In our previous paper it was shown that the two C-terminal Gln residues of a C-terminal 15-residue fragment, Mel(12-26)(GLPALISWIKRKRQQ-NH2), of melittin and a series of individual substituted analogues might not involved in the interaction withbacterial membranes. In this paper, peptides with one and two Gln residues deletion, respectively, Mel(12-25) and Mel(12-24), weresynthesized and characterized. Both of the deletion peptides showed higher antimicrobial activities than the parent peptide, Mel(12-26). If both of the Gln residues of Mel(12-26) were respectively replaced by a hydrophilic amino acid Gly, the antimicrobial activity increasedslightly. If the Gln residue of Mel(12-25) was replaced by a hydrophobic amino acid Leu, the antimicrobial activity changed little, althoughthe substituted peptide possessed much higher hydrophobicity and higher α-helical conformation percentage in 1,1,1,3,3,3-hexafluoro-2-propanol/water determined by circular dichroism spectroscopy (CD) than the parent peptide. These results indicated that the two C-terminalresidues might be indeed not involved in the binding to bacterial membranes. The antimicrobial activity increasing with the residue deletionmay be caused by the decrease of the translational and rotational entropic cost of the binding of the peptides to bacterial membranes becauseof the lower molecular weights of the deletion peptides.

Telomerase, a ribonucleoprotein enzyme, is expected to be a new marker for cancer diagnosis. Thedevelopment of the telomeric repeat amplification protocol (TRAP) and its modified versions have facilitatedthe detection of telomerase activity in small tissue and tumour samples. But most of these techniques requirecomplex post-PCR procedures. As for the two real-time quantitative methods (SYBR Green and Amplifluormethods) reported so far, both use fluorogenic probes without specificity. To overcome these problems wedeveloped a new real-time method for the detection of telomerase activity. In this method a duplex scorpionprimer and reverse primers with hairpin-like structures were used. The use of duplex scorpion primers showsa series of advantages: the target-specific probe sequence provides higher specificity than the SYBR Greenand Amplifluor methods; the unimolecular probing mechanism allow the assay to be conducted under fastcycling conditions and a single operation can be completed in 1.5h; the closed-tube system reduces the riskof carryover contamination and supports high throughput. This method may be a useful tool to rapidly,specifically and precisely quantify telomerase activity.

A novel method for duplex probes is designed to simulate the TaqMan probe during polymerase chain reaction(PCR). In this method, two partly complementary single-labelled oligonucleotide probes labelled with afluorophore or a quencher, respectively, are used. At lower temperature the two probes can bind to each otherand form a mismatched duplex, in which the fluorophore and quencher are in close proximity and the sameenergy transfer mechanism as in molecular beacons may occur between them; thus, a quenching efficiencybetter than conventional TaqMan probes is acquired. In the anneal-extend step of PCR, one single-labelledprobe hybridises to the predetermined target and is cleaved by Taq DNA polymerase. Increased fluorescentsignal can be observed at lower temperature. The fluorescent data analysis demonstrated that a significantlyhigher level of fluorescent signal and hence higher sensitivity of detection is obtainable using our duplex probesin place of conventional TaqMan probes. Combined with real-time PCR instruments, the assay can be used toquantify the input target molecules and the dynamic linear range is of at least six orders of magnitude.

The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservationbut their function is unknown. We have determined that these fingers mediate homodimerization andbinding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammaliancells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cellsresults in altered expression of HOX genes that are targets for regulation by MLL. These alterations aresuppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHDfingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.

The application of a new fluorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detectionof Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant ofduplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detectionspecificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive resultsfor the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probingmechanism of this probe makes the use of short target-specific probe sequence possible, which will render thisprobe applicable in some specific systems.

A modified molecular beacon that possesses a stem-hairpinstructure as seen in conventional molecular beacons and canbe cleaved during PCR is designed, and it can specificallyrecognize the presence of the target and was obviously moresensitive than conventional molecular beacons.

A novel method for duplex probes is designed to simulate theTaqMan probe during polymerase chain reaction (PCR); two single-labelled probes are used for this method, whichrelies on the 5'-exonuclease activity of nucleic acid polymerase.

A novel real-time quantitative method for measuring telomerase activity is described in which the duplex scorpionprimer is used to provide an intramolecular probing mechanism for specific detection of telomerase activity. Usingthis method, linearity from 10 to 104 cells expressing telomerase activity could be obtained (R2=0.994). Therequirement of post-PCR steps is thus obviated.

A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirableproducts arising throughout PCR amplification thereby giving better results than a manual hot-start method.

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based onthe use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resultingfluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize thepresence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combinedwith real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initialtarget molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.