[Unsolved Puzzles of Qß Replicase].

Qß phage replicase has been the first RNA-directed RNA polymerase purified to homogeneity and intensively studied in vitro. In the mid-sixties, papers on Qß and related replicases appeared in nearly every issue of the PNAS journal. By 1968, the mechanism of its action seemed to be almost completely understood. However, even now, a half of century later, a number of fundamental questions remains unanswered. How does the replicase manage to prevent the template and its complementary copy from annealing during the entire replication round? How does it recognize its templates? What is the function of the translation factors present in the replicase molecule? What is the mechanism the replicase uses to join (recombine) separate RNA molecules? Even the determination of the crystal structure of Qß replicase did not help much. Certainly, there remains a lot to discover in the replication of Qß phage, one of the smallest viruses known.

Thirty Years of Studies of Qß Replicase: What Have We Learned and What Is Yet to Be Learned?

Qß replicase (RNA-directed RNA polymerase of bacteriophage Qß) has an unsurpassed capacity to amplify polynucleotides in vitro. In 1986, the Group of Viral RNA Biochemistry was organized at the Institute of Protein Research in order to exploit this property for the synthesis of messenger RNAs to be used in cell-free translation systems. Although the task has not been implemented in full, this work has led to a number of unexpected important results including uncovering the nature of the "template-free" RNA synthesis by Qß replicase, discovering the ability of RNA molecules for spontaneous recombination, revealing the unusual mechanism Qß replicase uses to discriminate between its proper and improper templates, and discovering a new function of the largest ribosomal protein S1, that is also one of the replicase subunits. Finally, our work resulted in the invention of the molecular colonies technique that has become the basis for the next generation sequencing methods and provided a new insight into the origin of life. However, Qß replicase has not yet revealed all its secrets, and its studies promise further interesting findings.

Methods for Screening Live Cells.

Cell screening or, in other words, identification of cells with certain properties is now increasingly used in scientific and medical research, e.g., in diagnostics, drug testing, and production of cell clones with desired characteristics. In this review, we discuss existing methods of cell screening and their classification according to the cell presentation format. We describe the principles of the one-dimensional and two-dimensional formats and compare the main advantages and drawbacks of these formats. The first part describes the methods based on the 2D-format of cell presentation, when cells are immobilized in the same plane by various techniques. The second part describes the methods of the 1D-screening, when cells are aligned in a line in a stream of fluid and scanned one-by-one while passing through a detector. The final part of the review describes the method of high-performance cell analysis based on the merged gel technique. This technique combines the advantages of both 1D and 2D formats and, according to the authors, might become an effective alternative to many modern methods of cell screening.

The Contribution of Ribosomal Protein S1 to the Structure and Function of Qß Replicase.

The high resolution crystal structure of bacterial ribosome was determined more than 10 years ago; however, it contains no information on the structure of the largest ribosomal protein, S1. This unusual protein comprises six flexibly linked domains; therefore, it lacks a fixed structure and this prevents the formation of crystals. Besides being a component of the ribosome, protein S1 also serves as one of the four subunits of Qß replicase, the RNA-directed RNA polymerase of bacteriophage Qß. In each case, the role of this RNA-binding protein has been thought to consist in holding the template close to the active site of the enzyme. In recent years, a breakthrough was made in studies of protein S1 within Qß replicase. This includes the discovery of its paradoxical ability to displace RNA from the replicase complex and determining the crystal structure of its fragment capable of performing this function. The new findings call for a re-examination of the contribution of protein S1 to the structure and function of the ribosome.

[Paradoxes of replication of RNA of a bacterial virus].

The extraordinary ability of the bacteriophage Qbeta replicase to amplify RNA outside the cell attracted attention of molecular biologists in the late 60's-early 70's. However, at that time, a number of puzzling properties of the enzyme did not received a rational explanation. Only recently, Qbeta-replicase began to uncover its secrets, promising to give a key not only to understanding the mechanism of replication of the genome of the bacterial virus, but also to the solution of more general fundamental and applied problems.

Isolation and crystallization of a chimeric Qß replicase containing Thermus thermophilus EF-Ts.

Qß replicase is a protein complex responsible for the replication of the genomic RNA of bacteriophage Qß. In addition to the phage-encoded catalytic ß subunit, it recruits three proteins from the host Escherichia coli cell: elongation factors EF-Tu and EF-Ts and ribosomal protein S1. We prepared a chimeric Qß replicase in which the E. coli EF-Ts is replaced with EF-Ts from Thermus thermophilus. The chimeric protein is produced in E. coli cells during coexpression of the genes encoding the ß subunit and thermophilic EF-Ts. The developed isolation procedure yields a substantially homogeneous preparation of the chimeric replicase. Unlike the wild-type enzyme, the S1-less chimeric replicase could be crystallized. This result facilitates studies on the structure of Qß replicase and the mechanism of recognition of its templates that can replicate in vitro at a record rate.

Nanocolonies and diagnostics of oncological diseases associated with chromosomal translocations.

This paper reviews chromosomal abnormalities observed in oncological diseases, the history of discovery of chromosomal translocations (a widespread type of chromosomal abnormalities), and statistical data showing a correlation between translocations and emergence of oncological diseases (in particular leukemia). The importance of detection of minimal residual disease (MRD) in treatment of leukemia associated with translocations is discussed along with methods of MRD diagnosis, followed by description of a novel diagnostic procedure for the detection of single chimeric mRNA molecules serving as MRD markers. This procedure includes a number of improvements, of which the most important is the use of a PCR version of the method of nanocolonies (other names are molecular colonies, polonies) that provides for the determination of the absolute titer of RNA tumor markers, excludes false positive results in the detection of chimeric molecules, and significantly exceeds other methods in the sensitivity of MRD detection.

