Click here to switch to the mirror at domain Netscape

Нажмите, чтобы посмотреть русскую версию

	Molecular Colony Technique	Cell-free gene expression	RNA recombination
		Sequencing on oligonucleotide arrays	DNA colonies
		Plasmid isolation without RNases

New 5-year award from the Howard Hughes Medical Institute (click to read the HHMI press-release)

Click to see the meeting report

Alexander Chetverin, Ph.D., D.Sc. Laboratory Head Corresponding Member of the Russian Academy of Sciences alexch@vega.protres.ru Fax: +7(095) 924-0493 Phone: +7(0967) 73-2524

Helena Chetverina, Ph.D. Senior Scientist helena@vega.protres.ru Phone: +7(0967) 73-5755		Victor Ugarov, Ph.D. Scientist vugarov@vega.protres.ru Phone: +7(0967) 73-8916		Alexander Demidenko Junior Scientist dem@vega.protres.ru Phone: +7(0967) 73-5755

Timur Samatov Postgraduate tim@vega.protres.ru Phone: +7(0967) 73-5755		Damir Kopein Student kopein@vega.protres.ru Phone: +7(0967) 73-5755

Tatiana Popkova Leading Engineer t_popkova@mail.ru Phone:  +7(0967) 73-5755		Lyudmila Tolstova 1st Category Engineer Phone:  +7(0967) 73-8909		Nadezhda Androsova Senior Technician Phone: +7(0967) 73-8998		Larissa Shutova Senior Technician Phone: +7(0967) 73-8999

• Among the products of the spontaneous RNA synthesis, we found a 120 nt-long RQ RNA whose 2/3 matched a stretch of 80 nt of the Qb phage genome, whereas the other 1/3 matched the 3' half of an E. coli tRNA^Asp. It immediately followed that this RNA must had arisen in Qb phage-infected E. coli cells by an RNA-RNA recombination (inasmuch as the Qb phage genome never exists in a DNA form), and this was the first indication of the occurrence of RNA recombination in prokaryotic systems. Obviously, such an RNA could not had been generatedde novo but must had been synthesized on a template, and hence the apparently spontaneous synthesis was not really spontaneous (Munishkin et al, 1988).
• By using a high radioactivity 3'-terminal labeling, we have detected the same species in the RNA extracted from the purified wild-type Qb phage as those synthesized "spontaneously". This finding pinpointed Qb phage as the primary source of RQ RNAs which turned out to be the phage's satellites (Chetverin et al., 1991).
• In order to trace the immediate source of RQ RNAs in the apparently template-free reactions, we have invented a new format for nucleic acid amplification, the Molecular Colony Technique (MCT).

RNA colonies in an agarose gel containing Qb replicase and rNTPs (revealed by hybridization with a labeled probe; gel size 18?18 mm)

DNA colonies in a polyacrylamide gel containing PCR reaction components (revealed by hybridization with a labeled probe; gel diameter 14 mm)

Possible applications of MCT include:

• Nucleic acid-based diagnostics. MCT provides for detecting single molecules of a target (e.g., a viral genome or a cancer-related mRNA) and for direct determination of the target titer, greatly superseding the conventional PCR-based assays.
• Cell-free gene cloning. This is a true molecular cloning; there is no need in cloning vectors, in the transformation of cells (which is always very inefficient) or in purifying the cloned nucleic acids (each colony comprises a pure RNA or DNA preparation), and the clones can easily be screened (they comprise naked RNA or DNA that can be directly analyzed in situ). In particular, Qb replicase-MCT provides for direct screening of ribozymes and RNA aptamers.
• Protein selection and engineering. The invention further provides for gene expression in molecular colonies. To this end, the gel is supplemented with the components of a cell-free translation (or transcription-translation) system. The colonies are then screened in situ for properties of the synthesized proteins, such as the ability to perform an enzymatic reaction or to bind a ligand (including certain antigens, antibodies, or nucleic acids).

