Reverse transcriptase efficiency

RNA amplification by PCR has been facilitated by the use of a single heat-stable enzyme. Thus, DNA polymerase from Thermus thermophilus, which has enhanced reverse transcriptase (rT) activity in presence of manganese, can be used with one buffer system. The high temperature used for rT (70°C) to produce a complementary DNA copy from RNA, and the subsequent amplification of DNA at 60°C, increases efficiency by destabilizing secondary structures in the RNA template. This procedure has been used for the amplification of hepatitis C viral RNA (Yl). 

Initiation of reverse transcription in HIV-infected cells relies on a critical RNA-RNA interaction between tRNA y s, which is preferentially packaged into the viral particle, and a specific viral RNA seqnence. The 3 -terminaI 18 nucleotides of tRNA y are complementary to the primer binding site (PBS) sequence located in the 5 -Iong terminal repeat (LTR) of the viral RNA genome (Figure 10.3). The UUU anticodon of the tRNA is complementary to and binds to an adenosine rich loop located 8 nucleotides upstream (5 ) of the PBS. This RNA-RNA duplex which is formed when tRNA y s binds to the PBS fits within the active site of HIV-1 reverse transcriptase, bnt mnitiple interactions between the viral RNA and tRNA y are necessary for efficient initiation of reverse transcription. This interaction nucleates the reverse transcription complex which contains viral RNA, reverse transcriptase, tRNA y pl , nncleocapsid p7, and Vpr (Viral protein R), as well as multiple host factors." ... 

An efficient synthesis of the l-aUyl-6-(l, 2, 3 -triazolyl) analogue 170 of 1-[2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (KEPT), an anti-human immunodeficiency virus (HIV) reverse transcriptase inhibitor, was reported using an intermolecular 1,3-dipolar cycloaddition of the azide 169 with acetylenes (35) (Scheme 9.35). Azidouracil (169), when refluxed with an acetylene in equimolar proportions in toluene, gave the corresponding triazoles (170) in excellent yield. 

AZTTP on DNA polymerase of the host cell is much weaker. The effect of AZT is further increased by the accumulation of considerable amounts of AZTMP in cells. This monophosphate inhibits thymidylate kinase, with a consequent decrease in the concentration of dTTP in the cell. With less competition from dTTP there is more efficient incorporation of AZTTP into viral DNA by the reverse transcriptase. 2, 3 -Dideoxyinosine (ddl) is under trial for use against AIDS and is similarly incorporated into viral DNA by the reverse transcriptase with chain termination. 

Even with the sterically demanding coumarin derivative as nucleophile, the secondary ether could be formed in excellent regio- (92 8) and enantioselectivity (98% ee) by using the bicyclic ligand 135 (Scheme 8E.36). The alkylation product has efficiently served as a key intermediate in the synthesis of (-)-calanolides A and B, which have the most potent HIV-1-specific reverse-transcriptase inhibitory activity among the chromanol family. 

To secure a gene from a eukaryotic genome, the most efficient method among several is to obtain cDNA (complementary DNA) from mRNA via reverse transcriptase. Use of cDNA avoids dealing with the problem of introns, where a complete gene can stretch over thousands of base pairs but is divided into several small exons, alternating with introns of sometimes remarkable size [several thousand base pairs (kB) for one intron]. As, however, only a few eukaryotic genes are fully sequenced and deposited in one of the databases, one has to rely on ESTs (expressed... 

Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

A highly efficient, enantioselective industrial synthesis of the HIV reverse transcriptase inhibitor efavirenz is made available for the manufacture of this important compound. A novel, chiral Zn-alkoxide-mediated, enantioselective acetylide addition reaction is used to establish the chiral center in the target with a remarkable level of stereocontrol. The synthesis provides analytically pure efavirenz in an overall yield of 75 % in five steps from 4-chloroaniline. 

In the indirect labeling method, amine modified cDNA is first synthesised by incorporating aminoallyl-modified nucleotides in first strand cDNA by a reverse transcriptase. After hydrolysis of the RNA template, and purification of the amine-modified cDNA, chemical labeling with N-hydroxyl succinimidyl-ester derivative of the Cy dye is performed. A high excess of Cy dye NHS-ester is needed for an efficient reaction. The Cy dye-cDNA is then purified to remove Cy dye that is not incorporated into labeled cDNA. 

R-(R, S )]-p-Methyl-a-phenyl-1-pyrrolidineethanol is an important chiral mediator for the enantioselective addition of an acetylide to a prochiral ketone.2 3 This reaction has been successfully applied to the synthesis of the reverse transcriptase inhibitor efavirenz (DMP-266) (Scheme 1).3.4 Preparation of the enantiomer, (1S,2R)-N-pyrrolidinylnorephedrine, has been reported.2 The method used potassium carbonate (K2CO3) as base, but the yield of the product was only 33%. The submitters have extensively studied the formation of the pyrrolidinyl ring under various conditions as summarized in Table I. Eventually they found that the reaction was extremely efficient when it was run in toluene using sodium bicarbonate (NaHCC>3) as base (entry 8, Table I),5 which gave [R-(R, S )]-p-methyl-a-phenyl-1-pyrrolidineethanol quantitatively. Enantioselective (up to 99% ee) addition of cyclopropylacetylene to the ketoaniline 1 is achieved when the solution of [R-(R, S )]-p-methyl-a-phenyl-1-pyrrolidineethanol is used as a chiral additive.3 In addition, this method is also applicable to the preparation of a variety of alkylated norephedrines and other amino alcohols in excellent yields as Illustrated in Table II. These amino alcohols are potentially useful in asymmetric syntheses. 

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