Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Effect of deoxyribonuclease on adriamycin-polynucleotide complexes.

Recent interest in the use of adriamycin-DNA complex as an approach to improve the therapeutic effectiveness and to reduce toxicity of adriamycin for cancer chemotherapy requires an in-depth understanding of the physicochemical and biochemical properties of such complexes. The interactions of adriamycin with single-strand polydeoxyribonucleotides, double-strand DNA, and double-strand ribodeoxyribopolynucleotide hybrids were therfore investigated. Association constants (Kapp) of adriamycin and polynucleotides were obtained. These data showed that the inherent variable in such complex lies in the composition of the polynucleotides. Alternate deoxyguanylate (dG)-deoxycytidylate (dC) sequence binds 7-fold better than alternate deoxyadenylate (dA)-deoxythymidylate (dT) sequence. Comparative studies of the hydrolysis of DNA duplexes by deoxyribonucleases I and II with and without adriamycin were also carried out. The rate of hydrolysis decreased in the order poly(dA-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase I and poly(dA)-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase II. Intercalation of adriamycin to deoxyribopolynucleotide duplex resulted in inhibition of DNase II two to three times more than tat of DNase I. On the other hand, intercalation of adriamycin to homodeoxypolynucleotide duplex poly(dA)-poly(dT) and poly(dG)-poly(dC) enhanced the DNase I hydrolysis. If DNase I activity could be related to serum DNase and DNase II related to tumor lyososomal DNase as in the endocytosis mechanism proposed by Trouet et al. (Cancer Chemotherapy Rept., 59: 260, 1975), the best adriamycin carrier suggested by this investigation could be poly(dA)-poly(dT) and poly(dG-dC). It is also suggested in this study that adriamycin-RNA-DNA hybrid could be of interest as an antiviral agent by a similar release mechanism via RNase H, an enzyme associated with viral reverse transcriptase.

Position-specific inhibition of yeast mitochondrial transcription by a poly(T) sequence.

The 3' flanking nucleotide(s) of the octanucleotide promoter sequence regulates transcriptional efficiency of some mitochondrial genes in Saccharomyces cerevisiae. To understand this regulation the in vitro transcriptional activity of various synthetic mitochondrial promoters carrying different 3' flanking sequences was examined. The results presented here demonstrate that consecutive thymidine residues, but no other polynucleotides or secondary structure, in the promoter-proximal non-transcribed DNA strand inhibited mitochondrial transcription. The location and the number of T residues in the cluster as well as the concentration of UTP in the transcription reaction are the important factors determining this transcriptional inhibition. For example, a pair of thymidine nucleotides at positions +2 and +3 is sufficient for inactivation of mitochondrial transcription, whereas more than three consecutive thymidine nucleotides beyond these positions are required for inhibition of mitochondrial transcription. However, a cluster of six to 12 thymidine residues beyond position +11, a point where mtRNA polymerase has been shown to form a stable transcription complex, did not interfere with mitochondrial transcription. Interestingly, at low UTP concentration the mtRNA polymerase generates a large quantity of aborted initiation products on a template carrying promoter-proximal poly(T) sequence probably due to the inability of the polymerase to clear this promoter. On the other hand at high UTP concentration the same mtRNA polymerase on the same mitochondrial promoter produces a higher level of productive initiation complex. These observations suggest that the mechanism of poly(T) inhibition of mitochondrial transcription is a UTP-limited transcriptional attenuation at the promoter site, which might occur under specific physiological conditions (i.e. glucose repression-derepression, switching of aerobic-anaerobic conditions).

Molecular characterization, tissue distribution and expression analysis of TRIM25 in Gallus gallus domesticus.