    During his 2-year work (from August, 1990, to July, 1992) in the Public Health Research Institute (New York city, USA) as a visiting investigator at the laboratory of Dr. F. R. Kramer, Dr. Chetverin has invented novel types of oligonucleotide arrays and methods of their use for parallel sorting, isolating, manipulating, and sequencing thousands, and even millions, of nucleic acid species.
    By that time it was realized that nucleic acids can be sequenced by hybridizing them to an array of all possible oligonucleotides of a given length; this approach was called SBH (Sequencing By Hybridization: Bains and Smith, 1988; Lysov et al., 1988). An oligonucleotide array is comprised of a number of individual oligonucleotide species tethered to the surface of a solid support in a regular pattern, each one in a different area, so that the location of each species is known. Such an array can be prepared by a parallel synthesis of all the oligonucleotides, directly on the support, employing the methods of solid-phase chemical synthesis in combination with site-directed masks (Fodor et al., 1991). For example, an array of 4⁸ = 65,536 possible octanucleotides (8-mers) can be synthesized in just 32 reaction steps. Hybridization of a nucleic acid strand to such an array will provide the list of all 8-mers constituting the strand, and the strand sequence can, in principle, be reconstructed by overlapping the 8-mers at their 7 nt-long subsequences. However, if there are non-unique 7-mers in the strand (which is common for sequences longer than 200 nt), its sequence cannot be reconstructed unambiguously.
    The new methodology invented by Dr. Chetverin overcomes this problem (Chetverin and Kramer, 1993). Moreover, it allows the entire large diploid genomes (such as the human genome) to be sequenced in a fully automated fashion and without the need for cloning or mapping. The proposed strategy includes digestion of the genomic DNA by a restriction endonuclease and sorting the generated fragments into a large number of pools by hybridizing their termini with a comprehensive oligonucleotide array. The unseparated fragments in each pool are amplified in situ and converted into nested partials (i.e., strands truncated at one end to any possible extent) that are sorted on another oligonucleotide array by their truncated termini. Hybridization of partials sharing same terminal sequence, separately for each type of termini, reveals both the identity and the order of the oligonucleotides belonging to each fragment in the pool, including allelic variants of the fragment. Oligonucleotide arrays are then used to order the sequenced fragments and to allocate their allelic variations to sister chromosomes (Chetverin and Kramer, 1994). A series of patent applications has been submitted by the Public Health Research Institute, and the first of them has been approved. Recently, this invention has been licensed to Affymetrix, Inc., the world leader in manufacturing oligonucleotide arrays.
    After his return to Russia, Dr. Chetverin initiated a project supported by the U.S. Department of Energy, which resulted in the development of efficient computer algorithms allowing not only nucleotide sequences to be unambiguously reconstructed from "idealized" data sets produced by the proposed methods (Razgulyaev et al., 1995), but also most of the inevitable experimental errors to be filtered out.

    Plasmids are widely used in bioresearch laboratories, and a number of rapid and inexpensive methods for plasmid purification have been developed that employ pancreatic RNase to destroy cellular RNA. However, because of its high stability, RNase almost inevitably contaminates the laboratory, which may be detrimental for experiments employing RNA. Since a major part of our work involves RNAs and the isolation of hundreds of plasmids, there was an urgent need for a simple method that would ensure an RNase-free environment. Such a method has been developed in this laboratory (Ugarov et al., 1999).
 

The key step of the new protocol is degradation of RNA by heating in the presence of a high Mg2+ concentration rather than by the use of RNase. Conditions of the treatment ensure preservation of the native structure of plasmid DNA and result in plasmid DNA that is indistinguishable from that prepared by the CsCl method. The procedure can also be used in other applications that require removal of RNA from DNA samples.

    Recombinations between and within RNA molecules are rare, yet biologically important events that form a basis for the evolution and diversity of RNA viruses which account for most of the known viral pathogens and for the generation of defective interfering RNAs which attenuate viral infections. Our recent finding of the ability of RNAs to self-recombine (Chetverina et al., 1999) suggests that RNA recombination may also involve cellular RNAs resulting (by means of reverse transcription and integration) in alterations in the chromosomal DNA, and might have been a mechanism for evolution in the prebiotic RNA world.
 

Nonreplicative RNA recombination in the presence of Qb replicase and rNTPs, compared to recombination of the same RNAs by AMV reverse transcriptase which served as a template switch control. Yellow lines indicate the manner in which the fragment sequences become joined in the recombinant molecules. The nucleotides of the natural RQ RNA sequence are shown in black; the nucleotides of foreign extensions are colored, of which those from homologous segments are in red.

