Peptide nucleic acid (PNA) binding-mediated gene regulation

Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

Synthesis of Two New Chiral Blocks for the Construction of Peptide Nucleic Acid (PNA)

Protected (L) and (D)-lysine were used respectively as starting materials to synthesize two new types of chiral blocks for the construction of PNA. Nucleobase was linked to alpha-NH2 of lysine via -CH2C (O)- spacer in type I, and -C (O)- was used in type II. The corresponding oligomers were constructed in solution.

Synthesis and photo activity of phenylazonaphthalene peptide nucleic acid monomers

Phenylazonaphthalene peptide nucleic acid （PNA） monomers were successfully synthesized, and their photoisomerization was examined. The new PNA monomers showed reversible trans-cis isomerization with UVand visible light irradiation, which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA. Simultaneously, the fluorescence of the new PNA monomers might make them especially useful as structural probes.

Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

To investigate the effects of anti-sense peptide nucleic acids （PNAs） targeting Ki-67 gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations （1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L）. The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and ^3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide （ASODNs） targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs （16.9±0.7） was significantly inhibited as compared with that of the control groups （28.6±0.4） （P〈0.01）. The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs （42.1 ±2.2） was significantly reduced when compared with that of the control groups （83.6± 1.4） （P〈0.01）. Proliferation of 786-0 cells treated with anti-sense PNAs （20.7 ± 1.5） was significantly inhibited as compared with that of the control groups （58.6± 1.4） （P〈0.01）. The apoptosis rate of 786-0 cells treated with anti-sense PNAs （28.7 ± 2.3） was significantly increased higher compared with that of the control groups （13.8 ±1.0） （P〈0.01）. From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Acid-base and lipophilic properties of peptide nucleic acid derivatives

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid(PNA)derivatives,including eleven PNA monomers and two PNA decamers,is described.The acidity constants(pKa)of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution,and these values were found to be in agreement with those obtained by MoKa software.These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers.In addition,immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers,which showed the PNA derivatives had poor affinity towards membrane phospholipids,and confirmed their scarce cell penetrating ability.Overall,our study not only is of potential relevance to evaluate the pharmacokinetic properties of PNA,but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

Electrochemical investigation of DNA hybridization with ferrocene-labelled peptide nucleic acid on gold electrodes

An electrochemical DNA sensor based on ferrocene-labelled peptide nucleic acid （PNA-Fc） was prepared. The hybridization between PNA-Fc and DNA immobilized on a gold electrode was examined by cyclic voltammetry （CV） and differential pulse voltammetry （DPV）. PNA-Fc shows a good electrochemically activity and has a redox potential of 170 mV verus Ag/AgCl electrode after hybridization, representing the characteristic of ferrocene/ferrocenium （Fc/Fc＋） transformation. The results illustrate that PNA-Fe can be used as an effective electrochemical DNA probe sensor.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i>in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

In situ aneuploidy assessment in human sperm:the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques

Both the primed in situ(PRINS)and the peptide nucleic acid-fluorescence in situ hybridization(PNA-FISH) techniques constitute alternatives to the conventional(fluorescence in situ hybridization,FISH)procedure for chro- mosomal investigations.The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction.Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones.The two procedures present several advantages(specificity,rapidity and discriminating ability)that make them very attractive for cytogenetic purposes.Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.(Asian J Androl 2006 Jul;8:387-392)

Apoptosis Induced by Bcl-2 Antisense Peptide Acid in HL60 Cells

Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The expression levels of protein were assayed by immunofluorescence using fluoresce isothiocyanate label. The morphological changes in apoptotic cells were observed. Flow cytometric analysis of DNA fragmentation was also performed. Results Antisense peptide nucleic acid (PNA) targeting the coding region of the Bcl-2 mRNA could effectively inhibit the growth of HL60 cells, down-regulate the synthesis of Bcl-2 protein and induce apoptosis. After HL60 cells were treated with 10 μmol/L Bcl-2 antisense PNA or antisense oligonucleotide for 72 h respectively, apoptotic rates of HL60 cells were 17.80±1.53 and 13.17±1.12, respectively( P <0.05). Conclusion Antisense PNA targeting the coding region of Bcl-2 mRNA may have stronger antisense effects than the antisense oligonucleotides and could induce apoptosis of HL60 cells.

