A. M. Kopylov

The location of mRNA in the ribosomal 30S initiation complex; site-directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon.

Messenger RNA molecules 30–35 bases long, with sequences related to the 5′‐region of cro‐mRNA from lambda‐phage, were prepared by T7 transcription from synthetic DNA templates. Each mRNA contained five or six internal uridine residues, which were transcribed using a mixture of UTP and thio‐UTP. Initiation complexes were formed with Escherichia coli 30S ribosomes in the presence or absence of tRNA(fMet), and cross‐linking of the thio‐U residues was induced by UV irradiation at wavelengths greater than 300 nm. The cross‐linked ribosomal proteins were identified immunologically, and cross‐linked regions of the 16S RNA were isolated by excision with ribonuclease H and suitable deoxyoligonucleotides. In both cases, the particular thio‐U residue involved in the cross‐link was identified by ribonuclease T1 fingerprinting of the (radioactive) mRNA in the isolated cross‐linked complex. The principal results were that, at thio‐U positions upstream of the AUG codon, specific cross‐linking occurred to protein S7 and to the 3′‐terminus of the 16S RNA, in agreement with similar experiments using 70S ribosomes. Less specific cross‐linking was observed to proteins S1, S18 and S21 at various positions within the mRNA. Six bases downstream from the AUG codon, a tRNA‐dependent cross‐link was found to position approximately 1050 of the 16S RNA, but‐‐in contrast to similar experiments with 70S ribosomes‐‐no cross‐linking was found to the 1390–1400 region.

[Combinatorial chemistry of nucleic acids: SELEX].

A method of irrational oligonucleotide design, SELEX, is considered. Individual SELEX products, aptamers, are small molecules (40–100 nt) that have a unique three-dimensional structure, which provides for their specific and high-affinity binding to targets varying from low-molecular-weight ligands to proteins. Thus, the sophisticated biosynthesis of recognizing protein elements, antibodies, can be emulated in vitro via selection and synthesis of principally new recognizing elements based on nucleic acids.

Structural Dynamics of Thrombin-Binding DNA Aptamer d(GGTTGGTGTGGTTGG) Quadruplex DNA Studied by Large-Scale Explicit Solvent Simulations.

The thrombin-binding aptamer (15-TBA) is a 15-mer DNA oligonucleotide with sequence d(GGTTGGTGTGGTTGG). 15-TBA folds into a quadruplex DNA (G-DNA) structure with two planar G-quartets connected by three single-stranded loops. The arrangement of the 15-TBA-thrombin complex is unclear, particularly with respect to the precise 15-TBA residues that interact with the thrombin structure. Our present understanding suggests either the 15-TBA single stranded loops containing sequential thymidines (TT) or alternatively a single-stranded loop, containing a guanine flanked by 2 thymidines (TGT), physically associates with thrombin protein. In the present study, the explicit solvent molecular dynamics (MD) simulation method was utilized to further analyze the 15-TBA-thrombin three-dimensional structure. Functional annotation of the loop residues was made with long simulations in the parmbsc0 force field. In total, the elapsed time of simulations carried out in this study exceeds 12 microseconds, substantially surpassing previous G-DNA simulation reports. Our simulations suggest that the TGT-loop function is to stabilize the structure of the aptamer, while the TT-loops participate in direct binding to thrombin. The findings of the present report advance our understanding of the molecular structure of the 15-TBA-thrombin structure further enabling the construction of biosensors for aptamer bases and the development of anticoagulant agents.

Visualization of fibrinogen αC regions and their arrangement during fibrin network formation by high‐resolution AFM

Fibrinogen has been intensively studied with transmission electron microscopy and x‐ray diffraction. But until now, a complete 3D structure of the molecule has not yet been available because the two highly flexible αC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the αC regions can be visualized by high‐resolution atomic force microscopy.

DNA Aptamers as Radically New Recognition Elements for Biosensors

A fiber-optic biosensor based on DNA aptamers used as receptors was developed for the measurement of thrombin concentration. Anti-thrombin DNA aptamers were immobilized on silica microspheres, placed inside microwells on the distal tip on an imaging optical fiber, coupled to a modified epifluorescence microscope through its proximal tip. Thrombin concentration is determined by a competitive binding assay using a fluorescein-labeled competitor. The biosensor is selective and can be reused without any sensitivity change. The thrombin limit of detection is 1 nM, sample volume is 10 μl, and assay time per sample is 15 min including the regeneration step.

Modification of 18 S rRNA in the 40 S ribosomal subunit of yeast with dimethyl sulfate

A current study of a ribosome is rather restrained by a scarcity of data about rRNA topography in a ribosome [1 ]. The majority of up-to-date data concerns prokaryotic ribosomes [2-4] while almost nothing is known about eukaryotic rRNA topography. At the same time a comparison of these two types of ribosomes and their rRNAs should reveal common features which may be attributed to a ribosome as such, no matter of its origin.

Inhibition of Thrombin Activity with DNA-Aptamers

The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule.

Novel modular DNA aptamer for human thrombin with high anticoagulant activity.

Aptamers based on nucleic acids are a promising alternative to antibodies in therapy and diagnostics. Several DNA aptamers against human thrombin have been developed by selection from random libraries: a 15-mer and its derivatives, a 29-mer, and a 31-mer. Some of them are patented and already under clinical trial. The 15-mer structure was determined by X-ray and NMR and turned out to be a monomolecular antiparallel G-quadruplex. The other aptamers mentioned above have higher inhibitory activity than the initial 15-mer, but there are not yet structural data explaining this phenomenon. Here, the initial 15-mer, 31-mer, and novel RA-36 aptamers are compared to establish the structure-function correlation, providing a solid ground for further rational aptameric drug design. For the molecular dynamic simulation of aptamers, the force field parmbsc0 was ported onto GROMACS, and the main stabilizing parameters were revealed, leading to the novel DNA aptamer RA-36. The functional properties of the DNA aptamers were studied by conventional coagulation tests, which do not directly elucidate the mechanism of thrombin inhibition by aptamers. Improved turbidimetric measurements provided data to develop detailed kinetics showing that the 31-mer and RA-36, in contrast to the 15-mer, are competitive inhibitors. These data revealed RA-36 to be an efficient thrombin inhibitor with a dose-dependent effect. Animal tests of the studied DNA aptamers suggested an unexpected species-specificity of the novel RA-36.

Characteristics of a New DNA Aptamer, Direct Inhibitor of Thrombin

Characteristics of a new antithrombin DNA-aptamer RE31 were studied. This aptamer inhibited thrombin formation in human plasma catalyzed by exogenous (lengthening of thrombin time) and endogenous thrombin (lengthening of partial prothrombin time and activated partial thromboplastin time). In addition, the aptamer completely suppressed thrombin-induced aggregation of human platelets. On the other hand, RE31 did not reduce amidolytic activity of thrombin towards the short peptide substrate, in other words, did not modify the state of enzyme active center. By the capacity to inhibit clotting reactions, RE31 was superior to the previously described highly effective 31-component antithrombin aptamer 31TBA (thrombin binding aptamer, TBA). The effect of RE31 was species-specifi c: it inhibited human thrombin activity more effectively than activities of rat and rabbit thrombins.

Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.