慢性乙型肝炎患者血清HBV DNA水平与HBsAg和HBeAg水平的相关性分析

目的探讨慢性乙型肝炎患者血清HBV DNA水平与HBsAg和HBeAg滴度的关系。方法在951例慢性乙型肝炎患者,采用FQ-PCR法和Abbott化学发光微粒子免疫分析技术分别测定血清HBV DNA水平及HBsAg和HBeAg滴度,分析HBV DNA水平与HBsAg和HBeAg滴度的相关性。结果在951例患者中,HBVDNA阳性率为53.83%(512/951);患者血清HBV DNA水平与HBsAg和HBeAg滴度呈正相关(rs=0.45和re=0.49,P<0.05);在HBV DNA水平≥7lg拷贝/毫升患者,血清HBsAg和HBeAg滴度高于HBV DNA为3～7lg拷贝/毫升患者,HBV DNA为3～7lg拷贝/毫升患者血清HBsAg和HBeAg滴度大于HBV DNA<3lg拷贝/毫升患者,差异均有统计学意义(P<0.05);将HBsAg分为<1000 IU/ml、1000～10000 IU/ml和≥10000 IU/ml3组,结果不同HBsAg滴度患者血清HBV DNA水平差异有统计学意义(P<0.05)。结论在血清HBV DNA≥7lg拷贝/毫升和HBsAg滴度≥10000 IU/mL患者,HBV DNA水平与HBsAg滴度呈正相关,在HBV DNA>3 lg拷贝/毫升患者,血清HBV DNA水平与HBeAg滴度呈正相关。     

HBV cccDNA、HBsAg、HBV pgRNA联合检测对恩替卡韦治疗HBeAg阳性慢性乙型肝炎效果的预测价值

目的研究血清HBV共价闭合环状DNA(HBV cccDNA)、HBsAg、HBV前基因组RNA(HBV pgRNA)联合检测对恩替卡韦(ETV)治疗HBeAg阳性慢性乙型肝炎(CHB)患者的效果预测价值。方法将2015年5月-2017年5月经苏州大学附属第二医院确诊为HBeAg阳性的87例CHB患者纳入研究。根据患者ETV治疗48周后是否获得完全应答,分为获得组(n=38)和未获得组(n=49)。均采取ETV治疗,疗程48周,观测患者基线及治疗12、24、48周时血清HBV cccDNA、HBsAg、HBV pgRNA的水平。正态分布的计量资料组间比较采用t检验,非正态分布的采用Mann-WhitneyU检验。计数资料组间比较采用χ~2检验;等级资料组间比较采用Mann-WhitneyU检验。采用Pearson相关分析探讨肝组织HBV cccDNA与血清HBsAg、HBV pgRNA的相关性。采用多因素logistic回归分析完全应答的预测因素,并采用受试者工作特征曲线(ROC曲线)评价血清HBV cccDNA、HBsAg、HBV pgRNA单独或联合检测对完全应答的预测价值。结果相较于未获得组,完全应答获得组患者基线,治疗24、48周时的HBV cccDNA水平明显下降(Z值分别为-2.452、-2.518、-2.266,P值均0. 001);基线,治疗12、24及48周时的HBsAg水平明显下降(Z值分别为-2.431、-2.750、2.386、2.536,P值均0. 001);治疗12、24、48周时的HBV pgRNA水平明显下降(Z值分别为-2.674、-2.503、-2.528,P值均0. 001)。完全应答获得组患者HBV cccDNA在治疗24周时、HBsAg在治疗12周时、HBV pgRNA在治疗12周时的下降水平均明显高于未获得组(Z值分别为-2.352、-2.566、-2.389,P值分别为0. 006、0. 001、0. 004)。血清HBsAg、HBV pgRNA与肝组织HBV cccDNA均呈正相关(r值分别为0.553、0.757,P值均0. 001)。治疗24周时HBV cccDNA、治疗12周时HBsAg、治疗12周时HBV pgRNA的水平可作为预测完全应答的独立影响因素(OR分别为6. 248、5. 452、5. 670,95%CI分别为1574～14262、2. 048～16. 888、1. 201～16. 183,P值均0. 05)。HBV cccDNA、HBsAg、HBV pgRNA的ROC曲线下面积值分别为0.845、0741、0. 773,均明显小于三者联合检测的0. 913(Z值分别为-2.411、-2.712、-2.673,P值均0. 001),其临界值小于7时患者治疗48周时可获得完全应答。结论血清HBV cccDNA、HBsAg、HBV pgRNA联合检测对HBeAg阳性CHB患者的ETV疗效具有较好的预测价值。     