Synthesis of Peptoid Nucleic Acid with Thymine as Nucleic Base

The synthesis of peptoid nucleic acid bearing thymine as nucleobase has been achieved. This modified oligonucleotide showed good hybridization with DNA.

Selective Enrichment of Low-Abundance DNA Variants Based on Programmable Peptide Nucleic Acid Probes

Single-nucleotide variants(SNVs)are crucial in disease development,but their accurate detection is challenging due to their low abundance and interference from wild-type targets.Although nucleic acid analogs like peptide nucleic acids(PNAs)have been used for SNV detection,they often lack programmable sensitivity and specificity due to poorly calculated thermodynamics and kinetics.Here,we present a computational method for calculating the stacking energy of PNA and DNA hybrids,leveraging nearest neighbor parameters.Validation against experimental data from 16 sequences under varied hybridization conditions yielded good agreement using Bland-Altman analysis,with all data points falling within the confidence interval.Our findings indicate that PNA-DNA hybridization is thermodynamically more stable and exhibits kinetics 140-fold faster than DNA-DNA hybridization for identical sequences.Utilizing this computational framework,we designed PNA toehold probes,which were screened via simulations and experiments.This combined approach facilitated the identification of highly sensitive and specific PNA toehold probes for single point mutation detection via strand displacement reaction.Our results demonstrate the successful application of PNA toehold probes for detecting point mutations with high sensitivity and specificity,achieving a selective amplification of approximately 200-fold for variants with a variant allele frequency(VAF)of 0.5%using quantitative polymerase chain reaction.

探针浓度值对LSAW-bisPNA传感器检测系统的影响

目的探讨双体肽核酸(bisPNA)探针浓度值对漏声表面波(LSAW)-bisPNA基因传感器检测系统的影响。方法LSAW基因传感器表面分别固定终浓度为0.001、0.005、0.01、0.05、0.5、1.0、1.5、2.0、3.0、4.0μmol/L的bisPNA探针后,分别观察其与相应的HPV靶序列杂交时相位变化及反应所需时间。结果与HPV靶序列杂交所引起的相位变化值是先升后降,以1.0μmol/L探针浓度为分界线;杂交平衡时间上各组间未见明显的变化趋势。结论1.0μmol/L bisPNA探针固定浓度最为适宜。

缓冲液离子浓度对新型PNA-LSAW基因传感器杂交效应的影响

目的探讨PBS缓冲液的离子浓度对构建的肽核酸(PNA)漏声表面波(LSAW)基因传感器杂交效应的影响。方法将1.0μmol/L的PNA探针固定在LSAW基因传感器表面,分别加入10、20、50、100mmol/L的PBS缓冲液,观察不同离子浓度的PBS缓冲液对PNA-LSAW基因传感器杂交效应的影响,记录分析传感器相位的变化值及反应所需的时间。结果当钠离子浓度从10mmol/L增加至100mmol/L时,传感器的相位变化呈先升高后降低的趋势,以20mmol/L为分水岭。而杂交平衡时间增加显著。结论离子浓度极大地影响了杂交反应的时间,低盐离子浓度更有利于提高PNA和靶序列的杂交速率,高盐离子浓度则会大大降低PNA的结合速率。离子浓度为20mmol/L时杂交最为适宜。

反义端粒酶PNA对肺癌细胞株生长的体外抑制作用

目的 探讨反义端粒酶肽核酸 (PNA)片段对肺癌细胞株端粒酶活性及细胞株生长的抑制作用。方法 将人工合成的端粒酶反义PNA片段 ,应用脂质体转染法将反义端粒酶序列导入肺腺癌细胞株A5 49及小细胞癌细胞株NCI H44 6中 ,采用MTT法检测活细胞数 ,采用RT PCR ELLISA法检测端粒酶活性。结果 转染 72h后 ,A5 49、NCI H 44 6细胞株端粒酶活性 (A45 0值 )分别由 0 .5 82± 0 .0 3 9和 0 .5 71± 0 .0 43降低至 0 .2 94± 0 .0 48(P <0 .0 1)和 0 .2 76± 0 .0 5 1(P <0 .0 1) ;而两细胞株的活细胞数 (A5 80值 )分别由 0 .485± 0 .0 0 9和 0 .5 13± 0 .0 15降低至 0 .191± 0 .0 2 7(P <0 .0 1)和 0 .13 8± 0 .0 46(P <0 .0 1) ,细胞生长受到显著抑制。结论 反义端粒酶PNA片段具有抑制肺癌细胞株端粒酶活性的作用 。

