Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets.

Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia. Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives. Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative. The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate. The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C). As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases. It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.     

Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties.

We report here the site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides (ODNs). ODNs containing both 5-( N-aminohexyl)-carbamoyl-2'-deoxyuridine (H), which serves as a tether for the further conjugation of functional groups, and 5-(N,N-dimethylaminohexyl)carbamoyl-2'-deoxyuridine (D), which contributes to the thermal stability of the duplex and to the resistance to nucleolytic hydrolysis by nucleases, were synthesized. Functional groups such as folic acid and palmitic acid were site-specifically introduced into the terminus of the aminohexyl-linker of H. The thermal stability and resistance toward nuclease digestion of the modified ODNs were studied. We found that ODNs containing D and H formed stable duplexes with both the complementary DNA and RNA strands even when a bulky functional group such as folic acid, palmitic acid or cholesterol was attached to the terminus of the amino-linker. We also found that ODN analogues which contained D were more resistant to nucleolytic degradation by exo- and endonuclease than the unmodified ODN. Furthermore, duplexes formed by ODNs containing D and the complementary RNA could elicit RNase H activity.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Duplex recognition by oligonucleotides containing 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxy-2'-fluoro-D-ribose. Intermolecular 2'-OH-phosphate contacts versus sugar puckering in the stabilization of triple-helical complexes

To gain insight into the origins of the large binding affinity of RNA toward target duplexes, 2'-deoxy-2'-fluororibonucleic acid (2'F-RNA) and 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) were tested for their ability to recognize duplex DNA, duplex RNA, and RNA-DNA hybrids. 2'F-RNA, 2'F-ANA, and the corresponding control single-stranded (ss) DNA strands were shown to form triple-helical complexes only with duplex DNA and hybrid DNA (Pu)-RNA (Py), but not with duplex RNA and hybrid RNA (Pu)-DNA (Py). In contrast, an RNA third strand recognized all four possible duplexes (DD, DR, RD, and RR) as previously demonstrated by Roberts and Crothers [(1992) Science 258, 1463-1466]. The 2'F-RNA (C3'-endo) strand exhibited significantly reduced affinity for duplexes compared to an unmodified RNA (C3'-endo) strand. These findings are consistent with the intermolecular 2'-OH-phosphate contact mechanism proposed by Escudé et al. [(1993) Nucleic Acids Res. 24, 5547-5553], as a ribo 2'-F atom should not interact with a negatively charged phosphate. In addition, they emphasize the role of the 2'-OH ribose as a general recognition and binding determinant of RNA. The 2'-F arabino modification (2'F-ANA, C2'-endo) led to a considerable increase in the binding affinity for duplex DNA, as compared to those of DNA and 2'F-RNA third strands. This is likely to be the result of a greater population of C2'-endo pucker of the 2'F-ANA compared to DNA. The enhancement observed for 2'F-ANA strands toward duplex DNA is comparable to that observed with 2'-OMe RNA. Since 2'F-ANA has been shown to be more resistant to nuclease degradation than DNA, these results are likely to stimulate experimental work on arabinose derivatives in laboratories concerned with targeting DNA sequences in vivo ("antigene" strategy).     

Nucleosides and nucleotides. 204. Synthesis of oligodeoxynucleotides containing 6'alpha-[N-(Aminoalkyl)carbamoyloxy]-carbocyclic-thymidines and the thermal stability of the duplexes and their nuclease-resistance properties

To construct the nuclease-resistant oligodeoxynucleotides (ODNs) with natural phosphodiester linkages, we synthesized ODNs that contain 6'alpha-[N-(aminoalkyl)carbamoyloxy]-carbocyclic-thymidines (4, 5, and 6). The stability of these ODNs to nuclease hydrolysis was examined by using snake venom phosphodiesterase (3'-exonuclease) and nuclease S1 (endonuclease). It was found that the ODNs containing 4, 5, or 6 were more resistant to both the enzymes than the unmodified ODN. These nuclease-resistant properties are noteworthy, since they have natural phosphodiester linkages. Next, the thermal stabilities of duplexes consisting of these ODNs and either the complementary DNA or RNA were studied by thermal denaturation. The ODNs that contain 4 were found to enhance the thermal stability of the duplexes with the complementary DNA, while those containing 5 or 6 decreased the thermal stability of the ODN-DNA duplexes. On the other hand, all ODNs that contained 4, 5, or 6 decreased the thermal stability of the ODN-RNA duplexes.     

Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity.

The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.     

Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates.

Triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (2) was coupled to the 5' terminus of oligodeoxynucleotides via hydrogen phosphonate solid support DNA synthesis methodology. Duplex DNA oligomers with a single 5'-phospholipid melted at lower temperatures than the corresponding unmodified duplex, but duplexes bearing lipids at each 5' end had higher Tms. In uptake experiments with L929 cells, 8-10 times more lipid-DNA became cell-associated than did unmodified DNA. Unmodified antisense diesters were inactive in a VSV antiviral assay in L929 cells (at up to 200 microM). Attachment of a lipid to the oligomer, however, led to a greater than 90% at 150 microM (greater than 80% at 100 microM) reduction in viral protein synthesis. The antiviral activity depended on the sequence of the oligodeoxynucleotide, but some compounds having little or no base complementarity to the viral target were also effective. Phosphorothioate derivatives reduced viral protein synthesis by 20-30% at 100 microM in the VSV assay. The lipid-DNA compounds were not toxic to the cells at up to 100 microM.     

CD, absorption and thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes [d/r(AC)]12.[d/r(GT/U)]12 and the influence of phosphorothioate substitution.

Circular dichroism (CD) spectra and melting temperature (Tm) data for five duplexes containing phosphorothioate linkages were compared with data for four unmodified duplexes to assess the effect of phosphorothioate modification on the structure and stability of DNA. DNA and DNA.RNA duplexes. Nine duplexes were formed by mixing oligomers 24 nt long in 0.15 M K+(phosphate buffer), pH 7.0. Unmodified DNA.DNA and RNA.RNA duplexes were used as reference B-form and A-form structures. The CD spectra of the modified hybrids S-d(AC)12.r(GU)12 and r(AC)12.S-d(GT)12 differed from each other but were essentially the same as the spectra of the respective unmodified hybrids. They were more A-form than B-form in character. CD spectra of duplexes S-d(AC)12.d(GT)12 and d(AC)12.S-d(GT)12 were similar to that of d(AC)12.d(GT)12, except for a reduced long wavelength CD band. Sulfur modifications on both strands of the DNA duplex caused a pronounced effect on its CD spectrum. The order of thermal stability was: RNA.RNA > DNA.DNA > DNA.RNA > S-DNA.DNA > S-DNA. RNA > S-DNA.S-DNA. Phosphorothioation of one strand decreased the melting temperature by 7.8+/-0.6 degrees C, regardless of whether the substitution was in a hybrid or DNA duplex. Thermodynamic parameters were obtained from a multistate analysis of the thermal melting profiles. Interestingly, the destabilizing effect of the phosphorothioate substitution appears to arise from a difference in the entropy upon forming the DNA.DNA duplexes, while the destabilizing effect in the DNA.RNA hybrids appears to come from a difference in enthalpy.     

C5-(1-propynyl)-2'-deoxy-pyrimidines enhance mismatch penalties of DNA:RNA duplex formation

UV melting experiments show that C5-(1-propynyl)ation of seven pyrimidines to give a fully propynylated oligodeoxynucleotide (PrODN) heptamer increases the thermodynamic stability of six Watson-Crick paired DNA:RNA duplexes by 8.2 kcal/mol, on average, at 37 degrees C. About 2.5 kcal/mol of this enhancement is due to long-range cooperativity between the propynylated pyrimidines, Y(p)'s. On average, penalties for dU(p):rG, dC(p):rA, dU(p):rC, and dC(p):rC mismatches are enhanced by 2.9 kcal/mol in PrODN:RNA duplexes over those in unmodified duplexes. This results in penalties as large as 10 kcal/mol for a single mismatch. Removing a single propyne two base pairs away from a mismatch in a PrODN:RNA duplex eliminates the enhancement in specificity. Evidently, enhanced specificity is directly linked to long-range cooperativity between Y(p)'s. In most cases, the enhanced specificity is larger for internal than for terminal mismatches. PrODN:RNA duplexes are destabilized by full phosphorothioate backbone substitution to give S-PrODN:RNA duplexes. The S-PrODN:RNA duplexes retain enhanced mismatch penalties, however. These results provide insight for utilizing long-range cooperativity and enhanced specificity to improve nucleic acid based probe and drug design.     