TRIM25, a member of the tripartite motif-containing (TRIM) family of proteins, plays an important role in cell proliferation, protein modification, and the RIG-I-mediated antiviral signaling pathway. However, relatively few studies have investigated the molecular characterization, tissue distribution, and potential function of TRIM25 in chickens. In this study, we cloned the full-length cDNA of chicken TRIM25 that is composed of 2706 bp. Sequence analyses revealed that TRIM25 contains a 1902-bp open-reading frame that probably encodes a 633-amino acid protein. Multiple comparisons with deduced amino acid sequences revealed that the RING finger and B30.2 domains of chicken TRIM25 share a high sequence similarity with human and murine TRIM25, indicating that these domains are critical for the function of chicken TRIM25. qPCR assays revealed that TRIM25 is highly expressed in the spleen, thymus and lungs in chickens. Furthermore, we observed that TRIM25 expression was significantly upregulated both in vitro and in vivo following infection with Newcastle disease virus. TRIM25 expression was also significantly upregulated in chicken embryo fibroblasts upon stimulation with poly(I:C) or poly(dA:dT). Taken together, these findings suggest that TRIM25 plays an important role in antiviral signaling pathways in chickens.

Poly(dT) inhibition of globin synthesis in the rabbit reticulocyte lysate system. Reversal of the inhibition by poly(dT)-binding protein.

The mechanism of inhibition of globin synthesis by poly(dT) was studied in the rabbit reticulocyte lysate system. When the lysate was incubated with [14C]poly(dT), poly(dT) was found to bind with the native 40S ribosomal subunit and the "supernatant factor." But the binding to the native 40S ribosomal subunits was not directly related to the poly(dT) inhibition. Ribosomal subunits were prepared from rabbit reticulocytes and tested for their binding with poly(dT) and their effect on the poly(dT) inhibition. Poly(dT) was found to bind with the derived 40S ribosomal subunit, but not with the derived 60S subunit, and the poly(dT) inhibition was slightly reversed by the derived 40S ribosomal subunit. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of the 80S/Met-tRNAf complex was inhibited by poly(dT) and the inhibition was greater at high concentration of KOAc. However, the formation of the 40S/Met-tRNAf complex was inhibited to the same extent at 70 mM and 200 mM KOAc in the presence of GMPPCP. A factor (TF) that reverses the poly(dT) inhibition was partially purified from the KCl-wash of rabbit reticulocyte ribosomes by ammonium sulfate fractionation, and Sephadex G-150 and DEAE-cellulose column chromatographies. From the Sephadex G-150 and DEAE-cellulose column chromatography of TF, the molecular weight of TF was estimated to be 81,000-102,000. TF reversed the poly(dT) inhibition of 80S/[3H]mRNA/Met-tRNAf complex or that of 40S/[3H]mRNA/Met-tRNAf complex. TF bound to [14C]poly(dT) or 3H-labeled globin mRNA. SDS/polyacrylamide slab gel electrophoresis of the complexes between the factor and [14C]poly(dT) or [3H]mRNA showed common polypeptide bands of 22,500, 25,000, and 49,000 daltons. These data can be explained by assuming that poly(dT) binds to a factor which is required for the binding of 40S/Met-tRNAf complex with mRNA to form inactive complexes, and thus inhibits globin synthesis. The relationship between the poly(dT)-binding protein and known initiation factors is discussed.

Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8.

The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS) remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs) or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs) with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

A five-amino-acid motif in the undefined region of the TLR8 ectodomain is required for species-specific ligand recognition.

Toll-like receptors play important roles in regulating immunity against microbial infections. Toll-like receptor 8 (TLR8) belongs to a subfamily comprising TLR7, TLR8 and TLR9. Human TLR8 mediates anti-viral immunity by recognizing ssRNA viruses, and triggers potent anti-viral and antitumor immune responses upon ligation by synthetic small molecular weight ligands. Interestingly, distinct from human TLR8, mouse TLR8 was not responsive to ligand stimulation in the absence of polyT-oligodeoxynucleotides (polyT-ODN). The molecular basis for this distinct ligand recognition is still unclear. In the present study, we compared the activation of TLR8 from different species including mouse, rat, human, bovine, porcine, horse, sheep, and cat by ligand ligations. Only the TLR8s from the rodent species (i.e., mouse and rat TLR8s) failed to respond to ligand stimulation in the absence of polyT-ODN. Multiple sequence alignment analysis suggested that these two rodent TLR8s lack a five-amino-acid motif that is conserved in the non-rodent species with varied sequence. This small motif is located in an undefined region of the hTLR8 ectodomain, immediately following LRR-14. Deletion mutation analysis suggested that this motif is essential for the species-specific ligand recognition of hTLR8, whereas it is not required for self-dimerization and intracellular localization of this receptor.