• from RNA fragments possessing homologous segments

• from RNA fragments possessing nonhomologous segments

RNA recombination in the presence of Qb replicase does not occur
if the 3' terminus of the 5' fragment is modified

    The 3'-OH requirement has allowed us to separate the step of recombinant formation from its amplification, by incubating the fragment mixture under chosen conditions, and then oxidizing it with NaIO₄ to eliminate 3' hydroxyls and thereby to prevent further recombination by the above mechanism during colony growth. It turned out that (i) the above mechanism requires the presence of Qb replicase; (ii) in the absence of replicase, RNA fragments can self-recombine; (iii) the rate of self-recombination is several orders of magnitude lower; (iv) self-recombination does not involve 3' hydroxyls; instead, the fragments react by internal nucleotides which suggests the involvement of 2' hydroxyls; (v) the only requirement of self-recombination is the presence of Mg²⁺ ions; (vi) self-recombination is sequence-nonspecific, indicating that cryptic ribozyme structures are not involved; (vii) self-recombination can also occur in cis, resulting in a deletion of mRNA inserts from RQ-mRNAs (Chetverina et al., 1999).

• of RNA fragments possessing homologous segments

• of RNA fragments possessing nonhomologous segments

   Despite its low rate, RNA self-recombination might play an important evolutionary role both in the prebiotic RNA world and in the contemporary DNA world. At the rate of 10^–9 nt^-1h^-1 as observed in vitro, a new recombinant RNA would arise in a human cell every minute, yielding up to 10²⁰ recombinant molecules during the life span of the human body. Reverse transcription and integration of even a minute fraction of the rearranged sequences would change the genome significantly.
    The question inevitably arises: to what extent are the conclusions made from in vitro data valid for the natural systems? This issue has been recently addressed in similar experiments on recombination between nonreplicable and nontranslatable fragments of the poliovirus RNA, carried out in collaboration with Prof. V. I. Agol's laboratory at the Institute of Poliomyelitis and Viral Encephalitides, Moscow (Gmyl et al., 1999). Upon transfecting appropriate cells, the fragments did produce viable viral genomes demonstrating that nonreplicative (non-template switch) recombination can also occur in vivo. The results show that, similar to self-recombination, the 3' hydroxyls do not participate in the reaction, and suggest an involvement of the 2' hydroxyls and 2',3' cyclic phosphate intermediates. Importantly, the rate of the nonreplicative recombination in vivo is several orders of magnitude higher than that of self-recombination in vitro indicating that cell milieu may enhance RNA recombination by the 3' hydroxyl-independent mechanism.
    Currently, our efforts are aimed at deciphering the role of Qb replicase in RNA recombination. As seen from the above, Qb replicase not just accelerates recombination, but changes its mechanism. The questions under investigation are:

• Are nucleotides required?
• Is RNA synthesis required?
• Are parental RNA strands incorporated into the primary recombinant molecule?
• Is recombinant formed in a transesterification reaction or as a result of a primer-dependent RNA synthesis?
• Can other RNA polymerases (e.g., that of poliovirus) employ the same mechanism?

    The most obvious application of PCR-MCT is for nucleic acid diagnostics. The MCT format is able to overcome the conceptual problems inherent to the solution PCR (S-PCR, including RT-PCR), such as the sensitivity loss due to a competition from background amplification (caused by false priming on non-target sequences often present in the sample in a great excess over the target), preferential amplification of some templates over others, and template recombination (during the amplification of heterozygous samples and multigene families, and in multiplex PCR). In PCR-MCT, different molecules amplify at different locations that eliminates any competition and prevents recombination between them.
    Besides the sensitivity and reliability offered by PCR-MCT in detecting a target, its greatest power is in QUANTITATIVE diagnostic assays. There is an increasing demand for such assays, as one can judge from the current biomedical publications. Quantitative assay for a virus is important for observing the disease progression and the efficacy of the medicine being taken, and for optimizing the treatment. The need for a reliable quantitative assay is even more urgent in the case of cancer diagnostics. While the detection of a viral DNA or RNA is sufficient for diagnosing viral infection (provided that sample carryovers are excluded), the presence of a specific mRNA does not necessarily indicates malignancy; this is because many, if not all, cancer genes are also expressed in normal cells, albeit at a much lower level.
    There are several reasons to prefer PCR-MCT for quantitative assays.
 
