DNA-RNA heteroduplex

Despite these clear advantages, the efficiency of splinted RNA ligation reactions catalyzed by T4 DNA ligase is typically limited by the amount of enzyme one can introduce into the reaction. This problem is derived from the poor turnover of T4 DNA ligase on DNA/RNA heteroduplexes. More recently, a variant of T4 RNA ligase, called T4 RNA ligase 2 (Nandakumar et al, 2004), has been identified. This enzyme has a pronounced preference... 

Morgan MA, Hecht SM. Iron(II) bleomycin-mediated degradation of DNA—RNA heteroduplex. Biochemistry. 1994 33 10286. 

Ribonuclease H (RNase H), also termed hybridase in some earlier literature, is an endoribonuclease that specifically degrades the RNA strand of DNA RNA heteroduplexes (Scheme 3.3). The oligoribonucleotide products have 5 -P and 3 -OH termini. 

Exonuclease III (Exo III) of E. coli is a monomeric multifunctional enzyme (31 kDa) that catalyzes the hydrolysis of at least four different types of phosphoester bonds in dsDNA (Fig. 3.4). The main enzymatic activity of Exo III is the 3 — 5 -exonuclease activity that carries out the successive release of 5 -P-mononucleotides from the 3 ends of dsDNA. The second activity is the DNA 3 -phosphatase activity that hydrolyzes 3 -terminal phosphomonoesters. In fact, Exo III was initially discovered as a DNA 3 -phosphatase in E. coli (1,2). Exo III has a third activity which degrades the RNA strand in a DNA RNA heteroduplex, thus the RNase H activity. The fourth activity of Exo III is an AP endonuclease which cleaves phosphodiester bonds at apurinic or apyrimidinic sites. 

Exo III degrades not only the DNA strand(s) in duplex DNA and DNA-RNA heteroduplexes, but it also degrades the RNA strand in heteroduplexes. Poly(rA dT) is degraded at 20% of the rate for poly(dA dT) (24). The enzyme has no measurable activity on either homopolymer strand in the absence of the other. The products of RNase H activity are a mixture of 5 -mono- and 5 -oligonucleotides, although 5 -rAMP is the major product (25). This suggests... 

The reduced rate of cleavage by Hindlll on the DNA RNA heteroduplex compared with that on the DNA homoduplex has been utilized in the Okayama-Berg vector—primer cloning method (21). 

All replication-competent retroviruses possess a characteristic enzyme, reverse transcriptase (RT), which is present at 20-70 mol/virus particle (1-3). The enzyme is cleaved, and thereby activated, from an inactive precursor by the action of another retroviral enzyme, the viral protease. All RTs possess three distinct enzymatic activities (1) an RNA-dependent DNA polymerase, which is the RT in the strict sense of the word, (2) an RNase H, and (3) a DNA-dependent DNA polymerase. After infection of a new host cell, these different activities serve in turn to synthesize a cDNA of the viral RNA, to degrade RNA from the cDNA-RNA heteroduplex, and to duplicate the cDNA strand (reviewed in ref. 4). 

By reversing the polarity of the DNA strands, the opposite strand will be transcribed and a complementary or antisense RNA will be produced. When antisense RNA was introduced either in vitro (27) or in vivo (28), the presence of the antisense RNA inhibited the expression of the normal RNA transcript produced by the resident gene. Suppression of RNA translation by antisense RNA may be due to the formation of an RNA heteroduplex between the two complementary RNA strands thus blocking the attachment of ribosomes. An example of this application in the context of the Adh example would be the construction of a chimeric gene containing the antisense sequence for Adh linked with a female-specific promoter. Thus, Adh activity would be eliminated from females and make them lethally sensitive to treatment with ethanol. 

H-1 abstraction by Fe-BLM from ODN duplexes containing a ribonucleotide unit was evidenced (50), but degradation of DNA-RNA hybrids by BLM involves C-4 rather than C-1 chemistry (51) the DNA strand of this heteroduplex is preferentially cleaved (52). 

This section examines the synthesis of nucleosides that contain seven-membered sugar analogues in place of the deoxyribose component. Nucleosides from the last group have been further incorporated into ONs via solid-phase DNA synthesis. A physical and biochemical investigation of the oligomers thus prepared continues in the next section. The study under review culminated in the assessment of the ability of the oligomers to complex with single-stranded RNA and for the heteroduplexes so formed to serve as substrates of RNAseH. 

RNA viruses that cause tumors (oncogenic RNA viruses) are called retroviruses because their life cycle involves a DNA intermediate. The ability of retroviruses to use such a route for replication hinges on a viral-encoded enzyme called reverse transcriptase, an enzyme with three discrete activities (1) It catalyzes the synthesis of DNA from the viral plus-strand (2) it catalyzes the synthesis of DNA plus-strand from the viral minus-strand DNA and (3) it catalyzes the degradation of the viral RNA from an RNA-DNA heteroduplex. 

Hybridization can occur between complementary strands of nucleic acids derived from different sources. The double stranded nucleic acid that forms is a heteroduplex and the extent of heteroduplexes indicates homology between the two nucleic acid sources. For example, humans and mice mix with a very small fraction of the DNA renaturing but humans and chimpanzees give greater than 98% homology. DNA and RNA can also hybridize with one another to form heteroduplexes. 

The RNA helicase activity of the full-length NS3/4A enzyme (8) and of the NS3 C-terminal helicase domain has been demonstrated (10,11,17-19) by the unwinding of double-stranded RNA (dsRNA) molecules. The directionality of RNA helicase unwinding is 3 to 5 with respect to the template strand (8,19). Unlike most other helicases, the HCV NS3 helicase is capable of unwinding both RNA and DNA homoduplex and heteroduplex molecules (19). In common with other helicases, NS3 helicase requires divalent cations (Mg2+ or Mn2+) in conjunction with the hydrolysis of nucleoside triphosphates (NTPs) to provide the energy source for unwinding (11,12). 

Fig. 5. The helicase high-throughput assay can detect both DNA and RNA unwinding activity and gives similar results to the traditional gel-based helicase assay. (A-E) Gel-based assay showing unwinding of RNA and DNA heteroduplex and homoduplex substrates by action of 5-120 fmol of HCV NS3/4A enzyme. The positions of the dsNA and ssNA are labeled.

Batra et al. Q991) subtracted ( + ) cDNA against poly(A)+RNA, removed the heteroduplexes by the polyCA)" " tails and used the subtracted jj DNA as template for cloning. They observed an enrichment of > 300-fold in their system. 

To test and optimise the coupled reaction, RNA hydrolysis was simulated by the addition of AMP as a preformed product to a complete nucleic acid amplifrcation cocktail. Additions of AMP gave linear bioluminescent responses over a range of concentrations from 0.1 to 10 /tmol/L AMP (Fig. 1). The ability to detect RNA-DNA heteroduplex hydrolysis with this assay was tested using a proprietary exonuclease degradation system. 5 -AMP released from the poly rA/oligo dT probertemplate heteroduplex was measured using the triple enzyme coupled... 

---