Cutting edge: activation of murine TLR8 by a combination of imidazoquinoline immune response modifiers and polyT oligodeoxynucleotides.

Synthetic immune response modifiers (IRM) such as imidazoquinolines can selectively activate human TLR7 or TLR8. Although these endosomal TLRs are close relatives, TLR7-deficient mice are unresponsive to TLR8 agonist IRMs. Similarly, natural ssRNA cannot activate murine TLR8, leading to the belief that murine TLR8 is nonfunctional. In this study, we transfected HEK293 cells with murine TLR8 and NF-kappaB reporter constructs and stimulated them with combinations of IRM and oligodeoxynucleotides (ODNs). When stimulated with TLR7 or TLR8 agonists alone, no NF-kappaB response was observed. However, a combination of polyT ODN plus the TLR8 agonist activated NF-kappaB, whereas polyT ODN plus the TLR7 agonist did not activate. Primary mouse cells responded to the IRM/polyT ODN by secreting TNF. Cells from TLR7(-/-) and TLR9(-/-) mice responded to the IRM/polyT ODN combination, whereas MyD88(-/-) cells did not respond. In conclusion, this study demonstrates for the first time that mouse TLR8 is functional.

Protease-resistant prion protein amplification reconstituted with partially purified substrates and synthetic polyanions.

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27-30 and PrPC molecules at a molar ratio of 1:250 yielded approximately 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to approximately 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27-30, PrPC, and a stimulatory polyanionic compound.

A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen.

Highly purified SV40 large T antigen exhibits an ATPase activity which can be stimulated approximately 7-fold by the DNA homopolymer poly(dT). The poly(dT)-stimulated enzyme can hydrolyze various ribonucleotide and deoxyribonucleotide triphosphates, with ATP and dATP serving as the best substrates. Purified large T antigen hydrolyzes ATP to ADP and Pi, with a maximum specific activity of 13.5 mumol of inorganic phosphate released per h per mg of protein. Of the various natural and synthetic polynucleotides tested, poly(dT) was by far the best activator. Long chain poly(dT) molecules are much more effective activators than are short chain length oligo(dT) molecules. The highly purified large T antigen contains no detectable protein kinase activity.

Immunochemical detection of sequence-specific modifications to DNA induced by UV light.

Sequence specificity of antibodies to UV-damaged DNA has not been described previously. The antisera investigated here were specific for UV-modified DNA and were absolutely dependent upon the presence of thymine residues. Using a series of oligonucleotides in competition ELISA, increased inhibition was observed with increasing chain length of UV-polythymidylate. A minimum of three adjacent thymines was required for effective inhibition; alone, dimers of thymine were poor antigens. Although UV-irradiated poly(dC) was not antigenic, cytosines could partially replace thymines within the smallest effective epitope (T-T-T) with a high degree of sequence specificity, not previously described. The main epitope induced by UV was formed from adjacent thymines and either a 3' or a 5' pyrimidine.

Characteristics of the inhibition of poly(ADP-ribose) glycohydrolase by homopolypurines.

Poly(ADP-ribose) glycohydrolase partially purified from rat testis was markedly inhibited by the homopolypurines polyG, polyI and polyA. The inhibition was competitive with respect to poly(ADP-ribose) and the Ki for polyG and polyA was 2.8 uM and 5.5 uM, respectively. This inhibitory effect of the homopolypurines was practically eliminated when 250 mM KCl was present in the reaction mixture. Moreover, the inhibition exerted by polyI or polyA was markedly diminished after hybridization with polyC or polyT, respectively.

recA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor.

