A model compound for the chromophore within the purple nonfluorescent GFP-like chromoprotein asFP595 was synthesized. The postulated structure of the chromophore, 2-acetyl-4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone, was taken from the high-resolution crystal structure analysis of intact asFP595 [Quillin, M. L., Anstrom, D., Shu, X., O'Leary, S., Kallio, K., Lukyanov, K. A., and Remington, S. J. (2005) Kindling Fluorescent Protein from Anemonia sulcata: Dark-State Structure at 1.38 A Resolution, Biochemistry 44, 5774-5787]. Erlenmeyer lactonization and oxidation of the methylene group attached to the heteroaromatic moiety with selenium dioxide were used at the key stages of the synthesis. The spectral properties of the model chromophore in solution and their dependence on the pH and polarity of the solvent were investigated. In water, the chromophore was found to exist in two forms, neutral and anionic, with a pK(a) of 7.1. In a dimethylformamide solution, the spectral properties of the anionic form closely match those of the native protein, demonstrating that under these conditions, the compound is an excellent model for the chromophore within native asFP595.

SummaryGreen Fluorescent Protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Greento-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad.Here we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(phydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore TrpTyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore AsnTyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

Concatemer DNA duplexes which contain at the EcoRII restric-

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Background: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system.

Laser spectroscopy and kinetic mass spectrometry of polyatomic isolated molecules have been carried out by using stepwise laser ionization of molecules in a mass spectrometer. The optical absorption spectrum of NO(2) has been recorded, for example, by using pulsed dye-laser excitation and H(2) vacuum-ultraviolet laser ionization of molecules. The kinetics of excited electron states and of the production of molecular and fragmented ions of some polyatomic molecules have been investigated with high temporal resolution. The sensitivity achieved is 10(-10) cm(-1) of the absorption coefficient and 10(9) (10(5)) molecules in the ground (electronically excited) state in the irradiated volume.

Modified oligonucleotides containing 5-methylcytidine and/or 2-aminoadenosine form tighter hybrids with DNA and are, therefore, more efficient primers for DNA sequencing as compared to their natural counterparts. Strings of contiguous modified pentanucleotides can be used for DNA sequencing by primer walking.One of the promising approaches to speeding up large-scale DNA sequencing is a recently described "primer walking" technique (1). In this technique, the first primer complementary to a known portion of DNA under study is used for sequencing the adjacent region. The new sequence information obtained is then used to prompt a new primer for further sequencing beyond the known regions. By repeatedly performing this procedure, the sequence of long DNA stretches can be established. However, the approach includes expensive and time-consuming synthesis of new and rather long primers after each sequencing step. This shortcoming was elegantly circumvented by Studier's group (2). Instead of synthesizing long sequencing primers, they proposed to use strings of short contiguous oligonucleotides constituting together the sequence of a desirable primer. Oligonucleotide constituents of such composite primers could be simply chosen from a premade oligonucleotide library. The authors (2) have demonstrated that strings of three contiguous hexamers can serve as specific sequencing primers in the presence of single-stranded DNA-binding protein (SSB). The full library of all possible hexanucleotides should contain as many as 46 = 4096 individual components. Still this figure seems too high for routine sequencing, and a further decrease in size of the library is highly desirable.In principle, the decrease could be achieved by shortening oligomers that form composite primers. However, it was convincingly demonstrated (2) that six is the lowest limit of the length of natural oligomers capable of functioning as components of composite primers, whereas pentamer combinations provide only "weak and ambiguous" priming.We have previously described the use of modified oligonucleotides containing 5-methylcytidine (m5C) and 2-aminoadenosine (n2A) instead of their natural counterparts as primers for sequencing (3). Oligonucleotides with m5C (4) or n2A (see the discussion of the problem in ref. MATERIALS AND METHODS Phosphoramidites. 5-Methyl-2'-deoxycytidine and 2-amino-2'-deoxyadenosine were prepared according to refs. 6 and 7; protected using the 4,4'-dimethoxytrityl group for the 5' position, the benzoyl group for the N4 in 5-methyl-2'-deoxycytidine, and the dimethylacetamidine group for N2 and N6 in 2-amino-2'-deoxyadenosine (8); and converted to the corresponding 3 '-cyanoethyldiisopropyl phosphoramidites (9). Other phosphoramidites were synthesized by a standard procedure.Oligonucleotide Synthesis. Oligonucleotides were synthesized on a controlled porous glass support with a Milligene 7500 DNA synthesizer. After deprotection by aqueous ammonia at 60°C for 20 h, the oligonucleotides were purified by denaturing polyacrylamide gel ...

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