The synthesis of anti-fixed 3-methyl-3-deaza-2'-deoxyadenosine and other 3H-imidazo[4,5-c]pyridine analogs

Rotation of a heterocyclic base around a glycosidic bond allows the formation of syn and anti conformations in nucleosides. The syn conformation has been observed primarily in purine-purine mismatches in DNA duplexes. Such mismatches give rise to false positive oligonucleotide hybridization in DNA-based diagnostics. Here we describe the synthesis of an analog of 2'-deoxyadenosine that retains its Watson-Crick functional groups, but cannot form the syn conformation. In this analog, the N3 atom of 2'-deoxyadenosine is replaced by a C-CH3 group to give 7-methyl-1-beta-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine or 3-methyl-3-deaza-2'-deoxyadenosine (3mddA). This modification sterically prevents the syn conformation and 3mddA becomes an anti-fixed nucleoside analog of 2'-deoxyadenosine. The synthesis and conformational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-c]pyridine skeleton are described, as well as their potential applications.     

Rnai Activity of Sirnas Modified with 2′-Aminoalkyl-Substituted Fluorinated Nucleobases

We recently reported that a 1′-deoxy-1′-(4,6-difluoro-1H-benzimidazol-1-yl)-2′-(β-aminoethyl)-β-d-ribofuranose nucleoside appears to be a universal nucleoside which does not differentiate between the four natural nucleosides A, C, G, and U in duplexes. Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches.^{[ 1} Klöpffer, A. E. and Engels, J. W. 2004. Synthesis of 2′-aminoalkyl substituted fluorinated nucleobases and their influence on the kinetic properties of hammerhead ribozymes. ChemBioChem, 5: 100–109.  [Google Scholar] ^] Due to these data, we investigated the ability of this compound to tolerate Watson-Crick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.     

THE SYNTHESIS OF ANTI-FIXED 3-METHYL-3- DEAZA-2′-DEOXYADENOSINE AND OTHER 3H-IMIDAZO[4,5-c]PYRIDINE ANALOGS

ABSTRACT

Rotation of a heterocyclic base around a glycosidic bond allows the formation of syn and anti conformations in nucleosides. The syn conformation has been observed primarily in purine-purine mismatches in DNA duplexes. Such mismatches give rise to false positive oligonucleotide hybridization in DNA-based diagnostics. Here we describe the synthesis of an analog of 2′-deoxyadenosine that retains its Watson-Crick functional groups, but cannot form the syn conformation. In this analog, the N3 atom of 2′-deoxyadenosine is replaced by a C-CH₃ group to give 7-methyl-1-β-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine or 3-methyl-3-deaza-2′-deoxyadenosine (3mddA). This modification sterically prevents the syn conformation and 3mddA becomes an anti-fixed nucleoside analog of 2′-deoxyadenosine. The synthesis and conformational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-c]pyridine skeleton are described, as well as their potential applications.     

Influence of assembly of siRNA elements into RNA-induced silencing complex by fork-siRNA duplex carrying nucleotide mismatches at the 3'- or 5'-end of the sense-stranded siRNA element

RNA interference (RNAi) is a powerful method for suppressing the expression of a gene of interest, and can be induced by 21-25 nucleotide small interfering RNA (siRNA) duplexes homologous to the silenced gene, which function as sequence-specific RNAi mediators in RNA-induced silencing complexes (RISCs). In the previous study, it was shown that fork-siRNA duplexes, whose sense-stranded siRNA elements carried a few nucleotide mismatches at the 3'-ends against the antisense-stranded siRNA elements, could enhance RNAi activity more than conventional siRNA duplexes in cultured mammalian cells. In this study, we further characterized fork-siRNA duplexes using reporter plasmids carrying target sequences complementary to the sense- or antisense-stranded siRNA elements in the untranslated region of Renilla luciferase. The data presented here suggest that nucleotide mismatches at either the 3'- or 5'-end of the sense-stranded siRNA elements in fork-siRNA duplexes could influence assembly of not only the antisense-stranded siRNA elements but also the sense-stranded elements into RISCs. In addition, we further suggest the possibility that there could be a positional effect of siRNA duplex on RNAi activity.     

