Prediction of rho-independent transcriptional terminators in Escherichia coli.

A new algorithm called RNAMotif containing RNA structure and sequence constraints and a thermodynamic scoring system was used to search for intrinsic rho-independent terminators in the Escherichia coli K-12 genome. We identified all 135 reported terminators and 940 putative terminator sequences beginning no more than 60 nt away from the 3'-end of the annotated transcription units (TU). Putative and reported terminators with the scores above our chosen threshold were found for 37 of the 53 non-coding RNA TU and for almost 50% of the 2592 annotated protein-encoding TU, which correlates well with the number of TU expected to contain rho-independent terminators. We also identified 439 terminators that could function in a bi-directional fashion, servicing one gene on the positive strand and a different gene on the negative strand. Approximately 700 additional termination signals in non-coding regions (NCR) far away from the nearest annotated gene were predicted. This number correlates well with the excess number of predicted 'orphan' promoters in the NCR, and these promoters and terminators may be associated with as yet unidentified TU. The significant number of high scoring hits that occurred within the reading frame of annotated genes suggests that either an additional component of rho-independent terminators exists or that a suppressive mechanism to prevent unwanted termination remains to be discovered.

The inhibition of restriction endonucleases due to Z-DNA in negatively supercoiled plasmid.

Plasmid pGC20 containing the (dGC)9 insert in SmaI recognition site has been used to study the inhibition of cleavage by different restriction endonuclease due to Z-DNA formation in (dCG)10 sequence of the negatively supercoiled plasmid. Data obtained indicate the different sensitivity of restriction endonucleases to DNA conformational perturbations resulted from the Z-DNA formation. Therefore, the inhibition of DNA cleavage by a particular restriction endonuclease cannot serve as a criterion for the estimation of the length of B-Z junctions in circular supercoiled DNAs.

Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets.

"Uniformly" modified phosphodiester or phosphorothioate oligonucleotides incorporating 2'-deoxy-2'-fluoroadenosine, -guanosine, -uridine, and -cytidine, reported herein for the first time, when hybridized with RNA afforded consistent additive enhancement of duplex stability without compromising base-pair specificity. CD spectra of the 2'-deoxy-2'-fluoro-modified oligonucleotides hybridized with RNA indicated that the duplex adopts a fully A-form conformation. The 2'-deoxy-2'-fluoro-modified oligonucleotides in phosphodiester form were not resistant to nucleases; however, the modified phosphorothioate oligonucleotides were highly nuclease resistant and retained exceptional binding affinity to the RNA targets. The stabilizing effects of the 2'-deoxy-2'-fluoro modifications on RNA-DNA duplexes were shown to be superior to those of the 2'-O-methylribo substitutions. RNA hybrid duplexes with uniformly 2'-deoxy-2'-fluoro-modified oligonucleotides did not support HeLa RNase H activity; however, incorporation of the modifications into "chimeric" oligonucleotides has been shown to activate mammalian RNase H. "Uniformly" modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides afforded antisense molecules with (1) high binding affinity and selectivity for the RNA target and (2) stability toward nucleases.

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

2'-O-[2-[N,N-(dialkyl)aminooxy]ethyl]-modified antisense oligonucleotides.

[structure] Oligonucleotides with two novel modifications, 2'-O-¿2-[N, N-(dimethyl)aminooxy]ethyl¿ (2'-O-DMAOE) and 2'-O-¿2-[N, N-(diethyl)aminooxy]ethyl¿ (2'-O-DEAOE), have been synthesized. These modifications exhibit high binding affinity to target RNA (and not to DNA) and enhance the nuclease stability of oligonucleotides considerably with t(1/2) > 24 h as a phosphodiester.

Conformational peculiarities of polynucleotides with a nonrandom base sequence according to the 1H----3H exchange rate in C8H groups of purinic residues.

