Pyrazolo[3,4-d]pyrimidine Nucleic Acids: Adjustment of the dA-dT to the dG-dC Base Pair Stability

Abstract

The pyrazolo[3,4-d]pyrimidine-4,6-diamine nucleosides 2b-d stabilize the dA-dT base pair significantly when the dA-residue is replaced. Oligonucleotide duplexes incorporating 2b-d show a 4–6°C T _m increase per modification. The 7-bromo compound 2b harmonizes the stability of the dA-dT vs. the dG-dC pair. According to this the stability of such duplexes depends no longer on the base pair composition of a DNA molecule.     

Minor groove functional groups are critical for the B-form conformation of duplex DNA

Two analogue bases are described: 3-deazaadenine is a derivative of adenine from which N3 has been deleted and 3-methyl-2-pyridone is a C-nucleoside that mimics thymine but lacks the O2 carbonyl. The dc(3)A-dm(3)2P base pair is similar to dA-dT but eliminates the polar functional groups in the minor groove. The presence of this base pair in dA-dT rich sequences results in destabilized duplexes or conformational preferences for monomolecular hairpins rather than bimolecular duplexes. When present in dG-dC rich sequences, no significant differences in helix stability are observed. These differences are explained on the basis of hydration effects, most notably, the elimination of the minor groove spine of hydration normally present in dA-dT rich sequences. CD spectra suggest that sequences with a fully modified core (four analogue base pairs) are more A-like helices than B-like helices. Sequences containing two analogue base pairs can be transformed to A-like helices under conditions of high salt, or 65% trifluoroethanol. These conformational changes are also explained in terms of a loss of hydration in the minor groove that normally stabilizes the B-form conformation. In the absence of such hydration, the helices are conformationally mobile and adopt a more A-like helix form.     

Base dynamics of nitroxide-labeled thymidine analogues incorporated into (dA-dT)n by DNA polymerase I from Escherichia coli

Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template. All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base. The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry. The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands. The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization. A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems. Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier [Kao, S.-C., & Bobst, A.M. (1985) Biochemistry 24, 5465-5469]. The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Fluorescence properties and base pair stability of oligonucleotides containing 8-aza-7-deaza-2'-deoxyisoinosine or 2'-deoxyisoinosine

The fluorescence and the base pairing properties of 8-aza-7-deaza-2'-deoxyisoinosine (1) are described and compared with those of 2'-deoxyisoinosine (2). The corresponding phosphoramidites (11, 12) are synthesized using the diphenylcarbamoyl (DPC) residue for the 2-oxo group protection. The nucleosides 1 and 2 base pair with 2'-deoxy-5-methylisocytidine in DNA duplexes with antiparallel chain orientation and with 2'-deoxycytidine in a parallel DNA. These base pairs are less stable than the canonical dA-dT pair and that of 2'-deoxyinosine (4) with 2'-deoxycytidine. The fluorescence of the nucleosides 1 and 2 is quenched (approximately 95%) in duplex DNA. The residual fluorescence is used to determine the Tm-values, which are found to be the same as determined UV-spectrophotometrically.     

Solution Structure of Poly(dA-dT)· Poly(dA-dT) in Low and High Salt: A 500 MHz 1H NMR Study Using One-Dimensional NOE

