Cooperative amplification of templates by cross-hybridization (CATCH).

In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self-sustained sequence replication (3SR), also known as nucleic acid sequence-based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA-dependent RNA polymerase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single-stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3' end of each template. RT elongates the hybridized templates producing a double-stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.

[1]  E. Szathmáry,et al.  The integration of the earliest genetic information. , 1989, Trends in ecology & evolution.

[2]  U. Tangen,et al.  Identification of rodent specific primers for screening hamster-human cell lines. , 1993, Gene.

[3]  F. Oehlenschläger,et al.  Comparison of self-sustained sequence-replication reaction systems. , 1996, European journal of biochemistry.

[4]  R R Breaker,et al.  Emergence of a replicating species from an in vitro RNA evolution reaction. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Richard P. Haugland,et al.  Handbook of fluorescent probes and research chemicals , 1996 .

[6]  K. Mullis,et al.  Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. , 1986, Cold Spring Harbor symposia on quantitative biology.

[7]  D. van Strijp,et al.  NASBATM isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection , 1991 .

[8]  M. Salas,et al.  Protein-priming of DNA replication. , 1991, Annual review of biochemistry.

[9]  D. Richman,et al.  Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Walter Fontana,et al.  Fast folding and comparison of RNA secondary structures , 1994 .

[11]  S. Spiegelman,et al.  An approach to the experimental analysis of precellular evolution , 1971, Quarterly Reviews of Biophysics.

[12]  D. van Strijp,et al.  Distinct changes in HIV type 1 RNA versus p24 antigen levels in serum during short-term zidovudine therapy in asymptomatic individuals with and without progression to AIDS. , 1995, AIDS research and human retroviruses.

[13]  M. Eigen,et al.  The Hypercycle: A principle of natural self-organization , 2009 .

[14]  L. Blanco,et al.  Terminal protein-primed DNA amplification. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Paulien Hogeweg,et al.  Spiral wave structure in pre-biotic evolution: hypercycles stable against parasites , 1991 .

[16]  J. Szostak,et al.  Ribozyme-catalysed amino-acid transfer reactions , 1996, Nature.

[17]  N. Walter.  Modelling viral evolution in vitro using exo- Klenow polymerase: continuous selection of strand displacement amplified DNA that binds an oligodeoxynucleotide to form a triple-helix. , 1995, Journal of molecular biology.

[18]  C Bresch,et al.  Hypercycles, parasites and packages. , 1980, Journal of theoretical biology.

[19]  J. Compton,et al.  Nucleic acid sequence-based amplification , 1991, Nature.

[20]  R. Pal,et al.  Detection of HIV-1 infection in vitro using NASBA: an isothermal RNA amplification technique. , 1995, Journal of virological methods.

[21]  C. Biebricher,et al.  The role of RNA structure in RNA replication , 1994 .

[22]  J W Szostak,et al.  Structurally complex and highly active RNA ligases derived from random RNA sequences. , 1995, Science.

[23]  T. Gingeras,et al.  Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR. , 1991, PCR methods and applications.

[24]  John Maynard Smith,et al.  Hypercycles and the origin of life , 1979, Nature.

[25]  D. Shank,et al.  Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[26]  T. Cech.  The chemistry of self-splicing RNA and RNA enzymes. , 1987, Science.

[27]  J. McCaskill,et al.  Spatially resolved evolution studies in an open reactor , 1994 .

[28]  J. McCaskill,et al.  Images of evolution: origin of spontaneous RNA replication waves. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[29]  G. F. Joyce,et al.  Cleavage of an amide bond by a ribozyme , 1995, Science.