不同临床类型老年慢性乙型肝炎患者的HBsAg、HBV DNA水平及与肝功能的关系

目的探讨不同临床类型慢性乙型肝炎的表面抗原(HBsAg)、乙肝病毒(HBV)DNA水平及与肝功能的关系。方法收集该院2011年2月至2012年7月127例老年慢性乙型肝炎患者的临床资料及血清标本,采用酶联免疫吸附法检测其HBsAg水平,聚合酶链式反应检测HBV DNA的拷贝情况:分析不同临床类型(慢性重乙型肝炎、乙肝肝硬化及慢性乙型肝炎)的HBsAg和HBV DNA水平;两指标不同水平的肝功能情况。结果慢性乙型肝炎组的HBsAg和HBV DNA水平均高于慢性重乙型肝炎组和乙肝肝硬化组(P<0.05);三组两指标的分布差异有统计学意义(P<0.05)。慢性乙型肝炎组<100 IU/ml(HBsAg)、<4 log10/ml(HBV DNA)的例数少于其余两组(P<0.05),>200 IU/ml(HBsAg)、>5 log10/ml(HBV DNA)的例数多于其余两组(P<0.05);两指标不同HBsAg和HBV DNA水平的丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBIL)和直接胆红素(DBIL)水平存在差异(P<0.05),其中100²00 IU/ml和4200 IU/ml和4⁵ log10/ml的上述4个肝功能指标均高于<100 IU/ml和<4 log10/ml(P<0.05),而>200 IU/ml和>5 log10/ml的均高于其余两组(P<0.05)。结论不同临床类型慢性乙型肝炎的HBsAg和HBV DNA水平存在差异,且与肝功能相关。     

慢性乙型肝炎患者血清、肝内HBV DNA定量及肝组织HBV DNA载量与肝组织损害程度的关系

目的 了解慢性乙型肝炎患者血清、肝内HBV DNA定量及肝组织HBV DNA载量与肝组织损害程度的关系.方法 经血清学及肝穿刺病理证实为慢性乙型肝炎患者38例,分为2组(HBeAg阳性组及HBeAg阴性组).采用ELISA法测定HBV-M、实时荧光定量检测血清及肝内HBV DNA定量、肝组织HBV DNA载量.结果 HBeAg阳性组血清、肝内HBV DNA定量及肝组织HBV DNA载量均高于HBeAg阴性组(P均<0.001);血清HBV DNA定量随肝脏纤维化程度加重而升高(P<0.01),但与肝脏炎症程度未见明显相关;肝组织HBV DNA载量则随肝脏炎症及纤维化程度加重而降低(P<0.001).结论 血清HBV DNA定量与肝组织纤维化程度呈正相关,肝组织HBV DNA载量则与肝组织的炎症及纤维化程度呈明显负相关.     

国人慢性HBV携带者HBV复制水平与T细胞亚群变化的关系

目的:研究慢性HBV携带者(HBVc)HBV复制水平与T细胞亚群变化的关系.方法:应用流式细胞仪检测肝功能正常的HBvc 216例和正常人100例外周血T细胞亚群百分比,ELISA法检测血清乙肝标志物(HBsAg,HBsAb,HBeAg,HBeAb,HBcAb,Anti-HBcAb IgM),实时荧光定量PCR法检测血清HBV DNA,对T细胞亚群结果与血清HBV DNA载量和HBeAg表达的关系进行分析.结果:HBV c外周血CD3~ ,CD4~ 及CD4~ / CD8~ 较正常人显著降低(P<0.01),而CD8~ 显著升高(P<0.01).与HBV DNA(-)组比较,HBV DNA( )组CD3~ ,CD4~ 及CD4~ /CD8~ 分别降低20.4%,17.8%和35.7%,CD8~ 升高21.9%(P<0.0 1).与HBeAg(-)组比较,HBeAg( )组CD3~ ,CD4~ 及CD4~ /CD8~ 分别降低19.5%,14.0%和28.6%,CD8~ 升高19.6%(P<0.01).随着病毒载量的升高,CD3~ ,CD4~ 及CD4~ /CD8~ 显著下降,分别与病毒载量呈显著负相关(r=-0.67,-0.54,-0.67,P<0.01);CD8~ 显著升高,与病毒载量呈显著正相关(r=0.61,P<0.01).HBV DNA( )和HBeAg( )组与HBV DNA单阳性组和阴性组比较,CD3~ ,CD4~ 及CD4~ /CD8~ 显著降低(P<0.05,P<0.01),CD8~ 显著升高(P<0.01).有母亲感染史者CD3~ ,CD4~ 及CD4~ /CD8~ 较无母亲感染史者明显降低,CD8~ 则显著升高(P<0.01).在有母亲感染史者中,血清HBV DNA( )82.2%,HBeAg( ) 75.2%,HBV DNA水平>1×10~(10)copies/L者65.1%,均分别显著高于无母亲感染史者的34.5%,OR=8.65,95%CI:[4.45,17.33],28.7%,OR=7.44,95%CI:[3.91,14.56]和10.3%,OR=15.94,95%CI:7.13,39.66].有母亲感染史和无母亲感染史者中,与HBV DNA(-)组比较,HBV DNA( )组CD3~ ,CD4~ 及CD4~ /CD8~ 均显著降低(P<0.05,P<0.01),CD8~ 均显著升高(P<0.01);与HBeAg(-)组比较,HBeAg( )组CD3~ ,CD4~ 及CD4~ /CD8~ 比均显著降低(P<0.05,P<0.01).结论:肝功能正常的HBVc T细胞免疫功能紊乱与病毒复制水平之间存在显著相关性,HBV活跃复制进一步加重紊乱.     

