoogsteen strands with target 21-mer duplexes was investigated. Comparison of the triplex-forming abilities of the different building blocks revealed that: i) 5-allyl substitution has a negative influence on triplex stability, ii) a uniform backbone of the Hoogsteen strand stabilizes triplexes relative to mixed backbones; iii) RNA strands with 6-oxocytidine or 5-allyl-6-oxocytidine do not form a triple helix with the DNA target duplex, probably due to backbone torsional constraints; and (iv) a 15-mer DNA sequence with three isolated 2'-deoxy-6-oxocytidines has the highest T(m) of all cytidine analogues investigated in this study. CD experiments provided further evidence for the presence or absence of triplex structures. In the course of these temperature-dependent CD measurements we were able to detect duplex and triplex melting independent from each other at selected wavelengths. This methodology is especially interesting in cases where UV melting curves show only one transition owing to spectral overlap.     

Synthesis of 4-Substituted 1H-Benzimidazole 2′-Deoxyribonucleosides and Utility of the 4-Nitro Compound as Universal Base

The stereoselective synthesis of 4-substituted 1H-benzimidazole 2′-deoxyribonucleosides is described. Regioisomeric (N¹ and N³) β-D -deoxyribonucleosides 2a–c and 3a–c were formed. ¹³C-NMR Chemical shifts of the 1H-benzimidazole 2′-deoxy-β-D -ribofuranosides were correlated with point charges of C-atoms as well as with Hammett constants of the exocyclic substituents. Phosphonate and phosphoramidite building blocks of 4-nitro-1H-benzimidazole 2′-deoxyribofuranoside ( 2a ) were prepared (see 4a, b ). Oligonucleotides of the d(A₂₀) type were synthesized in which the two central dA bases were replaced by 4-nitro-1H-benzimidazole residues. They were hybridized with oligomeric dT and related oligomers having the other conventional bases opposite to the 4-nitro-1H-benzimidazole moieties. Within these duplexes ( 12·13, 12·14, 12·15 , and 12·16 ), the destabilization was almost independent of the mismatch which is required for a universal base. The thermodynamic data indicate that the 4-nitro-1H-benzimidazole residues do not form H-bonds with opposite bases but are stabilizing the duplex by stacking interactions and favorable entropic changes.     

Theoretical Studies on the Intermolecular Interactions of Potentially Primordial Base‐Pair Analogues

Recent experimental studies on the Watson–Crick type base pairing of triazine and aminopyrimidine derivatives suggest that acid/base properties of the constituent bases might be related to the duplex stabilities measured in solution. Herein we use high‐level quantum chemical calculations and molecular dynamics simulations to evaluate the base pairing and stacking interactions of seven selected base pairs, which are common in that they are stabilized by two N? H???O hydrogen bonds separated by one N? H???N hydrogen bond. We show that neither the base pairing nor the base stacking interaction energies correlate with the reported pK_a data of the bases and the melting points of the duplexes. This suggests that the experimentally observed correlation between the melting point data of the duplexes and the pK_a values of the constituent bases is not rooted in the intrinsic base pairing and stacking properties. The physical chemistry origin of the observed experimental correlation thus remains unexplained and requires further investigations. In addition, since our calculations are carried out with extrapolation to the complete basis set of atomic orbitals and with inclusion of higher electron correlation effects, they provide reference data for stacking and base pairing energies of non‐natural bases.     

Hydrogen Bonding or Stacking Interactions in Differentiating Duplex Stability in Oligonucleotides Containing Synthetic Nucleoside Probes for Alkylated DNA

Oligonucleotides that hybridize to modified DNA are useful chemical tools to probe the noncovalent interactions that stabilize DNA duplexes. In an effort to better understand the interactions that influence the specificity of hybridization probes for O⁶‐alkylguanine lesions, we examined a series of synthetic nucleoside analogues (BIM, Benzi, and Peri) with respect to their ability to stabilize duplex DNA comprised of native or damaged DNA oligonucleotides. The base‐modified nucleoside analogues contained systematically varied hydrogen‐bonding and π‐stacking properties. The nucleoside probes were incorporated into DNA and paired opposite canonical bases (A, T, C, or G), O⁶‐methylguanine (O⁶‐MeG), O⁶‐benzylguanine (O⁶‐BnG), or a stable abasic site analogue (tetrahydrofuran, THF). On the basis of the free energy of duplex formation, the highest degree of stabilization was observed when Peri was paired opposite O⁶‐MeG. The thermodynamic data suggest that the smaller probes stabilize DNA duplexes more through hydrogen bonding, whereas the larger probes, with a greater capacity to π stack, contribute to duplex stabilization more on the basis of base stacking. These results demonstrate that increased helix stability could be achieved when BIM, Benzi, or Peri were paired opposite damage‐containing DNA rather than unmodified DNA (that is, O⁶‐MeG rather than G). This knowledge is expected to be useful in the design and development of nucleoside analogues for uses in DNA‐based technologies.     

B-DNA Structure and Stability: The Role of Nucleotide Composition and Order

We have quantum chemically analyzed the influence of nucleotide composition and sequence (that is, order) on the stability of double-stranded B-DNA triplets in aqueous solution. To this end, we have investigated the structure and bonding of all 32 possible DNA duplexes with Watson–Crick base pairing, using dispersion-corrected DFT at the BLYP-D3(BJ)/TZ2P level and COSMO for simulating aqueous solvation. We find enhanced stabilities for duplexes possessing a higher GC base pair content. Our activation strain analyses unexpectedly identify the loss of stacking interactions within individual strands as a destabilizing factor in the duplex formation, in addition to the better-known effects of partial desolvation. Furthermore, we show that the sequence-dependent differences in the interaction energy for duplexes of the same overall base pair composition result from the so-called “diagonal interactions” or “cross terms”. Whether cross terms are stabilizing or destabilizing depends on the nature of the electrostatic interaction between polar functional groups in the pertinent nucleobases.     