 

PCR-MCT is more accurate PCR-MCT directly counts the number of colonies, whereas S-PCR relies on the signal intensity measurements which are inherently error prone. In S-PCR, the target is assayed by determining the cycle at which the signal becomes detectable; and, since there is an exponential relationship between the cycle number and the input number of target molecules, the assay error is an exponential function of the cycle determination error.

PCR-MCT is more reliable The results of S-PCR assays (which rely on the intensity measurements) are greatly affected by small variations in the reaction conditions (e.g., an uneven temperature distribution in the heating block, composition of the reaction mixture, the rate of DNA polymerase inactivation, etc.). In contrast, in PCR-MCT, such variation can only affect the size of DNA colonies, but not their number.

PCR-MCT prevents interference between DNA species While PCR-MCT directly determines the number of target molecules, S-PCR measures it indirectly, by referring to the signal produced by a known number of molecules of an internal standard (IS) added to the sample. However, because of mutual competition between the two species, even small difference in the amplification rates or in the input number of the IS and target results in a great assay error. In contrast, inasmuch as in PCR-MCT, DNA species are spatially separated which precludes any competition between them, different amplification rates will affect the size of DNA colonies, rather than their number.

    A laboratory protocol for PCR-MCT assay has been developed in the laboratory, and there should be no major difficulties in transferring the technology to the market (at least, in the form of kits that could be sold to research laboratories as the first step). We are not aware of any other competitive technology. Since PCR is a very popular technique, PCR-MCT should be readily accepted by research and diagnostic laboratories.

    One more our task is cell-free gene cloning. Now we can grow colonies of long (up to 2 Kbp) DNA fragments and transcribe them in situ. Currently our efforts are aimed to the gene translation in molecular colonies. Completion of this part of work will allow the entire gene cloning process, up to its identification according to the function of the encoded protein, to be performed in vitro. The developed protocol could be used, instead of the traditional in vivo protocols, for the cloning of any cDNAs, the locations of the sought DNA colonies being revealed by their ability to bind a specific ligand or antibody, or to perform some enzymatic reaction. In combination with the ribosome display or mRNA-protein fusion methodologies it can also be used for a direct screening of specific single-chain antibodies, catalytic antibodies and other engineered proteins or peptides as an alternative to the phage display. The advantages of the in vitro cloning format are the ease of screening, the homogeneity of the expression products, the absence of a natural (cell or phage) selection pressure, and the possibility of obtaining proteins or polypeptides that contain unnatural aminoacids.

Reported on finding in the products of the so-called spontaneous RNA synthesis of RQ120 RNA that turned out to be a recombinant RNA composed of pieces of the genomic Qb phage RNA and an E. coli tRNA^Asp. Marked the discovery of RNA-RNA recombination in prokaryotic systems.

Reported another natural recombinant, RQ135 RNA, mainly composed of pieces of E. coli 23S ribosomal RNA. This finding indicated that recombination could occur between cellular RNAs only, without the participation of a viral RNA, and that presence of viral sequences is not required for RNA replication. RQ135 RNA proved to be one of the best Qb replicase template, and since then was routinely used in this laboratory as an RNA vector.

Marked the invention of the molecular colony technique (MCT). Reported on the use of MCT in Pasteur-like experiments demonstrating that the "spontaneous" RNA synthesis is caused by airborne RQ RNAs. Also reported on the presence of RQ RNAs in the Qb phage particles, that marked the discovery of satellite RNAs in bacteriophages.

Described a greatly improved Qb replicase version of MCT. Directly demonstrated that RNA colonies comprise molecular clones.

Described the first cell-free system for a coupled replication-translation of a recombinant RQ-mRNA. Demonstrated that long foreign inserts inhibited replication of RQ RNAs, but that recombinant RNAs could be replicated if they were also involved in translation. Marked the invention of coupled replication-translation methods.

The first publication on principles of the Sequencing by Nested Strand Hybridization (SNSH) describing how unseparated pools of unknown nucleic acids can be sequenced by hybridization with oligonucleotide arrays without invoking any other information.

A review on various uses of oligonucleotide arrays, with a special attention to methods (sorting, manipulating and sequencing nucleic acids on oligonucleotide arrays, including direct sequencing of diploid genomes) invented by the authors.

Demonstrated that expression of mRNAs in cell-free E. coli translation systems is greatly enhanced as a result of their insertion into an RQ RNA vector, mainly due to protection of the recombinant RNAs against endogenous ribonucleases.