荧光光谱法研究PNA-DNA的杂交和解链

本文建立了一种以阳离子型荧光染料藏红 T(ST)作为荧光试剂的荧光猝灭新方法 ,ST首次被用于PNA- DNA杂交和解链的检测。实验结果发现当 PNA- DNA杂交形成双链时 ,可以在一定程度上猝灭 ST的荧光 ,而当双螺旋解链时 ,对 ST荧光的猝灭程度减小 ,逐渐接近于 ST本身的荧光 ,根据荧光强度的变化研究了 PNA- DNA的杂交和解链过程。通过 UV光谱实验证实了这种荧光猝灭由它们之间的静电作用以及

荧光探针法研究PNA-DNA双链和dsDNA结构的差异

运用荧光和紫外吸收光谱研究了Ru(bipy)2(dppz)2+和苯胺红T两种荧光试剂与PNA-DNA、dsDNA之间的相互作用。实验结果表明:相同条件下同一荧光试剂与PNA-DNA、dsD-NA两者之间发生不同的作用,从而说明了PNA-DNA、dsDNA的结构具有一定的差异,并对引起差异的原因进行了初步的探讨。

缓冲液pH值对LSAW-bisPNA传感器检测系统的影响

目的探讨PBS缓冲液pH值对LSAW-bisPNA基因传感器检测系统的影响。方法LSAW-bisPNA基因传感器表面固定终浓度为1.0μmol/L的bis-PNA探针,然后分别在pH5.8～8.0的PBS缓冲液中和相应的靶序列杂交,记录相位变化及反应所需时间。结果在不同pH值缓冲液中,bis-PNA与靶序列反应引起的相位变化有明显差异(P<0·01),pH6.6与pH6.8时传感器响应信号最大,但两者反应所达到的平衡时间有明显差异(P<0·05),pH值6.6时检测所需时间更短。结论选择pH6.6作为bis-PNA和靶序列dsDNA反应的最适pH值,在该pH值条件下传感器响应信号大,反应达平衡时间短。

CPG-PNA的合成、纯化和鉴定

目的 建立肽核酸 (CPG PNA)合成、纯化、分析鉴定的方法。方法 固相肽合成标准Fmoc方法进行合成 ;中压液相层析纯化 ;反向高压液相层析进行纯度分析 ,质谱法进行分子量的鉴定。结果 经纯化 ,CPG PNA的纯度达到 95 .5 % ,分子量测定值与理论值相符。结论 合成的肽核酸与预期的一致 。

177Lu-DOTA-anti-CITED1-PNA的制备、鉴定及其对甲状腺乳头状癌K1细胞增殖能力的影响

目的研究放射性镥核素(^177Lu)标记的结合转化激活因子(Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1,CITED1)mRNA反义肽核酸(antisense peptide nucleic acid,asPNA)即^177Lu-DOTA-anti-CITED1-PNA的制备,鉴定标记物的理化性质并探讨其在人甲状腺乳头状癌K1细胞系中的摄取能力及细胞毒性作用。方法实时荧光定量PCR(RT-PCR)鉴定肽核酸(peptide nucleic acid,PNA)的靶向特异性。通过间接标记法得到反义探针^177Lu-DOTA-anti-CITED1-PNA并测定其标记率,检测其体内外的稳定性。体外细胞实验测定反义探针的摄取能力及对K1细胞增殖能力影响。结果 RT-PCR显示反义肽核酸作用于K1细胞后能明显降低CITED1的表达;反义探针的放射化学纯度为(94.5±0.12)%,比活度为(8.7±0.53)MBq/μg,48 h的放射化学纯度仍大于90%;体外细胞摄取、滞留以及细胞增殖实验显示反义探针能被人K1细胞特异性摄取,且相较于未偶联的^177Lu和asPNA,^177Lu和asPNA偶联后联用具有显著的抗肿瘤细胞作用。结论成功制备了^177Lu标记的CITED1mRNA反义肽核酸探针,证实反义探针对K1细胞具有特异性、靶向性且反义探针的联合作用能有效降低细胞活力及增殖能力。