不同临床时期乙型肝炎病毒感染者血清病毒核糖核酸水平的比较

目的观察和分析慢性HBV感染者不同临床时期的血清乙型肝炎病毒核糖核酸(HBV RNA)、HBV DNA以及HBsAg水平。方法入组86例慢性HBV感染者,抽提血清HBV RNA并以Oligo(dT)为引物反转录mRNA为cDNA,Taqmen荧光定量PCR法检测cDNA。将慢性HBV感染者不同临床时期的测定结果进行统计分析。结果血清HBV RNA定量在免疫耐受期、免疫清除期、再活动期、非活动或低(非)复制期患者中依次降低,不同临床时期血清HBV RNA水平两两比较均差异有统计学意义(P0.05)。在慢性HBV感染者中,血清HBV RNA与HBV DNA呈正相关(HBeAg阳性,r=0.523,P=0.004;HBeAg阴性,r=0.340,P=0.034)。血清HBV RNA水平与HBsAg水平均呈正相关(HBeAg阳性,r=0.548,P0.01;HBeAg阴性,r=0.317,P=0.049)。结论随着慢性HBV感染病程的进展,血清HBV RNA水平逐渐下降。且血清HBV RNA水平与HBV DNA、HBsAg水平呈正相关。     

恩替卡韦治疗慢性乙型肝炎过程中应答不佳相关因素分析

目的探讨年龄、性别、体质量指数(BMI)、治疗前HBV DNA载量、ALT、AST、APTT、PT、HBsAg定量、HBeAg定量、P区基因变异、治疗过程中ALT、AST、HBV DNA下降幅度等因素在恩替卡韦治疗慢性乙型肝炎(CHB)过程中与应答不佳的关系。方法选择128例CHB患者,HBeAg阳性患者84例,HBeAg阴性患者44例。均初次使用恩替卡韦进行抗病毒治疗。治疗前检测上述基线因素及P区变异,治疗过程中ALT、AST、HBV DNA下降幅度等指标。在治疗后4、12、24周分别检测HBV DNA、ALT、AST、HBsAg定量、HBeAg定量。根据治疗24周时HBV DNA载量,分为完全应答组(HBV DNA最低检测值下限)和应答不佳组(HBV DNA下降2 lg,但仍﹥最低检测值下限)。统计分析上述指标与治疗应答的关系。结果年龄、性别、APTT、PT、ALT、AST与治疗应答无明显相关性(P0.05)。BMI、治疗前HBV DNA载量、HBsAg定量与治疗应答呈负相关,BMI高、高HBV DNA载量、HBsAg高水平患者多应答不佳(P0.05)。HBeAg阳性为应答不佳的危险因素(OR=5.431;r=0.358)。HBeAg阳性患者,治疗24周时,应答不佳组HBV DNA下降幅度大(P0.05),rtM204V/I变异率10%时易出现应答不佳(P0.05),rtL180M变异与应答无明显相关性(P0.05)。HBeAg阴性患者,治疗12周时,完全应答组HBV DNA下降幅度大(P0.05),rtM204V/I变异,rtL180M变异均与应答无明显相关性(P0.05)。结论 BMI高、治疗前高HBV DNA载量、HBsAg定量高水平、HBeAg阳性、rtM204V/I变异率10%的HBeAg阳性慢性乙型肝炎患者应用恩替卡韦抗病毒治疗时易出现应答不佳。     