Reported computer simulations of SNSH that demonstrated the potentials of the method and its advantages over the ordinary sequencing by hybridization (SBH).

A review on the properties of Qbreplicase and RQ RNAs, with a special attention to the problem of spontaneous RNA synthesis and to the problems and possible applications of the recombinant RNA technology.

A review on the studies of RQ RNAs in the author's laboratory, with a special attention to the problem of spontaneous RNA synthesis.

Reported the first purified cell-free system for RNA recombination. Provided the first direct evidence that RNAs can recombine without a DNA intermediate and do this by a mechanism different from template switch.

A review on the studies of RNA recombination in single-stranded RNA phages, with a special attention to the in vitro studies in the author's laboratory.

A review on the studies of single-stranded RNA phages, their replicases and satellite RNAs, also discussing the impact those studies have produced on the developments in molecular biology.

Reported a method for low-cost and rapid isolation of high quality plasmids without any use of RNases. This can be a method of choice for the laboratories where ensuring an RNase-free environment is important.

Demonstrated that RNAs can recombine at physiologically significant rates, both in cis and in trans, in the absence of any protein or ribozyme. Marked the discovery of the intrinsic ability of RNA molecules to recombine.

A critical review of the data and hypothesis on RNA recombination, discussing the differences between, and possible mechanisms of, the homologous and nonhomologous recombination.

Demonstrated that viral RNAs can recombine in vivo in the absence of a viral RNA replicase, i.e., by a mechanism different from template switch.

Described the vectors and techniques allowing significant amount of pure proteins to be produced in E. coli cell-free translation systems.

---

* The title and rights to the invention belong to the Institute of Protein Research. All inquiries regarding licensing the invention should be addressed to: William J. Hone, Esq., Fish & Richardson P. C., 45 Rockefeller Plaza, Suite 2800, New York, NY 10111; phone +1 (212) 641-2270,
fax +1 (212) 258-2291, e-mail hone@fr.com.

    Wu, Y., Ryabova, L. A., Kurnasov, O. V., Morozov, I. Yu., Ugarov, V. I., Volianik, E. V., Chetverin, A. B., Zhang, D., Kramer, F. R., and Spirin, A. S. (1996). Coupled replication-translation methods and kits for protein synthesis. U.S. Patent 5,556,769.

    Chetverin, A. B., and Kramer, F. R. (2000). Method of sorting a mixture of nucleic acid strands on a binary array. U.S. Patent 6,103,463.

Russian Foundation for Basic Research

U.S. Department of Energy, Human Genome Program

International Science Foundation

Howard Hughes Medical Institutes

International Association for the promotion of co-operation with scientists from the New Independent States of the former Soviet Union (INTAS)

• Bains, W., and Smith, G. (1988). A novel method for nucleic acid sequence determination. J. Theor. Biol. 135, 303-307.
• Biebricher, C. K., Eigen, M., and Luce, R. (1986). Template-free RNA synthesis by Qb replicase. Nature 321, 89-91.
• Fodor, S. P., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A. T., and Solas, D. (1991). Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767-773.
• Haruna, I., and Spiegelman, S. (1965). Autocatalytic synthesis of a viral RNA in vitro. Science 150, 884-886.
• Lewin, R. (1983). The birth of recombinant RNA technology. Science 222, 1313-1315.
• Lysov, Yu. P., Florentiev, V. L., Khorlin, A. A., Khrapko, K. R., Shik, V. V., and Mirzabekov, A. D. (1988). Determination of the nucleotide sequence of DNA using hybridization to oligonucleotides. A new method. Doklady Akademii Nauk SSSR [In Russian] 303, 1508-1511.
• Miele, E. A., Mills, D. R., and Kramer, F. R. (1983). Autocatalytic replication of a recombinant RNA. J. Mol. Biol. 171, 281-295.
• Schmidt, T. G. M., and Skerra, A. (1994). One-step affinity purification of bacterially produced proteins by means of the "Strep-tag" and immobilized recombinant core streptavidin. J. Chromatogr. A 676, 337-345.
• Sumper, M., and Luce, R. (1975). Evidence for de novo production of self-replicating and environmentally adapted RNA structures by bacteriophage Qb replicase. Proc. Natl. Acad. Sci. U.S.A. 72, 162-166.

Last updated March 28, 2001