Abstract

CD spectra of poly(dA-dT)· poly(dA-dT) in low salt (10–100 mM NaCl) and high salt (4–6 M CsF) are different i.e. 275 nm band gets inverted in going from low to high salt (Vorhickova et. al.MarJ. Mol. Biol. 166, 85, 1983). However, from CD spectra alone it is not possible to decipher any structural differences that might exist between the low and high salt forms of poly(dA-dT)? poly(dA-dT). Hence, we took recourse to high resolution NMR spectroscopy to understand the structural properties of poly(dA-dT)? poly(dA-dT) in low and high salt. A detailed analysis of shielding constants and extensive use of NOE studies under minimum spin diffusion conditions using C(8)-deuterated poly(dA-dT)? poly(dA-dT) enabled us to come up with the following conclusions (i) base-pairing is Watson-Crick under low and high salt conditions, (ii) under both the conditions of salt the experimental data can be explained in terms of an equilibrium blend of right and left-handed B-DNA duplexes with the left-handed form 70% and the right-handed 30%. In a 400 base pairs long poly(dA-dT)? polyidA-dT) (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis, (iii) However, there are other structural differences between the low and high salt forms of poly(dA-dT) ? poly(dA-dT); under the low salt condition, right-and left-handed B-DNA duplexes have mononucleotide as a structural repeat while under the high salt conditions, right-and left-handed B-DNA duplexes have dinucleotide as a structural repeat. In the text we provide the listing of torsion angles for the low and high salt structural forms, (iv) Salt (CsF) induced structural transition in poly(dA-dT)? poly(dA-dT) occurs without any breakage of Watson- Crick pairing, (v) The high salt form of poly(dA-dT)? poly(dA-dT) is not the left-handed Z-helix.

Although the results above from NMR data are quite unambiguous, a question still remains i.e. what does the salt (CsF) induced change in the CD spectra of poly(dA-dT)? poly(dA-dT) really indicate? Interestingly, we could show that the salt (CsF) induced change in poly(dA-dT)? poly(dA-dT) is quite similar to that caused by a basic polypeptide viz. poly-L(Lys₂-Ala)_n i.e. both the agents induced a ψ-structure in DNA. And it was also demonstrated that the changes in poly(dA-dT)? poly(dA-dT) as caused by CsF and poly-L-(Lys₂-Ala)_n could be reverted back by ethidium bromide-a relaxing agent.

To minimize complications from spin diffusion in this study we have used very small presaturation pulse lengths and C(8)-deuterated poly(dA-dT)? poly(dA-dT) of 400 ± 150 bp long. Even though deuteration of a primary site of diffusion such as C(8)H substantially decreases diffusion, in order to make sure that our conclusions are not compromised by possible diffusion in such a long fragment under small presaturation times, we have repeated our experiments using the six base pair long duplex of d(A-T-A-T-A-T) and found the results to be strikingly similar to that from the polymer.     

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

A series of nucleobase-modified siRNA duplexes containing "rare" nucleosides, 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D), were evaluated for their thermodynamic stability and gene silencing activity. The duplexes with modified units at terminal positions exhibited similar stability as the nonmodified reference. Introduction of the s(2)U or Psi units into the central part of the antisense strand resulted in duplexes with higher melting temperatures (Tm). In contrary, D unit similarly like wobble base pair led to the less stable duplexes (DeltaTm 3.9 and 6.6 degrees C, respectively). Gene-silencing activity of siRNA duplexes directed toward enhanced green fluorescent protein or beta-site APP cleaving enzyme was tested in a dual fluorescence assay. The duplexes with s(2)U and Psi units at their 3'-ends and with a D unit at their 5'-ends (with respect to the guide strands) were the most potent gene expression inhibitors. Duplexes with s(2)U and Psi units at their 5'-ends were by 50% less active than the nonmodified counterpart. Those containing a D unit or wobble base pair in the central domain had the lowest Tm, disturbed the A-type helical structure, and had more than three times lower activity than their nonmodified congener. Activity of siRNA containing the wobble base pair could be rescued by placing the thio-nucleoside at the position 3'-adjacent to the mutation site. Thermally stable siRNA molecules containing several s(2)U units in the antisense strand were biologically as potent as their native counterparts. The present results provide a new chemical tool for modulation of siRNA gene-silencing activity.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

Fluorescence Properties and Base Pair Stability of Oligonucleotides Containing 8-Aza-7-deaza-2′-deoxyisoinosine or 2′-Deoxyisoinosine

Abstract

The fluorescence and the base pairing properties of 8-aza-7-deaza-2′-deoxyisoinosine (1) are described and compared with those of 2′-deoxyisoinosine (2). The corresponding phosphoramidites (11,12) are synthesized using the diphenyl-carbamoyl (DPC) residue for the 2-oxo group protection. The nucleosides 1 and 2 base pair with 2′-deoxy-5-methylisocytidine in DNA duplexes with antiparallel chain orientation and with 2′-deoxycytidine in a parallel DNA. These base pairs are less stable than the canonical dA-dT pair and that of 2′-deoxyinosine (4) with 2′-deoxycytidine. The fluorescence of the nucleosides 1 and 2 is quenched (～95%) in duplex DNA. The residual fluorescence is used to determine the T_m-values, which are found to be the same as determined UV-spectrophotometrically.     

