DNA nanotechnology for nucleic acid analysis: multifunctional molecular DNA machine for RNA detection.

The Nobel prize in chemistry in 2016 was awarded for 'the design and synthesis of molecular machines'. Here we designed and assembled a molecular machine for the detection of specific RNA molecules. An association of several DNA strands, named multifunctional DNA machine for RNA analysis (MDMR1), was designed to (i) unwind RNA with the help of RNA-binding arms, (ii) selectively recognize a targeted RNA fragment, (iii) attract a signal-producing substrate and (iv) amplify the fluorescent signal by catalysis. MDMR1 enabled detection of 16S rRNA at concentrations ∼24 times lower than that by a traditional deoxyribozyme probe.

[Comparative study of the efficacy of modifying DNA polymerase and DNA matrix by different photoactive groups at the 3'-end of the DNA primer]. / Sravnitel'noe izuchenie éffektivnosti modifikatsii DNK-polimeraz i DNK-matritsy razlichnymi fotoaktivnymi gruppami na 3'-kontse DNK-praimera.

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoactivatable group and the length of the linker that joins the group with the heterocyclic base of the primer 3'-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3'-termini were obtained using the rat DNA polymerase beta or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent binding of the [5'-32P]primer to DNA polymerases and template was determined. The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.

Highly efficient labeling of DNA polymerases by a binary system of photoaffinity reagents.

A binary system of photoaffinity reagents was proposed earlier for highly efficient labeling of DNA polymerases by 5;-[32P]DNA primers. In the present study we demonstrate the feasibility of this approach to increase the efficiency of DNA polymerase labeling. A photoactive 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was incorporated at the 3;-end of 5;-[32P]DNA primers synthesized by DNA polymerase beta or Tte in the presence of one of the dTTP analogs--FAB-4-dUTP, FAB-9-dUTP, or FAB-4-ddUTP. The reaction mixture was irradiated by light with wavelength of 334-365 nm (direct labeling) or 365-450 nm in the presence of photosensitizer, one of dTTP analogs containing a pyrene moiety, Pyr-6-dUTP or Pyr-8-dUTP. In the case of the binary system of photoaffinity reagents, a FAB group is activated by energy transfer from sensitizer localized in the dNTP-binding site of DNA polymerase in the triple complex, comprised by reagent, DNA polymerase, and Pyr-6(8)-dUTP. Direct activation of the FAB group under these conditions is negligible. The most efficient photolabeling of DNA polymerases was observed with a primer containing a FAB-4-dUMP group at the 3;-end, and Pyr-6-dUTP as a photosensitizer. Using 10-fold molar excess of photoreagent to DNA polymerase beta, the labeling efficiency was shown to achieve 60%, which is 2-fold higher than the efficiency of the direct DNA polymerase labeling under harsher conditions (334-365 nm).

Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP.

The interaction of dNTPs with the active site of HIV-1 reverse transcriptase (HIV RT) has been investigated. The kinetic parameters of primer elongation catalyzed by wild-type HIV-1 RT and two of its mutants with substitutions for Tyr115 using dTTP and two of its photoreactive analogs were determined. The substitution for Tyr115 with alanine or tryptophan resulted in an increase in K(m) values of dTTP and its analogs. Wild-type RT and its mutants were photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photocross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensitizer. The use of the sensitizer decreased the number of modification products and increased selective labeling of the catalytic subunit of both the mutant and wild-type RT.

[Interaction of human replication protein A with DNA-duplexes, containing gaps of varying sizes]. / Issledovanie vzaimodeistviia replikativnogo belka A cheloveka s DNK-dupleksami, soderzhashchimi breshi razlichnogo razmera.

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3' end of a gap-flanking oligonucleotide with DNA polymerase beta, and an oligonucleotide containing a 5'-photoreactive group was chemically synthesized. The 5' end predominantly interacted with the large RPA subunit (p70) regardless of the gap size, whereas interactions of the 3' end with the RPA subunits depended both on the gap size and on the RPA concentration. Subunit p32 was mostly labeled in the case of a larger gap and a lower RPA concentration. The results confirmed the model of polar RPA-DNA interaction, which has been advanced earlier.

A binary system of photoreagents for high-efficiency labeling of DNA polymerases.

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs.