Syntheses of 4'-thioribonucleosides and thermodynamic stability and crystal structure of RNA oligomers with incorporated 4'-thiocytosine

A facile synthetic route for the 4'-thioribonucleoside building block (4'S)N (N = U, C, A and G) with the ribose O4' replaced by sulfur is presented. Conversion of l-lyxose to 1,5-di-O-acetyl-2,3-di-O-benzoyl-4-thio-d-ribofuranose was achieved via an efficient four-step synthesis with high yield. Conversion of the thiosugar into the four ribonucleoside phosphoramidite building blocks was accomplished with additional four steps in each case. Incorporation of 4'-thiocytidines into oligoribonucleotides improved the thermal stability of the corresponding duplexes by approximately 1 degrees C per modification, irrespective of whether the strand contained a single modification or a consecutive stretch of (4'S)C residues. The gain in thermodynamic stability is comparable to that observed with oligoribonucleotides containing 2'-O-methylated residues. To establish potential conformational changes in RNA as a result of the 4'-thio modification and to better understand the origins of the observed stability changes, the crystal structure of the oligonucleotide 5'-r(CC(4'S)CCGGGG) was determined and analyzed using the previously solved structure of the native RNA octamer as a reference. The two 4'-thioriboses adopt conformations that are very similar to the C3'-endo pucker observed for the corresponding sugars in the native duplex. Subtle changes in the local geometry of the modified duplex are mostly due to the larger radius of sulfur compared to oxygen or appear to be lattice-induced. The significantly increased RNA affinity of 4'-thio-modified RNA relative to RNA, and the relatively minor conformational changes caused by the modification render this nucleic acid analog an interesting candidate for in vitro and in vivo applications, including use in RNA interference (RNAi), antisense, ribozyme, decoy and aptamer technologies.     

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.     

Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera

Antisense DNA oligodeoxynucleotides can selectively inhibit the expression of individual (undesirable) genes and thus, have potential in the treatment of cancer and viral diseases. A prerequisite to their use as therapeutic agents is information on the stability of oligodeoxynucleotides, and their structurally modified analogs, in the biological milieu. To this end, degradation of 5' end and internally [32P] labelled unmodified DNA oligodeoxynucleotide (D-oligo) and analogs containing phosphorothioate (S-oligo), methylphosphonate (MP-oligo), and novel alternating methylphosphonate and phosphodiester (Alt-MP-oligo) internucleoside linkages was studied in Hela cell nuclear extract, S100 cytoplasmic extract, normal human serum and calf serum at 37 degrees C. Both 5' end and internally labelled D-oligos showed complete degradation within 30 min incubation in human serum at 37 degrees C. In any given medium, the D-oligo was the least stable oligodeoxynucleotide to nuclease degradation whereas the Alt-MP, MP and S-oligos were generally of comparable stability and all relatively more stable than D-oligo. Interestingly, MP and Alt-MP-oligos also exhibited greater resistance to phosphatases in cellular extracts compared to D and S-oligos. Under the conditions of the experiments, increasing degradation for any given oligonucleotide was observed in the order: S100 cytoplasmic extract less than nuclear extract less than normal human serum less than calf serum. In a study involving alpha-MEM cell culture medium containing 10% heat inactivated fetal calf serum (heated to 56 degrees C for 1 hour), the D-oligo was found to be rapidly degraded (degradation evident within 10 mins) whereas degradation products for the S-oligo were observed within 1 hour. In contrast, the Alt-MP oligo remained stable throughout the 3 hour experiment. These results indicated that in cell culture medium containing heat inactivated serum Alt-MP oligo was more stable than D- and S-oligos.     

Thermodynamic, counterion, and hydration effects for the incorporation of locked nucleic acid nucleotides into DNA duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.     