Assignment and NOE analysis of 2'-hydroxyl protons in RNA: implications for stabilization of RNA A-form duplexes

The ribose 2'-OH hydroxyl group distinguishes RNA from DNA. The 2'-OH hydroxyl protons are responsible for differences in conformation, hydration, and thermodynamic stability of RNA and DNA oligonucleotides. Additionally, the 2'-OH group plays a central role in RNA catalysis. This important group lies in the shallow groove of RNA, where it is involved in a network of hydrogen bonds with water molecules stabilizing RNA A-form duplexes. Structural and dynamical information on 2'-OH hydroxyl protons is essential to understand their respective roles. Here we report the 2'-OH hydroxyl proton assignments for a 30mer RNA, the HIV-2 transactivation region, in water using solution NMR techniques. We provide structural information on 2'-OH hydroxyl groups in the form of orientational preferences contradicting the paradigm that the 2'-OH hydroxyl typically points away from the ribose H1' proton.     

Long RNA dangling end has large energetic contribution to duplex stability

Long terminal unpaired nucleotides known as dangling ends play interesting roles in biological systems. Previous studies, however, only dealt with the energy contributions of single dangling bases. The energy contributions of long dangling ends on the stability of duplexes have not been systematically studied. We now report a quantitative increase in stability of RNA-RNA and DNA-DNA duplexes containing a long dangling end. We found a larger enhancement of the stability by the long RNA dangling end of the RNA-RNA duplex than has been observed for the DNA duplexes. It is also found that structural stabilizations by long dangling ends seem to originate from the single-stranded stacking interactions of nucleotides. These results indicate that RNA stability can be achieved by increasing the length of the dangling end. The thermodynamic parameters of the long dangling ends are useful for designing ribozymes and antisense oligonucleotides, and for the prediction of the RNA secondary structure like the pseudoknot.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

Bipyridyl- and biphenyl-DNA: a recognition motif based on interstrand aromatic stacking

The synthesis and incorporation into oligonucleotides of C-nucleosides containing the two aromatic, non-hydrogen-bonding nucleobase substitutes biphenyl (I) and bipyridyl (Y) are described. Their homo- and hetero-recognition properties in different sequential arrangements were then investigated via UV-melting curve analysis, gel mobility assays, CD- and NMR spectroscopy. An NMR analysis of a dodecamer duplex containing one biphenyl pair in the center, as well as CD data on duplexes with multiple insertions provide further evidence for the zipper-like interstrand stacking motif that we proposed earlier based on molecular modeling. UV-thermal melting experiments with duplexes containing one to up to seven I- or Y base pairs revealed a constant increase in T(m) in the case of I and a constant decrease for Y. Mixed I/Y base pairs lead to stabilities in between the homoseries. Insertion of alternating I/abasic site- or Y/abasic site pairs strongly decreases the thermal stability of duplexes. Asymmetric distribution of I- or Y residues on either strand of the duplex were also investigated in this context. Duplexes with three natural base pairs at both ends and 50 % of I pairs in the center are still readily formed, while duplexes with blunt ended I pairs tend to aggregate unspecifically. Duplexes with one natural overhang at the end of a I-I base pair tract can both aggregate or form ordered duplexes, depending on the nature of the natural bases in the overhang.     

Measurement of nucleobase pKa values in model mononucleotides shows RNA-RNA duplexes to be more stable than DNA-DNA duplexes

To understand why the RNA-RNA duplexes in general has a higher thermodynamic stability over the corresponding DNA-DNA duplexes, we have measured the pK(a) values of both nucleoside 3',5'-bis-ethyl phosphates [Etp(d/rN)pEt] and nucleoside 3'-ethyl phosphates [(d/rN)pEt] (N = A, G, C, or T/U), modeling as donors and acceptors of base pairs in duplexes. While the 3',5'-bis-phosphates, Etp(d/rN)pEt, mimic the internucleotidic monomeric units of DNA and RNA, in which the stacking contribution is completely absent, the 3'-ethyl phosphates, (d/rN)pEt, mimic the nucleotide at the 5'-end. The pK(a) values of the nucleobase in each of these model nucleoside phosphates have been determined with low pK(a) error (sigma = +/-0.01 to 0.02) by (1)H NMR (at 500 MHz) with 20-33 different pH measurements for each compound. This study has led us to show the following: (1) All monomeric DNA nucleobases are more basic than the corresponding RNA nucleobases in their respective Etp(d/rN)pEt and (d/rN)pEt. (2) The pK(a) values of the monomeric nucleotide blocks as well as Delta pK(a) values between the donor and acceptor can be used to understand the relative base-pairing strength in the oligomeric duplexes in the RNA and DNA series. (3) The Delta G*(pKa) of the donor and acceptor of the base pair in duplexes enables a qualitative dissection of the relative strength of the base-pairing and stacking in the RNA-RNA over the DNA-DNA duplexes. (4) It is also found that the relative contribution of base-pairing strength and nucleobase stacking in RNA-RNA over DNA-DNA is mutually compensating as the % A-T/U content increases or decreases. This interdependency of stacking and hydrogen bonding can be potentially important in the molecular design of the base-pair mimics to expand the alphabet of the genetic code.