乙型肝炎病毒感染者不同临床时期血清HBsAg水平的比较

目的观察和分析乙型肝炎病毒感染者不同临床时期的血清HBsAg和HBV DNA水平。方法采用雅培化学发光微粒子免疫法和聚合酶链式反应（PCR）技术检测315例乙型肝炎病毒感染者不同临床时期的血清HBsAg和血清HBV DNA定量水平,并对HBsAg、HBV DNA结果进行对数转换;对不同临床时期测定结果进行统计分析。结果患者临床时期分为非肝硬化期[包括免疫耐受期、免疫清除期、非活动或低（非）复制期]和肝硬化代偿期,除非活动或低（非）复制期和代偿期肝硬化期患者的HBsAg比较差异无统计学意义外,不同临床时期HBsAg和HBV DNA定量水平两两比较差异均有统计学意义。在非肝硬化期患者中,HBeAg阳性的患者血清HBV DNA水平与HBsAg水平有相关性（r=0.176,P=0.005）,HBeAg阴性患者的血清HBV DNA水平与HBsAg水平无相关性（r=0.003,P=0.823）。在肝硬化代偿期患者中,无论HBeAg是否阳性,血清HBV DNA水平与HBsAg均有相关性（HBeAg阳性,r=0.072,P=0.01;HBeAg阴性,r=0.101,P=0.004）。结论免疫耐受期和免疫清除期患者血清HBsAg和血清HBV DNA定量水平显著高于非活动或低（非）复制期和肝硬化代偿期患者,且HBsAg与HBV DNA水平正相关。     

恩替卡韦治疗慢性乙型肝炎患者早期出现ALT一过性升高的临床意义

目的探讨恩替卡韦初治慢性乙型肝炎患者早期出现ALT升高的临床意义。方法选取2015年1月至2016年10月住院的恩替卡韦初治的慢性乙型肝炎患者86例,其中出现ALT升高的28例设为A组,未出现ALT升高的58例设为B组,比较两组患者抗病毒治疗24、48、96周的ALT复常率、HBsAg下降幅度、HBV DNA及HBeAg转阴率。结果治疗24周,A、B组的HBV DNA转阴率分别为75.0%、51.7%,A、B组的HBsAg下降幅度分别为0.47(0.19,0.52)、0.25(0.07,0.41)lg IU/mL,差异有统计学意义(P0.05);治疗48周,A、B组的HBV DNA转阴率分别为89.3%、67.2%,A、B组的HBsAg下降幅度分别为0.62(0.27,0.74)、0.32(0.11,0.47)lg IU/mL,差异有统计学意义(P0.05);治疗96周,A、B组HBsAg下降幅度分别为0.69(0.30,0.99)、0.36(0.20,0.60)lg IU/mL,A、B组HBsAg水平分别为(3.30±0.29)、(3.48±0.44)lg IU/mL,差异有统计学意义(P0.05);其余各时间段其他指标,两组比较差异无统计学意义。组内比较,两组内各项指标均较前明显改善。结论恩替卡韦治疗慢性乙型肝炎患者早期出现一过性ALT升高者,可能促进了HBV DNA的清除及HBsAg水平的下降。     

基于社区人群的慢性乙型肝炎病毒感染孕妇产前血清学及病毒学特征北大核心CSCD

目的了解中国部分地区基于社区人群的未经抗病毒治疗的乙型肝炎(乙肝)病毒(HBV)表面抗原(HBsAg)阳性孕妇产前HBV血清学和病毒学特点。方法该研究从广西、江苏、河南地区入组1741例HBsAg阳性孕妇。所有孕妇的临产前血清样本采用Abbott Architect i2000和Abbott Architect m2000分别检测HBV血清学标志物及HBV DNA水平;采用型特异性引物巢式聚合酶链反应(n PCR)法进行HBV基因型分型。结果 1741例HBsAg阳性孕妇中,HBeAg阳性占37.0%(645/1741),HBeAg阴性孕妇占63.0%(1096/1741)。HBeAg阳性孕妇产前HBsAg滴度、HBV DNA水平以及B基因型孕妇所占比例均高于HBeAg阴性孕妇,但年龄低于HBeAg阴性孕妇。HBeAg阳性孕妇的HBsAg滴度和HBV DNA水平呈正相关(r=0.790,P<0.001),HBeAg滴度和HBV DNA水平也呈正相关性(r=0.564,P<0.001)。而HBeAg阴性孕妇的HBsAg滴度和HBV DNA水平无相关性(r=0.020,P=0.517)。结论未经抗病毒治疗的HBsAg阳性孕妇在不同HBeAg状态下,血清HBV DNA水平及HBsAg滴度分布各不相同。对于HBeAg阳性孕妇人群,HBeAg滴度、HBsAg滴度可能成为HBV DNA水平的替代指标。     

