Replicable and recombinogenic RNAs

Abstract

This paper summarizes results of the 40-year studies on replication and recombination of RNA molecules in the cell-free amplification system of bacteriophage Q. Special attention is paid to the molecular colony technique that has provided for the discovery of the nature of "spontaneous" RNA synthesis by Q replicase and of the ability of RNA molecules to spontaneously rearrange their sequences under physiological conditions. Also discussed is the impact of these data on the concept of RNA World and on the development of new in vitro cloning and diagnostic tools.

Fig. 1

Detection of airborne RQ RNAs using two-layer agarose sandwiches prepared in 35 mm Petri dishes . The lower layer contained rNTPs, the upper layer contained Qβ replicase. The upper layer was cast without exposure (A, C) or after a one-hour exposure of the lower layer to air (B). The experiments were carried out in a room where RQ RNAs were often used (A, B) or in a remote room (C).

Fig. 2

Recombination between supplementing 5^′ and 3^′ fragments of an RQ RNA. A: Nucleotides that become joined (connected with yellow lines) in the recombinants generated in the presence of Qβ replicase (Samatov, T.R. and Chetverin, A.B., unpublished data). B: Same in the absence of any protein . Black letters indicate sequences derived from RQ135 RNA , colored letters indicate artificial extensions of the fragments, red letters indicate homologous segments. C: Time course of the generation of replicable RNAs by self-recombination from the 5^′ and 3^′ fragments at 37 °C in the presence of 10 mM MgCl₂ and buffer (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA) . RNA colonies are detected by hybridization with ³²P-labeled probes.

Fig. 3

Assay of RNA and DNA targets with the PCR version of the molecular colony technique. A: Lack of competition between targets in a multiplex assay. Colonies produced by 300 molecules of human immunodeficiency virus type-1 (HIV-1) RNA in the presence of the indicated number of concurrently amplifying molecules of human hepatitis B virus (HBV) DNA. Each of the nine gels was blotted with a nylon membrane that was first hybridized with an HIV-1-specific ³²P-labeled probe (top row) and then with an HBV-specific probe (bottom row). B: Detection of HBV DNA molecules in human blood. Total nucleic acids were isolated from 60-μl aliquots of the whole human blood to which diluted samples, expected to contain the indicated number of HBV DNA molecules, had been added. The blood aliquots contained nucleic acids equivalent by weight to 10¹³ molecules of the target. (Reprinted from by permission of BioTechniques/Eaton Publishing.)