We have determined the 1H----3H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT).poly(dA-dT), poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT) as well as homopolynucleotides poly(dA).poly(dT) and poly(dG).poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4-6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25 degrees C. The rate constants for adenine (kA) and guanine (kG) of polynucleotides were compared with corresponding constants for E. coli DNA. dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution. Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT).poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating "wrinkled" DNA model. The conformations of poly(dG-dC).poly(dG-dC) and poly(dA-dC).poly(dG-dT), according to the exchange data obtained are within the B form. For homopolynucleotides in 0.15 M NaCl, the KA value for poly(dA).poly(dT) is nearly the same as kA for B-DNA, which indicates the similarity of their conformations, whereas the kG value for poly(dG).poly(dC) is 1.7-fold lower in comparison with the kG value in B-DNA. This seems to be connected with the existence of B = A conformation equilibrium for poly(dG).poly(dC) in solution. The increase of NaCl concentration to 3 M results in a B----Z transition in the case of poly(dG-dC).poly(dG-dC) and in the shift of B = A equilibrium towards the A-form in the case of poly(dG).poly(dC) as is evidenced by alterations of their KG values. Poly(dA-dT).poly(dA-dT) in 6 M CsF and poly(dA-dC).poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the "X-type" CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA).poly(dT) in 6 M CsF corresponds to the "heteronomous" DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.

[The effect of Z-conformation on enzymatic activity of restriction endonucleases]. / Vliianie Z-konformatsii na fermentativnuiu aktivnost' restriktsionnykh éndonukleaz.

Recombinant plasmid pGC20 containing (GC)9-insert into SmaI site of pUC19 has been used to study the inhibition of cleavage by six restriction endonucleases; KpnI, SacI, EcoRI and also BamHI, XbaI and SalI, due to Z-DNA formation in negatively supercoiled plasmid. The recognition sites of these enzymes were located at different distances on both sides of the (CG)10-sequence. It was shown that the inhibition of the cleavage by KpnI, SacI and EcoRI was decreased in this series as fast as the distance between recognition site and B-Z junction was increased, and no inhibition of cleavage by EcoRI was found. However, such a correlation was not found in the series of BamHI, XbaI and SalI. In contrast with EcoRI the cleavage by SalI was inhibited completely. These results indicate the difference for "sensitivity" of restriction endonucleases to the structural perturbations of DNA associated with B-Z junctions. It seems to depend on features of the enzyme-substrate interaction mechanisms and also on recognition and flanking sequences of DNA. Consequently, experiments with the inhibition of the cleavage by any enzyme can not help to determine the dimension of the region of DNA with altered structure.

[Reaction capabilities and structure of poly(rG) and poly(rG)-poly(rC) in solution by the method of the kinetics of hydrogen ion exchange]. / Issledovanie reaktsionnoi sposobnosti i struktury poli(rG) i poli(rG).poli(rC) v rastvore metodom kinetiki izotopnogo obmena vodoroda

Data on the kinetics of 1H greater than 3H exchange between water and C(8)H groups of guanylic residues in the poly(rG) and poly poly(rG)-poly(rC) are presented. Furthermore, optical properties (CD spectra and hyperchromism) of neutral solutions of these polymers from 20 to 100 degrees C are described. It is shown that the exchange in poly(rG) within the temperature range from 20 to 80 degrees C proceeds faster than in rGMP. Within the temperature range from 20 to 40 degrees C such an acceleration of the exchange is observed also in poly(rG)-poly(rC). According to the ylide mechanism of the exchange reaction the observed accleration of the exchanged in in C(8)H groups of guanylic residues is considered as a consequence of an increase of the positive charge at N(7) atoms. This effect is due to formation of additional hydrogen bonds in which N(7) atoms take part. The exchange in poly(rG)-poly(rG) at temperatures hihger than 75 degrees C, when these additional hydrogen bonds are absent, proceeds more slowly than in rGMP. Such picture is usual in other previously studied polynucleotides whose structure in solution is stabilized only by Watson - Crick hydrogen bonds and stacking interactions. The data obtained support a Guschelbauer's model of the four-stranded stranded poly(rG). They also indicate the posibility of associates formation in poly(rG)-poly(rC) solutions at temperature lower than 40 degrees C being stabilized by hydrogen bonds in which N(7) atoms of guanylic residues take part.

