Trehalose Plays Role in cDNA Research

The advent of thermostable enzymes has led to great advances in molecular biology, such as the development of PCR and ligase chain reaction. However, isolation of naturally thermostable enzymes has been restricted to those existing in thermophylic bacteria. Here, we show that the disaccharide trehalose enables enzymes to maintain their normal activity (thermostabilization) or even to increase activity at high temperatures (thermoactivation) at which they are normally inactive. We also demonstrate how enzyme thermoactivation can improve the reverse transcriptase reaction. In fact, thermoactivated reverse transcriptase, which displays full activity even at 60°C, was powerful enough to synthesize full length cDNA without the early termination usually induced by stable secondary structures of mRNA. The usefulness of thermostable enzymes is indisputable; they allowed the development of outstanding techniques such as PCR and ligase chain reaction (1, 2). However, the isolation of thermostable enzymes has been restricted to those existing in thermophylic organisms. To expand the availability of thermostable enzymes, we explored a completely new way—the thermal stabilization of those enzymes that are normally thermolabile by the addition of structure stabilizing molecules. In particular, we explored the properties of molecules that are normally involved in heat shock response, and we anticipated being able to confer thermal stability to enzymes. Among them, there is the disaccharide trehalose. Trehalose synthesis is induced by heat shock in the Saccharomyces cerevisiae yeast (3), suggesting its possible role in this response and in desiccation tolerance (4). In fact, yeast mutants defective in trehalose synthesis show a significant reduction in thermotolerance (5). It has been reported that enzymes could be protected from irreversible heat aggregation–heat denaturation in vitro by trehalose, suggesting its chaperonin-like function (6). Trehalose also has been used to confer stability to dried enzymes (7). In the present study, we discovered that trehalose can be used as a reaction additive to stabilize or stimulate enzymatic activity at unusually high temperatures, enabling the use of thermosensitive enzymes as though they would be thermostable. This property should be useful for converting a wide range of thermosensitive enzymes to thermostable and thermoactive ones for wide applications in biological, medical, and industrial fields. To show the power of trehalose-mediated thermal activation, we subsequently applied this new method to the synthesis of full length cDNA. The major obstacle to preparing high quality cDNA libraries has been the low efficiency of reverse transcriptase (RT) to synthesize full length cDNA, which is due to the strong secondary structure of mRNA, which cause the RT to stop the synthesis and subsequently to be released from the hybrid mRNA/incomplete cDNA. To overcome problems associated with the secondary structure of mRNA, both denaturing of sample before the reaction and increased temperature reaction would be advisable. However, attempts to overcome this problem by heat destabilization of the secondary structures of mRNA before reaction or treatment of mRNA with methylmercury hydroxide (8) were not successful, especially to obtain full length cDNA from very long transcripts (9). Although the increase of the reaction temperature might be useful for destabilizing the secondary structures of mRNA for high efficiency, full length cDNA synthesis (9), no thermostable enzymes with RT activity have been reported except for Tth DNA polymerase (10). However, this enzyme shows RT activity only in the presence of Mn , which readily degrades RNA before full length cDNA can be 2+ synthesized. In this report, we demonstrate the usefulness of trehalose to render a Moloney murine leukemia virus RT thermostable, which becomes then very highly effective to synthesize full length cDNAs. Assay of Thermal Activity of Enzymes. Enzymatic reactions were performed in the recommended buffer, with and without the addition of trehalose (or other sugars where specified) at 0.6 M unless otherwise specified. Samples were incubated at the recommended temperature (37°C, except 40°C for agarase) and as specified in the text. The activities of restriction nucleases in the presence or absence of trehalose were tested on ë DNA by using 0.5–1 units to follow the activation and 1–3 units/ìg DNA to follow the stabilization. DNase (RQ1 RNase-free DNase, Promega) was tested by using 0.025 units/ìg DNA. RNase I (Promega) was tested by digesting 10 ìg of RNA with 0.65 units of RNase I for 1 h. Changes in the activity of nucleic acid-cleaving or –degrading enzymes were followed by agarose electrophoresis and densitometric analysis of the intensity of the produced bands. The activity of agarase (New England Biolabs) was tested by measuring the residual amount of undigested agarose after incubating 200 ìl of 0.8% low melting point agarose with 0.8 units of agarase for 1 h. Polynucleotide kinase was tested by measuring the incorporation labeling by ã[ P]ATP on a 30-mer. Klenow fragment activity was tested by measuring the 32 efficiency of preparation a probe by random primer technique (11). RNA polymerase activity was measured by preparing in vitro transcripts.