Oligonucleotide--minor groove binder 1:2 conjugates: side by side parallel minor groove binder motif in stabilization of DNA duplex

Synthetic polycarboxamides consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and beta-alanine (beta) show strong and sequence-specific interaction with the DNA minor groove when they form hairpin structures with side-by-side antiparallel motifs. In the present paper, new conjugates containing two ligands linked to the same terminal phosphate of DNA strand were constructed. The paper describes optimized synthesis and properties of oligonucleotide-linked polyamide strands that insert into the minor groove of a duplex in a parallel or antiparallel orientation. Strong stabilization of DNA duplexes by two attached minor groove ligands is demonstrated by the thermal denaturation method. The unmodified duplex 5'-CGTTTATTp-3'/5'-AATAAACG-3' melts at 20 degrees C. When one tetra(Py) residue was attached to the first strand of this duplex, denaturation temperature was increased to 46 degrees C; attachment of the second tetra(Py) in a parallel orientation resulted in denaturation temperature of 60 degrees C. It is even higher than in case of "classic" octapyrrole hairpin ligand (Tm = 58 degrees C). Sequence-specific character of stabilization by two conjugated ligands was demonstrated for G:C-containing oligonucleotides attached to tetracarboxamide and octacarboxamide ligands constructed from Py, Im and beta units according to established recognition rules (deltaTm = 20 degrees C). The two-strand parallel minor groove binder constructions attached to addressing oligonucleotides could be considered as site-specific ligands recognizing single- and double-stranded DNA similarly to already described hairpin MGB structures with antiparallel orientation of carboxamide units.     

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.     

Properties of isonucleotide-incorporated oligodeoxynucleotides and inhibition of the expression of spike protein of SARS-CoV

Antisense oligonucleotides are recognized to be very efficient tools for the inhibition of gene expression in a sequence specific way. For the discovery of a novel efficient way to modify oligonucleotides, a series of single isonucleotide-incorporated antisense oligodeoxynucleotides have been synthesized, in which an isonucleotide was introduced at different positions of the sequences. The binding behaviors of modified oligodeoxynucleotides to the complementary sequence were studied by UV, CD, and molecular dynamics simulation. The results showed that although the incorporated isonucleotides at certain positions of the sequence interfere with the binding ability to a different extent, B-form duplexes were maintained and the binding abilities of the 3'-end-modified duplexes were better than the corresponding mismatched duplexes. The digestion of modified oligodeoxynucleotides by snake venom phosphodiesterase showed that an isonucleotide strongly antagonizes hydrolysis. The DNA/RNA hybrid formed by a modified oligodeoxynucleotide and its target RNA could activate RNase H. The 3'-end-modified antisense oligodeoxynucelotides inhibited S-glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. This study indicated the possibility of designing a novel and effective antisense oligodeoxynucleotide by incorporating an isonucleotide at the 3'-end of the sequence.     

Branch migration mediated DNA labeling and cloning

The sequence-dependent attachment (capture) of an oligodeoxynucleotide duplex containing a single-stranded tail can be mediated by branch migration into the end of a DNA molecule. Substitution of bromodeoxycytidine (BrdC) for deoxycytidine (dC) increased DNA-DNA hybrid stability. BrdC-containing oligodeoxynucleotides displaced dC-containing strands from duplexes with blunt ends or 3'-overhangs. In the later case the rate of displacement was of the same order of magnitude as DNA reassociation. A BrdC-containing displacer oligodeoxynucleotide was used for transient sequence-specific invasion at a particular PstI site. The product was captured by use of T4 DNA ligase and a linker oligodeoxynucleotide. The capture rate was more than 300 times the rate observed for an unrelated PstI site. This high degree of specificity required BrdC substitution. In addition, deliberate incorporation of an incorrect nucleotide into a displacer strand demonstrated that branch migration was terminated at a mismatch. A branched, BrdC-containing ligated product of a capture reaction was cloned and sequenced. The specific capture reaction may be used to label a particular DNA fragment prior to electrophoresis, to mark the specific fragment for affinity chromatography, or to facilitate cloning by introducing a new overhanging sequence compatible with a restriction endonuclease site in a cloning vector.