Base pair hydrogen bonds are essential for proofreading selectivity by the human mitochondrial DNA polymerase

We have characterized the role of Watson-Crick hydrogen bonding in the 3'-terminal base pair on the 3'-5' exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3-6 s(-1) fast phase, 0.16-0.3 s(-1) slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04-0.22 s(-1)), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3'-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition.     

Synthesis and biochemical evaluation of Aminopropanolyl-Thymine tri-Phosphate (ap-TTP)

Abstract

Deoxyribonucleoside triphosphates (dNTPs) are building blocks for the biosynthesis of DNA. Various modified dNTPs’ analogs have synthesized by structural changes of nucleoside’s susgar and nucleobases and employed for synthesis of modified DNA. A very few modified dNTPs have prepared from non-sugar nucleoside analogs. This report describes the synthesis of acyclic nucleoside triphosphate (NTP) analog from amino acid L-Serine as aminopropanolyl-thymine triphosphate (ap-TTP) and demonstrate its biochemical evaluation as enzymatic incorporation of ap-TTP into DNA with DNA polymerases with primer extension methods. Alanyl peptide nucleicacids (Ala-PNA) are the analogs of DNA which contains alanyl backbone. Aminopropanolyl – analogs are derivatives of alanyl back bone. Ap-TTP analog is nucleoside triphosphate analog derived from Ala-PNA. Importantly, this report also sheds light on the crystal packing arrangement of alaninyl thymine ester derivative in solid-state and reveals the formation of self-duplex assembly in anti-parallel fashion via reverse Watson-Crick hydrogen bonding and π–π interactions. Hence, ap-TTP is a useful analog which also generates the free amine functional group at the terminal of DNA oligonucleotide after incorporation.     

MALDI-TOF mass spectral analysis of siRNA degradation in serum confirms an RNAse A-like activity

Synthetic siRNA duplexes are used widely as reagents for silencing of mRNA targets in cells and are being developed for in vivo use. Serum stability is a major concern if siRNA is to be used for therapeutic delivery within blood circulation. We have developed the use of MALDI-TOF mass spectrometry as a rapid and convenient analytical tool to identify the most vulnerable sites within siRNA to serum degradation. Using this approach, we found that one siRNA duplex (Dh3) with UpA sequences close to one end was particularly vulnerable to rapid cleavage. This produced a fragment of mass consistent with the presence of a 2',3'-cyclic phosphate that was slowly hydrolysed to a 2'-(3'-)phosphate on extended incubation. Substitution of these sites with 2'-O-methyl U residues prevented cleavage and confirmed that the major pathway for initial degradation is via cleavage by an RNAse A-like activity. Mass spectral analysis was used to follow the serum degradation of siRNA over more prolonged periods to show the accumulation of many fragments, almost all showing cleavage following pyrimidine nucleoside residues. Overall, the MALDI-TOF mass spectral analysis technique should prove useful for preliminary screening of the serum stability of siRNA duplexes and for identification of the most vulnerable cleavage sites.     

Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line

In order to study nucleoside analog activation in the CEM cell line, a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation. Hematopoetic cell lines can be difficult to transfect with traditional lipid-based transfection, so the electroporation technique was used. Field strength, pulse length, temperature, electroporation media, siRNA concentration, among other conditions were tested in order to obtain approximately 70–80% mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase (dCK), necessary for nucleoside analog activation. Downregulation was assessed at mRNA and enzyme activity levels. After optimizing the protocol, a microarray analysis was performed in order to investigate whether the downregulation was specific. Additionally two genes were differentially expressed besides the downregulation of dCK. These were however of unknown function. The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs. Three of these pools were increased compared to untreated, unelectroporated cells. The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected. The multidrug resistance to other drugs, as seen in nucleoside analog-induced resistant cells, was not seen with this model.     

Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.     

Synthesis of 5-(2-amino-2-deoxy-beta-D-glucopyranosyloxymethyl)-2'- deoxyuridine and its incorporation into oligothymidylates

The protected 5-(2-amino-2-deoxy-beta-D-glucopyranosyloxymethyl)-2'- deoxyuridine phosphoramidite 15 has been prepared from uridine in 12 steps. When incorporated into oligodeoxyribonucleotides the novel nucleoside analog 5 leads to decreased binding affinity. This decrease is larger with a complementary RNA-strand than with a DNA complement.     

Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.     

Improving the fidelity of Thermus thermophilus DNA ligase.

The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single-strand breaks (nicks) in DNA duplex substrates. The specificity and thermostability of this enzyme are exploited in the ligase chain reaction (LCR) and ligase detection reaction (LDR) to distinguish single base mutations associated with genetic diseases. Herein, we describe a quantitative assay using fluorescently labeled substrates to study the fidelity of Tth DNA ligase. The enzyme exhibits significantly greater discrimination against all single base mismatches on the 3'-side of the nick in comparison with those on the 5'-side of the nick. Among all 12 possible single base pair mismatches on the 3'-side of the nick, only T-G and G-T mismatches generated a quantifiable level of ligation products after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nucleoside analog at the third position of the discriminating oligonucleotide. Finally, two mutant Tth DNA ligases, K294R and K294P, were found to have increased fidelity using this assay.     

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.     

Free energy contributions of G.U and other terminal mismatches to helix stability

Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.     

GDP does not mediate but rather inhibits hormonal signal to adenylate cyclase

This study was aimed to elucidate whether GDP can mediate hormonal signal to adenylate cyclase in hepatic glucagon sensitive adenylate cyclase with ATP as substrate. Conversion of added GDP to GTP catalyzed by nucleoside diphosphate kinase was suppressed to less than 0.3% of added GDP by including UDP. Inhibition of this enzyme activity by UDP was accompanied by a preferential loss of the stimulatory effect of glucagon plus GDP on cyclase activity without changes in effects of glucagon plus GTP, glucagon plus guanosine 5'-(beta, gamma-imino)triphosphate, and NaF. Under this condition, i.e. in the presence of UDP, GDP competitively inhibited the actions of GTP (Ki for GDP, 1 microM) and guanosine 5'-(beta, gamma-imino)triphosphate in the presence of glucagon, the inhibition being complete at high GDP concentrations. GDP also inhibited cyclase activity stimulated by NaF with UDP but did only slightly without UDP. It was demonstrated that nucleoside diphosphate kinase is located in membranes in addition to cytosol fraction. However, the activity of membrane-associated enzyme was not affected by the addition of glucagon. Based on these observations, it is concluded that GDP is unable to mediate hormonal signal to adenylate cyclase and that it acts as an inhibitor of cyclase activity stimulated by GTP or its analog along with hormone. The results suggest a possible role of membrane-associated nucleoside diphosphate kinase in determining GTP and GDP levels at or near their binding site so as to replenish GTP and, thereby, decrease the inhibitory action of GDP when hormone is present.     

A Metal-containing Nucleoside That Possesses Both Therapeutic and Diagnostic Activity against Cancer

Nucleoside transport is an essential process that helps maintain the hyperproliferative state of most cancer cells. As such, it represents an important target for developing diagnostic and therapeutic agents that can effectively detect and treat cancer, respectively. This report describes the development of a metal-containing nucleoside designated Ir(III)-PPY nucleoside that displays both therapeutic and diagnostic properties against the human epidermal carcinoma cell line KB3-1. The cytotoxic effects of Ir(III)-PPY nucleoside are both time- and dose-dependent. Flow cytometry analyses validate that the nucleoside analog causes apoptosis by blocking cell cycle progression at G₂/M. Fluorescent microscopy studies show rapid accumulation in the cytoplasm within 4 h. However, more significant accumulation is observed in the nucleus and mitochondria after 24 h. This localization is consistent with the ability of the metal-containing nucleoside to influence cell cycle progression at G₂/M. Mitochondrial depletion is also observed after longer incubations (Δt ∼48 h), and this effect may produce additional cytotoxic effects. siRNA knockdown experiments demonstrate that the nucleoside transporter, hENT1, plays a key role in the cellular entry of Ir(III)-PPY nucleoside. Collectively, these data provide evidence for the development of a metal-containing nucleoside that functions as a combined therapeutic and diagnostic agent against cancer.     

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.