The thermal stability of RNA duplexes containing modified base pairs placed at internal and terminal positions of the oligoribonucleotides

The presence of various modifications within oligomers changes their thermodynamic stability. To get more systematic data, we measured effects of 5- and 6-substituted uridine on thermal stability of (AUCU(Mod.)AGAU)2 and (AUCUAGAU(Mod.))2. Collected results lead to the following conclusions: (i) 5-halogenated and 5-alkylated substituents of the uridine affect thermal stability of the RNA duplexes differently. Moreover, the 5-fluorouridine changes stability of the RNA duplexes opposite to remaining 5-halogenouridines; (ii) for oligomers containing 5-chloro, 5-bromo or 5-iodouridine stronger hydrogen bond formed between oxygen-4 of the 5-halogenated uracil and 6-amino group of the adenine is presumably responsible for stabilizing effect; (iii) placing of A-U(5R) base pairs closer to the end of the duplex enhance thermal stability relatively to oligomer with central position of this base pair; (iv) the effects of 5-substituents are additive, particularly for substituents which stabilize RNA duplexes; (v) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine present at internal position of A-U(Mod.) inhibit duplexes formation; (vi) 6-methyluridines (N1 and N3 isomers) as well as 3N-methyluridine placed as terminal base pairs stabilize the duplexes mostly via 3'-dangling end effect.     

The stability of duplexes involving AT and/or G4EtC base pairs is not dependent on their AT/G4EtC ratio content. Implication for DNA sequencing by hybridization.

Sequencing by the recently reported hybridization technique requires the formation of DNA duplexes with similar stabilities. In this paper we describe a new strategy to obtain DNA duplexes with a thermal stability independent of their AT/GC ratio content. Melting data were acquired on 35 natural and 27 modified duplexes of a given length and of varying base compositions. Duplexes built with AT and/or G4EtC base pairs exhibit a thermal stability restrained to a lower range of temperature than that of the corresponding natural compounds (16 instead of 51 degrees C). The 16 degrees C difference in thermal stability observed between the least stable and the most stable duplex built with AT and/or G4EtC base pairs is mainly due to the sequence effect and not to their AT/G4EtC ratio content. Thus N -4-ethyl-2'-deoxycytidine (d4EtC) hybridizes specifically with natural deoxyguanosine leading to a G4EtC base pair whose stability is very close to that of the natural AT base pair. Oligonucleotide probes involving d4EtC can be easily prepared by chemical synthesis with phosphoramidite chemistry. Modified DNA targets were successfully amplified by random priming or PCR techniques using d4EtCTP, dATP, dGTP and dTTP in the presence of DNA polymerase. This new system might be very useful for DNA sequencing by hybridization.     

Synthesis and properties of cationic 2'-O-[N-(4-aminobutyl)carbamoyl] modified oligonucleotides

2'-O-[N-(4-Aminobutylcarbamoyl)]uridine (U(abcm)) was synthesized and incorporated into oligonucleotides. The oligonucleotides incorporating U(abcm) formed more stable duplexes with their complementary and mismatched RNAs than those containing 2'-O-carbamoyluridine (U(cm)). The stability of duplex with a U(abcm)-rG base pair showed higher thermostability than the duplex having unmodified U-rG base pair. The U(abcm) residue showed enhanced resistance to snake venome phosphodiesterase.     

Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.     