[Kinetics and mechanism of the 3H to 1H in C(8)H groups of purine derivatives]. / Kinetika i mekhanizm 3H to 1H obmena v C(8)H-gruppakh proizvodnykh purina

The pH-dependence of the 3H to 1H exchange between water and C(8)H groups of purine, adenine, 9-methyladenine, 7-methyladenine, hypoxanthine, guanine, xanthine as well as C(2)H groups of imidazole and benzimidazole was studied. It was shown that within the pH-ranges, where the majority of molecules under study are non-ionized, the values of observed rate constant (kobs) do not depend on pH. Beyond these ranges the values of k(obs) are increased or decreased depending on the type of ionizaiton of the compound under study in appropriate pH range. The observed pH dependence of the 3H to 1H exchange is in a good quantitative agreement with ylide mechanism of the exchange reaction. According to this mechanism the 3H--1H exchange takes place in N(7)-protonated forms of the purine derivatives and in zwitterions with positive charge on N(7). The ylide mechanism of the exchange reaction is also suggested by the fact that the true exchange rate constants (k+) of protonated forms of the studied compounds, calculated from the values of k(ods), rises linearly with the increase of their protonation constant (Ka1)--the tenfold increase of Ka1 leads to about four-fold rise of k+. The knowledge of 1h to 3H exchange mechanism in C(8)H groups of purine derivatives allows to estimate alterations of reactivity of the purine residues in polynucleotides and nucleic acids depending on their conformation.

[Study of conformation characteristics of polydeoxyribonucleotides with non-random base sequence by slow 1H----3H exchange]. / Issledovanie konformatsionnykh osobennostei polidezoksiribonukleotidov s nesluchainoi posledovatel'nost'iu osnovanii metodom medlennogo obmena 1H----3H.

The rate constants of 1H----3H exchange between water and C8H-groups of purinic residues of alternating polynucleotides: poly[d(A-T)].poly[d(A-T)] (I), poly[d(G-C)].poly[d(G-C)] (II), poly[d(A-C)].poly[d(G-T)] (III) and homopolynucleotides: poly(dA).poly(dt) (IV), poly(dG).poly(dC) (V), as well as DNA E. coli, was determined in 0.15 M NaCl at 25 degrees C. The retardation of exchange observed at these conditions (compared to that of the B-form DNA) is in agreement with the model of B-alternating structure for the (I) and is attributed to the co-existence of B- and A-conformers for the (V) in solution. Absence of distinguishable differences in exchange rate constants for purinic residues of the (II), (III) and (IV) (compared to that of the B-form DNA) evidences that conformations of these polynucleotides in solution are similar to "canonical" B-form DNA and don't correlate with the model of "heteronomous" DNA which was proposed for (IV).

[Study of conformation characteristics of "X-form" of alternating polynucleotides by the method of slow 1H----3H transition]. / Issledovanie konformatsionnykh osobennostei "X-formy" al'terniruiushchikh polinukleotidov metodom medlennogo 1H----3H obmena.

The rate constants of 1H----3H exchange between water and C8H-groups of purine residues of alternating polynucleotides: poly[d(A-C)].poly[d(G-T)] and poly[d(A-T)].poly[d(A-T)], as well as Escherichia coli DNA, dAMP and dGMP, in solutions with high concentration (4.3 or 6 M) CsF, in water ethanol (60%) solution and (in comparison) in 0.15 M NaCl were determined at 25 degrees C. The 1H----3H exchange rate exchange rate constants for adenylic (kA) and guanylic (kG) residues of polynucleotides were compared with the corresponding constant for DNA and mononucleotides. It was shown that at conditions when poly[d(G-T)] and poly[d(A-T)].poly[d(A-T)] exhibit the "X-form" CD spectrum, alteration of exchange rates in polynucleotides (approximately 2-fold increase in kA in CSF and approximately 1.5-fold decrease in kA and kG in 60% ethanol with 0.15 M NaCl) is due to the effect of solvents on the chemical reactivity of purine residues, but does not reflect a conformational transition. The analysis of these results allows us to conclude, that alternating polynucleotides under the above mentioned conditions retain roughly the conformations inherent in them in 0.15 M NaCl: poly[d(A-C)].poly[d(G-T)] conformation in 4.3 m CsF or 60% ethanol differs only insignificantly from the "canonic" B-DNA, whereas the poly[d(A-T)].poly[d(A-T)] conformation in 6 M CSF corresponds to B-alternating DNA.