To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant. In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment. In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present. However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide. The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present. Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand.

Specific in vitro adenylylation of the simian virus 40 large tumor antigen.

Incubation of the simian virus 40 (SV40) large tumor antigen (T) from either transformed or lytically infected cells with adenosine [8-3H]-, [alpha-32P]-, or [alpha-[35S]thio]-triphosphate in the presence of Mg2+ resulted in its labeling as defined by the appearance of an intact, appropriately immunoreactive band in NaDodSO4/polyacrylamide gels. Radioactivity remained associated with the protein after boiling in buffer containing 3% NaDodSO4, and 2-mercaptoethanol as well as after heating in 0.1 M HCl, 0.1 M NH4OH, or hydroxylamine, but it was dissociated after incubation in 0.1 M NaOH at 37 degrees C. After limited boiling of gel-purified [alpha-32P] ATP + T complex in 5.6 M HCl, o-[32P]phosphoserine was released, and snake venom phosphodiesterase or 0.5 M piperidine treatment of such a complex resulted in the liberation of [alpha-32P]AMP. The reaction proceeded when either purified, soluble T or insoluble, specifically immunoprecipitated antigen was used as substrate. ATP and dATP were the preferred nucleotide substrates by comparison with the other six standard ribonucleoside or deoxynucleoside triphosphates. Partial tryptic digests of T + [alpha-32P]ATP complexes revealed the presence of a single labeled peptide of Mr approximately equal to 12 - 14 X 10(3), and after exhaustive digestion, there was a single radioactive spot in the fingerprint. These data indicate that T can be adenylylated at a specific seryl residue(s) in a limited portion of the protein surface. Furthermore, adenylylation appears to be reversible and to proceed by a pyrophosphorylytic mechanism, since the nucleotide was released from the protein following incubation of adenylylated T with Mg2+, sodium pyrophosphate, and poly(dT).

Factors controlling the size of DNA loops in frog embryos and Friend erythroleukemia cells.

Measurements were made of the average size of fluorescent halos of nucleoids of developing frog embryos, mouse fibroblasts (A9 cells) and Friend erythroleukemia cells cultured with various compounds. These halos are thought to represent relaxed lengths of loops of DNA attached to the nuclear matrix. Measurement of transcription of RNA from nuclei in vitro suggested that cells with more loops (smaller halos) had more initiation sites for transcription. DNA loop number decreases during development of the frog embryos, and red blood cells of adults have even fewer and larger loops. One or two days of culture of Friend cells in various compounds that induce differentiation resulted in more DNA loops than for control cells, but after 5 days of culture the differentiated erythrocytes had fewer DNA loops than control cells at stationary phase. Stationary phase cells have almost twice as many DNA loops as log phase cells. Various compounds that stall cells at the G1/S border induced more DNA loops, but subsequently the number of DNA loops decreased as the cells entered S phase. A number of compounds which slow the rate of cell division cause the formation of more DNA loops, perhaps due to longer G1 periods.

Purification and characterization of two forms of DNA-dependent ATPase from yeast.

Two forms of DNA-dependent ATPase activity have been purified from yeast extracts. The two ATPases differ from each other in chromatographic properties and heat stabilities but have similar molecular weight and reaction properties. DNA-dependent ATPase I has been purified to near homogeneity, while DNA-dependent ATPase II is only partially purified. The two ATPases from yeast are related structurally since antiserum raised against ATPase I cross-react against ATPase II. Yeast DNA-dependent ATPase I has a native molecular weight of about 68,000 and consists of a single polypeptide chain. ATPase II also sediments on sucrose gradient as a 68,000-dalton protein. Both yeast DNA-dependent ATPases hydrolyze dNTPs and rNTPs to their corresponding nucleoside diphosphates and orthophosphate, but dATP and ATP are preferred substrates. In addition to nucleoside triphosphates, both enzymes require a divalent cation and a polynucleotide for activity. Single-stranded DNAs and polydeoxynucleotides are the most effective co-substrates for yeast DNA-dependent ATPases. Addition of yeast DNA-dependent ATPases to DNA synthesis system containing yeast DNA polymerases does not significantly stimulate the rate of DNA synthesis.