The flexibility of alternating dA-dT sequences

The flexibility of alternating poly (dA-dT) has been investigated by the technique of transient electric dichroism. Rotational relaxation times, which are very sensitive to changes in the end-to-end length of flexible polymers, are determined from the field free dichroism decay curves of four, well defined fragments of poly (dA-dT) ranging in size from 136 to 270 base pairs. Persistence lengths, calculated from the results of Hagerman and Zimm (Biopolymers (1981) 29, 1481-1502), are in the range 200-250 A. This makes alternating dA-dT sequences about twice as flexible as naturally occurring, "random" sequence DNA. Considering a bend around a nucleosome, for example, this difference in persistence length translates to an energy difference between poly (dA-dT) and random sequence DNA of 0.17 kT/base pair or 1 kcal per 10 base pair stretch. This energy difference is sufficiently large to suggest that dA-dT sequences could serve as markers in DNA packaging, for example, at sites where DNA must tightly bend to accommodate structures.     

Sequence-dependent changes in the chiroptical properties of DNA upon interaction with dipyrandium

Interaction of dipyrandium with DNA and its dependence on the base sequence was studied using circular dichroism. It was found that calf thymus DNA and polynucleotide duplexes with alternating purine-pyrimidine sequences containing GC basepairs underwent similar alterations in the chiroptical properties upon binding of dipyrandium. The alterations suggest that these DNAs have similar B-type structures which may kink at the dipyrandium binding sites. On the other hand, poly(dA-dt)·poly(dA-dT) and especially poly(dA-dU)·poly(dA-dU) exhibit some features of A-type structure. Poly(dA-dT)·poly(dA-dT) changes its chiroptical properties little when complexed with dipyrandium, as if it contained some type of kinks as equilibrium structural elements.     

Towards a DNA-like duplex without hydrogen bonds

The inverse quadrupolar moments of the phenyl and pentafluorophenyl residues in the base pair P-F5 promotes strong intramolecular stacking interactions in DNA duplexes. The more natural base pairs are replaced by this novel pair the higher the thermodynamic stability of the resulting duplex if they are arranged in an alternating fashion.     

Effects of functional group changes in the EcoRI recognition site on the cleavage reaction catalyzed by the endonuclease

Oligodeoxynucleotides have been prepared that contain changes in the functional group pattern present in the EcoRI recognition site. These changes involve "functional group deletions", "functional group reversals", and "displaced functional groups". Steady-state kinetic parameters have been used to characterize the interaction of these modified recognition sites with the EcoRI endonuclease. Changes in the functional group pattern have varying effects upon the cleavage reaction. Both the exocyclic amino groups of the two adenine residues and the methyl groups of the thymine residues appear to interact with the endonuclease quite differently. In both cases efficient catalysis was observed when these functional groups were present at the "outer" dA-dT base pair. Selectivity was decreased by over an order of magnitude largely via increases in Km when these functional groups were deleted. Similar modifications at the "inner" dA-dT base pair did not alter the kinetic parameters significantly from those observed with the native sequence. Addition of an amino group to the minor groove at the outer dA-dT base pair resulted in a modified recognition site that interacted with the enzyme, on the basis of observed competitive inhibition kinetics, but was not cleaved.     

Thermodynamics and kinetics for base-pair opening in the P1 duplex of the Tetrahymena group I ribozyme

The thermodynamics and kinetics for base-pair opening of the P1 duplex of the Tetrahymena group I ribozyme were studied by NMR hydrogen exchange experiments. The apparent equilibrium constants for base pair opening were measured for most of the imino protons in the P1 duplex using the base catalysts NH3, HPO4(2-) or TRIS. These equilibrium constants were also measured for several modified P1 duplexes, and the C-2.G23 base pair was the most stable base pair in all the duplexes. The conserved U-1*G22 base pair is required for activity of the ribozyme and the data here show that this wobble base pair destabilizes neighboring base pairs on only one side of the wobble. A 2'-OMe modification on the U-3 residue stabilized its own base pair but had little effect on the neighboring base pairs. Three base pairs, U-1*G22, C-2*G23 and A2*U21 showed unusual equilibrium constants for opening and possible implications of the opening thermodynamics of these base pairs on the undocking rates of the P1 helix with catalytic core are discussed.     

Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch.

Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.