[Comparative study of slow 1H to 3H exchange in synthetic polynucleotides of A- and B-type conformations]. / Sravnitel'noe issledovanie skorostei medlennogo 1H 3H-obmena v sintesinteticheskikh polinykleotidakh s konformatsiiami A- i V-tipov.

The rate of 1H leads to 3H exchange between water and C(8)H-groups of purinic residues in synthetic polynucleotides in wide temperature range measured. At temperatures below their Tm the rate of the exchange is shown to be lower as compared with that in corresponding mononucleotides. In the case of polynucleotides of A-conformation (poly(A).poly(U), poly(A).2poly(U) and poly(dA).2poly(dT), and poly(G).poly(C) the exchange is retarded by a factor of 5.7--7.5, whereas in the case of those of B-conformation (poly(dA).poly(dT), poly(dA--dT).poly(dA--dT) and poly(dG).poly(dC)) the exchange is retarded only by a factor of 2.3--2.5. Assuming the ylide mechanism of exchange the retardation is interpreted as a consequence of sterical hidrance in polynucleotides helical structure, which hampers contacts between purinic C(8)H-groups and OH-ions of solvent. Analysis of atomic arrangement around C(8)H-group and interatomic distances calculated on the basis of published atomic coordinates support our general conclusion that the sterical hindrance is more significant in the A-form as compared with that in the B-form. Elucidated correlation between the degree of the retardation in purine-containing polynucleotides and their conformation in solution allows to estimatf the type of conformation of polynucleotide with unknown structure on account of the slow 1H leads to 3H exchange data.

[Structure of polyriboguanylic acid in solution]. / Struktura poliriboguanilovoi kisloty v rastvore.

The electron microscopic data and the CD spectra of poly(G) have shown that the transition of a freeze-dried preparation of poly(G) from a "metastable" to a "stable" form is the transition of the four-stranded poly(G) from the globular form to the linear one. The observed phenomenon of the formation of long four-stranded poly(G) fibers (4--5 nm in diameter and 200-2000 nm in length) is suggested to be a result of a joint of the initial poly(G) molecules (50--80 nm in length) due to noncovalent binding ("sticking") of their free one-, double- or three-stranded ends. This phenomenon has permitted us not only to compare the thickness of low molecular weight poly (G) preparations under different conditions but to observe directly the existence of a large number of double-stranded (2--3 nm in diameter) regions ("defects") in four -stranded poly(G) fibers. The data obtained have shown that under certain conditions (high temperature, low pH, etc.) the four-stranded poly(G) fibers break down with the formation of two-stranded poly(G) fibers whose hydrogen bond systems depend on the conditions of their formation.

[Study of the intermolecular association of poly(G).poly(c) and poly(dG).poly(dC) in solutions by methods of 1H to 3H exchange and electron microscopy]. / Issledovanie mezhmolekuliarnoi assotsiatsii kompleksov poli (G). poli (C) i poli (dG). poli (dC) v rastvorakh metodami obmena 1H leads to 3H i elektronnoi mikroskopii.