[Use of nanocolonies for detection of minimal residual disease in patients with leukemia t(8;21)].

A complete diagnostic procedure was developed that allows single molecules of mRNA AML1-ETO to be detected in samples of whole blood and bone marrow of the leukemia t(8;21)(q22;q22) patients. The procedure includes: a method for preservation of biological samples ensuring the RNA integrity; an improved method for isolation of RNA from the unfractionated whole blood and bone marrow; an optimized reverse transcription; and the use of nanocolonies for detection and enumeration of RNA target molecules. The developed procedure is the first one that provides for determination of the absolute titer of an RNA target without reference to a control (housekeeping) gene, and significantly increases sensitivity, precision and reliability of detection of the minimal residual disease at a leukemia associated with known chromosomal translocation.

Nanocolonies: detection, cloning, and analysis of individual molecules.

Nanocolonies (other names molecular colonies or polonies) are formed upon template nanomolecule (DNA or RNA) amplification in immobilized medium with efficient pore size in the nanometer range. This work deals with the principle, invention, development, and diverse nanocolony applications based on their unique abilities to compartmentalize amplification and expression of individual DNA and RNA molecules, including studying reactions between single molecules, digital molecular diagnostics, in vitro gene cloning and expression, as well as identification of the molecular cis-elements including DNA sequencing, analysis of single-nucleotide polymorphism, and alternative splicing investigation.

[Express hybridization of molecular colonies with fluorescent probes].

DNA colonies formed during PCR in a polyacrylamide gel and RNA colonies grown in an agarose gel containing Qbeta replicase can be identified using the procedure of transfer of molecular colonies onto a nylon membrane followed by membrane hybridization with fluorescent oligonucleotide probes. The suggested improvements significantly simplify and accelerate the procedure. By the example of a chimeric AML1-ETO sequence, a marker of frequently occurring leukemia, the express hybridization method was shown to allow the rapid identification of single molecules and the determination of titers of DNA and RNA targets. Hybridization with a mixture of two oligonucleotide probes labeled with different fluorophores complementary to components of the chimeric molecule ensures the identification of molecular colonies containing both parts of the chimeric sequence and improves the specificity of diagnostics.

[Scientific and practical applications of molecular colonies].

Reviewed are the history of invention of the molecular colony technique, also known under name "polony technology", applications of this method to studies of reactions between single RNA molecules, ultrasensitive diagnostics, gene cloning and screening in vitro, and also concepts on the origin of life that consider molecular colonies as a prototype of living organisms.

[Retention of nucleic acid integrity in guanidine thiocyanate lysates of whole blood].

High-molecular-mass RNA and DNA have been shown to retain their integrity for three days at room temperature, no less than two weeks at +4 degrees C, and more than a year at -20 degrees C when whole blood samples are stored as lysates containing 4 M guanidine thiocyanate. Storage time at room temperature can be prolonged at least up to 14 days if nucleic acids were precipitated by two volumes of isopropanol. This preservation technique allows storage and transportation of samples at ambient temperature and is completely compatible with the procedure of subsequent isolation of nucleic acids.

[Accurate diagnostic methods with the use of molecular colonies]. / Tochnaia diagnostika s pomoshch'iu molekuliarnykh kolonii.

The accuracy and reliability of diagnostic tests for infections and cancer can be substantially improved by using a gel, rather than a liquid medium, to amplify nucleic acids and thereby to obtain molecular colonies.

Cell-free synthesis and affinity isolation of proteins on a nanomole scale.

The performance of conventional cell-free gene expression systems based on the Escherichia coli S30 extract can be significantly improved by using expression vectors that encode viral structural elements known to enhance translation in vivo and to protect mRNA from ribonuclease action. The expression vectors reported here are designed to produce a functionally active protein carrying the Strep-tag oligopeptide at its C-terminus. They can be used in translation, transcription-translation or replication-translation reactions. Depending on its type, the reaction yields up to 40 micrograms per mL, or about 1 nmol of a standard protein. The presence of Strep-tag allows the synthesized protein to be easily isolated on a streptavidin-agarose column under mild conditions and the entire procedure to be completed within one working day. The results show that standard low-cost, cell-free systems can serve for rapid preparation of purified proteins in amounts that can satisfy a number of needs of a research laboratory.

The puzzle of RNA recombination.

For more than three decades, RNA recombination remained a puzzle and has only begun to be solved in the last few years. The available data provide evidence for a variety of RNA recombination mechanisms. Non-homologous recombination seems to be the most common for RNA. Recent experiments in both the in vitro and the in vivo systems indicate that this type of recombination may result from various transesterification reactions which are either performed by RNA molecules themselves or are promoted by some proteins. The high frequency of homologous recombination manifested by some RNA viruses can be easier explained by a replicative template switch.

Nonreplicative RNA recombination in poliovirus.

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.

Spontaneous rearrangements in RNA sequences.

The ability of RNAs to spontaneously rearrange their sequences under physiological conditions is demonstrated using the molecular colony technique, which allows single RNA molecules to be detected provided that they are amplifiable by the replicase of bacteriophage Qbeta. The rearrangements are Mg2+-dependent, sequence-non-specific, and occur both in trans and in cis at a rate of 10(-9) h(-1) per site. The results suggest that the mechanism of spontaneous RNA rearrangements differs from the transesterification reactions earlier observed in the presence of Qbeta replicase, and have a number of biologically important implications.

Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure.

Extensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.