Cyclization of short DNA fragments and bending fluctuations of the double helix

Cloutier and Widom [Cloutier, T. E. & Widom, J. (2004) Mol. Cell 14, 355-362] recently reported that the cyclization efficiency of short DNA fragments, about 100 bp in length, exceeds theoretical expectations by three orders of magnitude. In an effort to resolve this discrepancy, we tried modifying the theory. We investigated how the distribution of the angles between adjacent base pairs of the double helix affects the cyclization efficiency. We found that only the incorporation of sharp kinks in the angle distribution provides the desired increase of the cyclization efficiency. We did not find a model, however, that fits all cyclization data for DNA fragments of different lengths. Therefore, we carefully reinvestigated the cyclization of 100-bp DNA fragments experimentally and found their cyclization efficiency to be in remarkable agreement with the traditional model of DNA bending. We also found an explanation for the discrepancy between our results and those of Cloutier and Widom.     

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ^−/− mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ.     

p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation

It is well established that p53 contacts DNA in a sequence-dependent manner in order to transactivate its myriad target genes. Yet little is known about how p53 interacts with its binding site/response element (RE) within such genes in vivo in the context of nucleosomal DNA. In this study we demonstrate that both distal (5') and proximal (3') p53 REs within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are localized within a region of relatively high nucleosome occupancy. In the absence of cellular stress, p53 is prebound to both p21 REs within nucleosomal DNA in these cells. Treatment of cells with the DNA-damaging drug doxorubicin or the p53 stabilizing agent Nutlin-3, however, is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 REs. We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 RE, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal RE within the p21 gene is located close to the end of the nucleosome. Thus, low- and high-resolution mapping of nucleosome boundaries around p53 REs within the p21 promoter have provided insight into the mechanism of p53 binding to its sites in cells and the consequent changes in nucleosome occupancy at such sites.     

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.     

Single-molecule denaturation mapping of DNA in nanofluidic channels

Here we explore the potential power of denaturation mapping as a single-molecule technique. By partially denaturing YOYO®-1-labeled DNA in nanofluidic channels with a combination of formamide and local heating, we obtain a sequence-dependent “barcode” corresponding to a series of local dips and peaks in the intensity trace along the extended molecule. We demonstrate that this structure arises from the physics of local denaturation: statistical mechanical calculations of sequence-dependent melting probability can predict the barcode to be observed experimentally for a given sequence. Consequently, the technique is sensitive to sequence variation without requiring enzymatic labeling or a restriction step. This technique may serve as the basis for a new mapping technology ideally suited for investigating the long-range structure of entire genomes extracted from single cells.     

Mutational specificity and genetic control of replicative bypass of an abasic site in yeast

Abasic (AP) sites represent one of the most frequently formed lesions in DNA, and they present a strong block to continued synthesis by the replicative DNA polymerases (Pols). Here we determine the mutational specificity and the genetic control of translesion synthesis (TLS) opposite an AP site in yeast by using a double-stranded plasmid system that we have devised in which bidirectional replication proceeds from a replication origin. We find that the rate, the genetic control, and the types and frequencies of nucleotides inserted opposite the AP site are very similar for both the leading and the lagging DNA strands, and that an A is predominantly inserted opposite the AP site, whereas C insertion by Rev1 constitutes a much less frequent event. In striking contrast, in studies that have been reported previously for AP bypass with gapped-duplex and single-stranded plasmids, it has been shown that a C is the predominant nucleotide inserted opposite the AP site. We discuss the implications of our observations for the mechanisms of TLS on the leading versus the lagging DNA strand and suggest that lesion bypass during replication involves the coordination of activities of the replicative Pol with that of the lesion-bypass Pol.     

Sequence dependence of DNA bending rigidity

For many aspects of DNA–protein interaction, it is vital to know how DNA bending rigidity (or persistence length, a) depends on its sequence. We addressed this problem using the method based on cyclization of short DNA fragments, which allows very accurate determination of a. Our approach was based on assigning specific values of a to each of 10 distinct dinucleotide steps. We prepared DNA fragments, each about 200 bp in length, with various quasi-periodic sequences, measured their cyclization efficiencies (j factors), and fitted the data by the theoretical equation to obtain the values of a for each fragment. From these data, we obtained a set of a for the dinucleotide steps. To test this set, we used it to design DNA sequences that should correspond to very low and very high values of a, prepared the corresponding fragments, and determined their values of a experimentally. The measured and calculated values of a were very close to one another, confirming that we have found the correct solution to this long-standing problem. The same experimental data also allowed us to determine the sequence dependence of DNA helical repeat.