The kinetic of 1H leads to 3H exchange between water and C(8)H-groups of the guanylic residues in poly(G) . poly(C) and poly(dG) . poly(dC) was investigated within the temperature range from 30 to 90 degrees in 0.5 M NaCl (pH 7.2). It was shown that the exchange in freshly dissolved preparations at temperatures lower than 50 degrees proceeds faster than that in the case of GMP. According to the ylide mechanism of the exchange reaction the observed acceleration of the exchange is considered as a consequence of associates formation in poly(G) . poly(c) and poly(dG) . poly(dC) solutions at temperatures lower than 50 degrees. Associates are stabilized by intermolecular hydrogen bonds in which N(7) atoms of guanylic residues take part. The increase of the temperature is accompanied by gradual disappearance of the exchange acceleration. The retardation of exchange, which is characteristic of most non-associated double-stranded polynucleotides and nucleic acids is observed at the temperatures above 60 degrees. The retardation points to thermal destruction of the associates at temperatures higher than 50 degrees. The associates which are characterized by ordered structure including several "side by side" arranged double-stranded molecules were observed by electron microscopy. The addition of EDTA to solutions as well as the increase of temperature leads to destruction of the associates whereas the addition of Mg2+ makes the associates more stable.

[Structural characteristics and availability of DNA for solvent molecules in nuclei of Physarum polycephalum]. / Strukturnaia kharakteristika i dostupnost' DNK dlia molekul rastvoritelia v iadrakh Physarum polycephalum.

The rate constants of 1H--3H exchange between water and C8H-groups of purinic residues of DNA in solution and in nuclei of synchronous Physarum polycephalum at different phases of the cell cycle were measured. The nuclear membranes and nonhistone proteins were shown not to create additional hindrances and not to diminish the availability of DNA in nuclei for solvent molecules. A small increase of the rate of the 1H--3H exchange between water and the DNA of nuclei upon transition from the S-phase to the late G2-phase seems to reflect the process of chromatin decondensation connected with activation of the transcription and local changes of the secondary structure of DNA at the late G2-phase of the cell cycle.

Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure.

Fourteen oligonucleotides 8-21 nucleotides in length and their complements were synthesized as DNA and RNA. For each sequence, four kinds of duplexes, DNA:DNA, RNA:RNA, DNA:RNA, and RNA:DNA, were prepared. Twelve sequences had A.T/U content varying from 25 to 80% and dPy content in the DNA strands varying from 0 to 100%. Thermodynamic stabilities of four duplexes for each sequence were determined in solution containing 100 mM Na+, 10 mM phosphate, and 0.1 mM EDTA, pH 7.1. CD spectra and electrophoretic mobility on native polyacrylamide gel were measured for most duplexes. Quantitative correlations of hybrid stability both with deoxypyrimidine content and, at fixed dPy content, with the fraction of A.T/U in duplexes were found. We also demonstrated that hybrids with 70-80% deoxypyrimidine DNA strand and a high or moderate A.T/U fraction displayed the highest relative stability compared to their RNA counterparts. Relationships of relative intensities of CD bands at 210 nm and relative electrophoretic mobilities of hybrids with relative hybrid stability suggested that hybrid conformation varies continuously between A- and B-form and is the decisive factor in relative hybrid stability.

What affects the effect of 2'-alkoxy modifications? 1. Stabilization effect of 2'-methoxy substitutions in uniformly modified DNA oligonucleotides.

The thermostability of hybrid duplexes with uniformly 2'-methoxy modified DNA strands (D'R and RD'), their unmodified DNA:RNA counterparts (DR and RD), and corresponded RNA:RNA (RR) duplexes for six sequences with different GC and deoxypyrimidine (dPy) content was measured. The linear correlation between the total stabilization effect of 2'-methoxy modifications (Delta DeltaG(o)37(D'R-DR)) and the relative stability of corresponding unmodified hybrids compared to the RR counterparts (Delta DeltaG(o)37(RR-DR)) suggests that the initial conformational and the thermodynamic state of the "parent" unmodified hybrid governs the effect of 2'-methoxy (and may be other 2'-alkoxy) modifications whose mechanism of action includes an S --> N conformational shift resulting in an RNA-like A-form duplex. We also found a correlation between the "hydrophobic" part of the total effect (Delta DeltaG(o)37(D'R-RR)) and the dA fraction in the modified DNA strand, suggesting that the "hydrophobic" effect of the 2'-methoxy groups results mainly from intraresidue steric effects increasing rigidity of the modified sugar rings. The correlations observed enabled us to predict the stability of hybrids with 2'-methoxy modified DNA strands for any sequence except for sequences with (dU)10 and (dA)10 strings.