Mechanisms of polyclonal B-cell activation in autoimmune B6-lpr/lpr mice.

The influence of the lpr gene on spontaneous and lipopolysaccharide (LPS)-induced immunoglobulin production was studied in B6 mice homozygous for the mutant lpr gene (B6-lpr/lpr). Male and female mice of this congenic strain were followed for 1 year and sera serially tested by the enzyme-linked immunosorbent assay (ELISA) for the production of antibodies to single-stranded DNA (anti-sDNA), immunoglobulin (anti-IgG), and keyhole limpet hemocyanin (anti-KLH), models of autoantibody and non-autoantibody responses, respectively. Female B6-lpr/lpr mice demonstrated marked spontaneous responses to all three antigens; the responses of male B6-lpr/lpr mice were significantly lower but still exceeded those of the congenic B6-+/+ controls. These results demonstrate a generalized influence of sex on lpr associated responses. To determine whether this sex difference could be demonstrated with other forms of B-cell activation, young B6-+/+ and B6-lpr/lpr male and female mice were immunized with lipopolysaccharide and the induced responses determined. This immunization caused significant increases in the IgM response only. The levels of the induced responses produced after LPS treatment were comparable for +/+ and lpr/lpr mice. These results indicate that the enhanced responsiveness of female mice to lpr action is not reflected in the polyclonal response to LPS, which, furthermore, was unaffected by the presence of lpr. The differential influence of sex on lpr and LPS-induced responses and their apparent independence suggests that lpr and LPS promote B-cell activation by dissimilar mechanisms.

Sequence-targeted chemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins.

Oligo-heptathymidylates covalently linked to porphyrins bind to complementary sequences and can induce local damages on the target molecule. In dark reactions, iron porphyrin derivatives exhibited various chemical reactivities resulting in base oxidation, crosslinking and chain scission reactions. Reactions induced by reductants, such as ascorbic acid, dithiothreitol or mercapto-propionic acid, led to very localised reactions. A single base was the target for more than 50% of the damages. Oxidising agents such as H2O2 and its alkyl derivatives induced reactions that extended to a wider range of altered bases. The specificity of the chemical modifications observed in these systems is discussed from a mechanistic point of view.

Sequence-specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-[alpha]-thymidylate covalently linked to an azidoproflavine derivative.

A 3-azidoproflavine derivative was covalently linked to the 5'-end of an octathymidylate synthesized with the [alpha]-anomers of the nucleoside. Two target nucleic acids were used for this substituted oligo-[alpha]-thymidylate: a 27-mer single-stranded DNA fragment containing an octadeoxyadenylate sequence and a 27-mer duplex containing eight contiguous A.T base pairs with all adenines on the same strand. Upon visible light irradiation the octa-[alpha]-thymidylate was photocrosslinked to the single-stranded 27-mer. Chain breaks were induced at the crosslinked sites upon piperidine treatment. From the location of the cleavage sites on the 27-mer sequence it was concluded that a triple helix was formed by the azidoproflavine-substituted oligo-[alpha]-thymidylate with its complementary oligodeoxyadenylate sequence. When the 27-mer duplex was used as a substrate cleavage sites were observed on both strands after piperidine treatment of the irradiated sample. They were located at well defined positions which indicated that the octathymidylate was bound to the (dA)8.(dT)8 sequence in parallel orientation with respect to the (dA)8-containing strand. Specific binding of the [alpha]-octathymidylate involved local triple strand formation with the duplex (dA)8.(dT)8 sequence. This result shows that it is possible to synthesize sequence-specific molecules which specifically bind oligopurine-oligopyrimidine sequences in double-stranded DNA via recognition of the major groove hydrogen bonding sites of the purines.