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5'-flap strands of the branched DNA structure and possesses 5'-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3'-end of the primer were synthesized in situ by the action of DNA polymerase beta using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5'-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5'- and 3'-ends of the nick or with primer-template systems containing 5'- or 3'-protruding DNA strands was shown. Formation of a structure with the 5'-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3'-end and the apurine/apyrimidine site at the 5'-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.

Localization of the large subunit of replication factor C near the 5' end of DNA primers.

Replication factor C (RFC) is a heteropentameric sliding clamp loader protein essential for processive synthesis of DNA by eukaryotic DNA polymerases delta and epsilon. To study the interaction of RFC with 3' and 5' ends of the DNA primer, we have developed chemical photocrosslinking assay using a synthetic DNA gap and DNA primer-template structures. We have found that the radioactively labeled primers containing a photoreactive group at their 5' end could crosslink with the largest RFC subunit (RFC140) on primer-templates and DNA gap structures, but that 3' end photoreactive primers could only crosslink with RFC140 within the DNA gap structure. Addition of replication protein A (RPA) to the reaction mixture resulted in the crosslinking of RPA subunits and inhibited crosslinking of RFC140 using 3' but not 5' photoreactive primers present at the gap. The results suggest specific contacts between RFC140 and the 5' end of the DNA primer. Together with previous data, these experiments allow us to propose a model for the DNA polymerase switch during eukaryotic DNA replication.

[Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins]. / Poluchenie fotoreaktsionnosposobnykh oligonukleotidnykh dupleksov i ikh primenenie dlia fotoaffinnoi modifikatsii DNK-sviazyvaiushchikh belkov.

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.

Polarity of human replication protein A binding to DNA.

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5'-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5'-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3'-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.

Synthesis of base-substituted dUTP analogues carrying a photoreactive group and their application to study human replication protein A.

Analogues of dUTP bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group linked via spacers of varying length (n = 2, 4, 7-13 atoms) to the 5-position of the uridine ring (NAB-n-dUTP) were synthesized and characterized. DNA polymerase beta efficiently incorporated these analogues into synthetic primer-template substrates in place of TTP, which allowed us to selectively introduce a photoreactive group at the 3' primer terminus. After completing photoreactive primer synthesis, the reaction mixtures were irradiated with monochromatic UV light (315 nm) in the presence of human replication protein A (RPA), a heterotrimer consisting of three subunits with molecular mass 70 kDa (p70), 32 kDa (p32), and 14 kDa (p14), and were separated by SDS-PAGE. The photoreactive primers cross-linked directly with p70 and p32, but cross-linking of p14 was not achieved even by varying the length of the spacer group. The data speak in favor of the protection of p14 by other RPA subunits from the interaction with 3'-end of the primer. Cross-linking of substrates to pol beta is inhibited when the analogue bears a short spacer (n = 2, 4, 7, and 8), but this is abrogated somewhat when longer spacers (n = 9-13) are examined. On the basis of these observations, we suggest that RPA and pol beta form a complex on primer-template substrates.

[A photoreactive analogue of 2'3'-dideoxyuridine 5'-triphosphate: preparation and use for photoaffinity modification of human replication protein A]. / Fotoreaktsionnosposobnyi analog 2',3'-didezoksiuridin-5'-trifosfata: poluchenie i ispol'zovanie dlia fotoaffinnoi modifikatsii faktora replikatsii A cheloveka.

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2',3'-dideoxyuridine 5'-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2'-deoxyuridine 5'-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase beta and to terminate DNA synthesis. A 5'-32P-labeled primer with a photoreactive group at the 3'-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.

Study of interaction of human replication factor A with DNA using new photoreactive analogs of dTTP.

Replication factor A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells; it participates in replication, repair, and recombination of DNA. RPA is composed of three subunits with molecular masses 70 (p70), 32 (p32), and 14 kD (p14). The photoaffinity labeling method was used to study the interaction of RPA with the 3;-end of duplex DNA containing extended 5;-end of a single strand. We have synthesized dTTP analogs containing photoreactive 2,3,5,6-tetrafluoro-4-azidobenzoyl group attached to the 5th position of the uracil residue with linkers of variable length (9, 11, and 13 atom chains). Using these analogs and dTTP analog containing the same photoreactive residue attached to the 5th position of the uracil residue with a 4-atom linker, a number of oligonucleotide primers carrying a single photoreactive group on the 3;-end were enzymatically synthesized. Using the complex of the photoreactive primers with DNA template containing extended 19-base 5;-end, human RPA was photoaffinity modified. The primers were covalently bound to the p70 and p32 subunits of RPA and the p14 subunit was not labeled by the primers. The data are discussed considering the previously suggested model of interaction of RPA with DNA during replication.

Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents.

The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5;-[32P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.

RPA subunit arrangement near the 3'-end of the primer is modulated by the length of the template strand and cooperative protein interactions.

To analyze the interaction of human replication protein A (RPA) and its subunits with the DNA template-primer junction in the DNA replication fork, we designed several template-primer systems differing in the size of the single-stranded template tail (4, 9, 13, 14, 19 and 31 nt). Base substituted photoreactive dNTP analogs-5-[ N -(2-nitro-5-azidobenzoyl)- trans -3-amino-propenyl-1]-2'-deoxyuridine-5'-triphosphate (NAB-4-dUTP) and 5-[ N -[ N -(2-nitro-5-azidobenzoyl)glycyl]- trans -3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (NAB-7-dUTP)-were used as substrates for elongation of radiolabeled primer-template by DNA polymerases in the presence or absence of RPA. Subsequent UV crosslinking showed that the pattern of p32 and p70 RPA subunit labeling, and consequently their interaction with the template-primer junction, is strongly dependent on the template extension length at a particular RPA concentration, as well as on the ratio of RPA to template concentration. Our results suggest a model of changes in the RPA configuration modulating by the length of the template extension in the course of nascent DNA synthesis.

[New reagents for affinity modification of biopolymers. Photoaffinity modification of Tte-DNA polymerase]. / Novye reagenty dlia affinnoi modifikatsii biopolimerov. Fotoaffinnaia modifikatsiia Tte-DNK-polimerazy.

Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) and N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesized and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb). The resulting azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effectively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase). Photoaffinity modification of Tte DNA polymerase was carried out by dTTP analogues (IIa, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetrafluorobenzyol)-trans-3- aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) using two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.

Synthesis of new photocross-linking 5-C-base-substituted UTP analogs and their application in highly selective affinity labelling of the tick-borne encephalitis virus RNA replicase proteins.

A new photocross-linking 5-C-base-substituted UTP analogs, carrying 4-azidoperfluorobenzoyl and 4-azidoaniline residues were synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation (lambda > 300 nm) and subsequent [alpha-32P]NTP incorporation.

Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3'-end of nascent DNA.

Human replication protein A is a heterotrimeric protein involved in various processes of DNA metabolism. To understand the contribution of replication protein A individual subunits to DNA binding, we have expressed them separately as soluble maltose binding protein fusion proteins. Using a DNA construct that had a photoreactive group incorporated at the 3'-end of the primer strand, we show that the p70 subunit on its own is efficiently cross-linked to the primer at physiological concentrations. In contrast, crosslinking of the p32 subunit required two orders of magnitude higher protein concentrations. In no case was the p14 subunit labelled above background. p70 seems to be the predominant subunit to bind single-stranded DNA and this interaction positions the p32 subunit to the 3'-end of the primer.

5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-2'-deoxy- uridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction.

The DNA targets may be labeled and simultaneously amplified in the polymerase chain reaction (PCR) using a pair of respective primers after elongation with nucleoside-5'-triphosphates carrying photoreactive groups. The amplified DNA may be subsequently photoactivated by irradiation above 300 nm, resulting in photo-cross-linking of the strands. For this goal 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)propenyl-1]-, 5-{N-[N'-(4-azido-2,3,5, 6-tetrafluorobenzoyl)-3-aminopropionyl]aminomethyl}-, and 5-{N-[N'-(2-nitro-5-azidobenzoyl)-3-aminopropionyl]aminomethyl}-2'-de oxyuridine-5'-triphosphate (VII, VIa, and VIb) derivatives have been synthesized. It was found that VII is capable of efficiently elongating DNA primers with both Klenow fragment DNA polymerase I and Thermus aquaticus DNA polymerase. Thereto, it turned out to provide quantitative incorporation in DNA as revealed by the formation of the full-length amplificate by PCR in the presence of this photoreactive analogue without any dilution with natural dTTP. On the contrary, it was found, that incorporation of VIa and VIb do not permit further DNA replication.